Biological Effects of Laser Radiation1

Biological Effects of Laser Radiation1

BIOLOGICAL EFFECTS OF LASER RADIATION1 By Samuel Fine Northeastern University, Boston, Massachusetts, and Edmund Klein Department of Dermatology, Ro...

10MB Sizes 0 Downloads 15 Views

BIOLOGICAL EFFECTS OF LASER RADIATION1 By Samuel Fine Northeastern

University, Boston, Massachusetts,

and Edmund Klein Department of Dermatology, Rosweli Park Memorial Institute, Buffalo, New York

I. Introduction II. General Physical Principles and Technology A. Fundamentals of Laser Action B. Solid State Lasers C. High Peak Power Pulses D. Gaseous Lasers E. Semiconductor Lasers F. Nonlinear Optical Effects III. Paramagnetic Resonance Studies of Laser-Irradiated Biological Systems.. IV. Spectroscopic Ultramicroanalysis A. Soft Tissues B. Calcined Tissues V. Interactions with Macromolecular Preparations A. Enzymes B. Blood Group Substances C. 7-Globulin D. Discussion VI. Microscope-Coupled Laser Radiation A. Unicellular Organisms and Plant Cells B. Studies on Microcirculation C. Studies on Formed Blood Elements VII. Tissue and Cell Culture Studies A. Tissue Culture Studies B. Studies on Microorganisms VIII. Embryological Studies A. Effects on Chick Embryos B. Intrauterine Irradiation I X . Studies on Normal Animals 1

Page 150 151 151 156 158 159 159 160 160 161 161 162 162 163 165 166 166 167 169 170 171 173 173 175 175 175 175 176

Studies at Northeastern University, Boston, Massachusetts, and at Rosweli Park Memorial Institute, Buffalo, New York, were supported in part by Research Contracts DA-49-193-MD-2436 and DA-49-193-MD-2437 from the Surgical Research Branch, U.S. Army Medical Research and Development Command, Office of the Surgeon General, U.S. Department of the Army. 149

150

SAMUEL F I N E AND EDMUND K L E I N A. B. C. D. E.

Skin and Subcutaneous Tissue Abdominal and Pelvic Regions Irradiation of the Thorax Irradiation of Exposed Abdominal, Pelvic, and Thoracic Orga Irradiation of the Head and Brain

F. Effects of Dyes G. Studies at High Power Densities H. Miscellaneous Studies I. Comparative Studies J. Discussion X . Experimental Tumors X I . Clinical Studies

176 177 180 180 181 183 184 184 185 185 188 194

A. Studies on Normal Skin, Benign Skin Lesions, and Cytological Prep-

XII.

XIII. XIV. XV.

arations B. Studies on Basal Cell Carcinoma C. Studies on Nevi and Melanoma D . Exploration of Fiber Optics Ophthalmological Studies A. Absorption by Ocular Media B. Threshold Studies C. Studies on Ocular Lesions D . Temperature Measurements in Experimental Retinal Burns E. Protection of the Eye from Laser Radiation F. Retinal Coagulator Modes of Interaction Summary and Conclusions Addendum A. Oral Tissues B. Microscopy and Holography References I.

194 195 196 196 197 197 199 201 205 206 207 212 216 218 218 219 220

INTRODUCTION

The recent development of lasers has stimulated interest in the interactions of relatively coherent monochromatic electromagnetic radiation with biological systems. T h e relatively high degree of coherence and monochromaticity of laser radiation facilitates the attainment of high energy and power densities in the ultraviolet, visible, and infrared regions. Studies on the effects of laser radiation on biological materials have been under w a y for three years. Reviews on biological effects of laser radiation include those b y Litwin and G l e w (113), M a l t and Townes {125), and Fine, Klein, and Scott (46), and the proceedings of recent conferences on the interaction of biological systems with laser radiation (1,2,3). Biological studies of the interactions of relatively coherent m o n o chromatic radiation, as made available b y lasers, have been oriented toward four main areas: correlation of effects on biological systems

BIOLOGICAL EFFECTS OF LASER RADIATION

151

with those o n physical systems in order to assist in an understanding of factors underlying the interaction; application of lasers to assist in an understanding of biological systems; exploration of the adaptability of laser devices for medical diagnosis and therapy; assessment of hazards of laser radiation on both a short- and long-term basis, and the subsequent development of safeguards. Since investigations on biological effects of laser radiation have been in progress for only three years, the data are still largely preliminary or incomplete. D u e to the "state-of-the-art" of laser technology, consistent laser output has been difficult to obtain, and methods for the accurate measurement of various parameters of laser radiation, particularly during irradiation of a biological system, have y e t to be established. H o w e v e r , laser technology is advancing at a rapid pace. T h e expansion of available wavelengths and the development of improved instruments and techniques provided b y rapid advances in laser research and development should facilitate further studies of in vivo and in vitro effects. T h e studies on biological effects of laser radiation have required and will continue to depend upon close liaison between physical and biological scientists. II.

G E N E R A L P H Y S I C A L P R I N C I P L E S AND T E C H N O L O G Y

This review is concerned with biological effects of laser radiation. Discussion of physical principles and of advances in laser technology a n d will be limited in this has been reviewed elsewhere (140, 14$) presentation to provide general orientation. A. Fundamentals

of Laser

Action

T h e term "laser" is an a c r o n y m derived from the first letters of "Zight amplification b y stimulated emission of radiation." Another term in c o m m o n usage is "optical maser." T h e basis for this alternate terminology is the extension of the principle of " m i c r o w a v e amplification b y stimulated emission of radiation" to the optical region. Coherent electromagnetic radiation has been previously obtained at high power levels but not in the visible region of the spectrum prior to the development of lasers. Although radiation of a relatively high degree of spatial and temporal coherency at visible wavelengths can be obtained b y the use of a small pinhole and a number of reflectors ( F a b r y - P e r o t etalons of successively increasing s p a c i n g ) , the p o w e r transmitted tends toward zero as the coherency is increased. Lasers provide relatively coherent radiation at high power density levels in the ultraviolet, visible, and infrared regions of the spectrum. Laser radi-

152

SAMUEL FINE AND EDMUND KLEIN

ation m a y , at some wavelengths, provide power levels up to 1 0 9 W and, b y means of focusing, power densities exceeding 10 9 W / p e r m m 2. Present-day lasers operate primarily as oscillators, although oscillator-amplifier combinations have been used. T w o factors of importance, particularly for solid state lasers, are atomic energy levels and resonant cavities. 1. Atomic Energy Levels. In 1913, Niels B o h r indicated that atomic systems can exist only in a set of discrete energy states (see Born, 18). Transition between states can occur, associated with emission or absorption of energy as radiation, or with transfer of energy to another system. If radiative transition occurs, the frequency of the emitted or absorbed radiation is given b y (1)

f

where E12 is the difference between the initial and final energy levels, and h is Planck's constant. In 1917, Einstein (40) discussed not only absorption and spontaneous emission, but stimulated emission. H e showed [ E q . ( 2 ) ] that in a given radiation field of density uf at the frequency / , the probability of stimulated emission from an excited atom in state m to a level n(ufBmn) is equal to that of absorption of radiation b y an atom in level n excited to level m (ufBnm): (2)

Interaction of a photon of energy therefore result in the presence of two the interaction with concomitant return energy level. T h e stimulated radiation external radiation.

E12 with an excited atom can photons of energy E12 following of the excited atom to the lower is in phase with the stimulating

In the absence of excitation, the relative populations at the various energy levels are related b y E q . ( 3 ) : exp

(3)

where N2 is the number of atoms in state 2 (the higher energy state), A ri is the number of atoms in state 1, K is Boltzmann's constant, and T is the absolute temperature. F o r optical and near infrared frequencies E12 is of the order of 1 to 2 eV. A t r o o m temperature KT is 0.025 eV, and at liquid nitrogen temperature KT is correspondingly less. C o n -

BIOLOGICAL EFFECTS OF LASER RADIATION

153

sequently, in the absence of excitation, the entire population is primarily in the ground state. Under these circumstances, when quanta of an energy E12 are incident on the media, some of the quanta are absorbed, and the intensity of light I (at l o w power density levels), at a depth x, follows the relationship given b y E q . ( 4 ) : / = I0e~k* where I0 is the incident radiation, is consequently less than J 0.

(4)

and k the attenuation constant.

I

Consider a two-level system with a ground state Ely an excited level E2, and an energy difference E12. If the system has been excited, and a greater proportion of atoms are in an excited state E2 than at the ground state Elf "population inversion" is said to have occurred. Since the probability of interaction of a photon of energy E12 with an excited atom at an energy level E2 relative to the ground state is equal to the probability of interaction of that photon with an atom in the ground level Ex (i.e., Bmn = Bnm), when quanta at an energy E12 are incident on a medium where population inversion exists, more radiation is emitted than absorbed and the attenuation constant k becomes negative. Since Eq. ( 4 ) is still valid under these circumstances, the intensity of emitted light, I, is greater than I0. Thus electromagnetic amplification b y stimulated emission occurs. In the visible region, this is called light amplification b y stimulated emission. Although population inversion is therefore required for light amplification, a resonant cavity is not necessary. Usually, spontaneous decay of the system to the ground state occurs within fractions of a microsecond following excitation, thus making p o p u lation inversion difficult to achieve. H o w e v e r , in some systems a metastable state exists in which spontaneous decay from the metastable state is of the order of milliseconds. Under these circumstances, if light at a quantum energy E12 is incident on the media, sufficient time exists for light amplification to occur and be observed. T h e existence of loss mechanisms, such as scattering and absorption of photons b y impurities or other atomic and molecular energy levels, requires that there be sufficient density of E12 levels to provide the system with a net gain. 2. Resonant Cavity. A second factor of importance in a consideration of laser operation, or rather "light oscillation b y stimulated emission," is the resonant structure formed b y the lasing medium and reflecting surfaces. A simplified understanding of the resonant c a v i t y can be o b tained from a consideration of resonance and resonant length. T h e concept of resonance and resonant systems is c o m m o n to m a n y fields of

154

SAMUEL FINE AND EDMUND KLEIN

physical science. A n inductor-capacitor combination, a mass-spring system, and a vibrating string represent simple resonant systems which will oscillate at a frequency determined b y the physical parameters. Some characteristics of a resonant cavity are illustrated b y a consideration of a vibrating string model. Standing waves due to resonances, dependent on constructive interference, are obtained in a string rigidly held at one end and vibrated at the other end at the resonant frequency. These standing waves, occurring at one of a large number of natural frequencies for the string, are due to constructive interference between the w a v e traveling down the string and the w a v e reflected from the rigid support. A t resonance, an integral number of half wavelengths are present on the string of length T h e distance between two adjacent nodes where the amplitude of the transverse oscillations is minimum, or the distance between t w o adjacent antinodes, where the transverse oscillation is maximum, is A / 2 , where A, the wavelength, is: A

n = 1,

2, 3, 4,

• •

If the string is rigidly clamped at both ends, as in a violin, set vibrating, and left to itself, similar resonances occur. H o w e v e r , the oscillations die out because of energy dissipation through the end supports and the resistance of the air to motion. Q, the figure of merit of a resonant system or cavity, is dependent on the energy stored/rate of energy dissipation. A small resistance to motion (small damping) relative to the other parameters, which can be considered analogous to a high Q cavity, will result in slow d e c a y of the standing w a v e oscillations. A high resistance to motion (large damping) relative to the other parameters, which can be considered analogous to a low Q cavity, will result in highly damped oscillations or no oscillation at all. T h e vibrating string with low energy loss per cycle supplied with initial energy storage b y plucking is analogous to the laser media with reflecting end surfaces, pumped to a level sufficient to obtain population inversion. If sufficient energy is supplied near the peaks (antinodes) of the oscillations of the string at the correct frequency and phase, the resonant oscillations will continue and in fact can be increased in amplitude for the vibrating string and, similarly, for the resonant laser cavity. In a manner similar to the vibrating string, the laser c a v i t y must be an integral number of half wavelengths in length to have constructive electromagnetic interference between waves which propagate parallel to the rod axis. Unlike the vibrating string, the distance between successive nodes is of the order of 1 micron. T h e laser cavity consequently oscillates in a very high order m o d e ( n » l ) . For example, for a 6-inch ruby

BIOLOGICAL EFFECTS OF LASER RADIATION

155

crystal, lasing at 6943 A, n > 10 7. T h e frequency difference between two adjacent resonant modes is consequently very small (of the order of 1 micron for the example g i v e n ) . It has been shown in the previous section that, if an optical disturbance is allowed to propagate through a dielectric medium in which a population inversion has been attained between two energy levels, the process of stimulated emission will cause an exponential growth in the amplitude of the initial disturbance (in the photon density) via in-phase contributions along the direction of propagation. A similar final photon density can be obtained with shorter physical dimensions of the active material b y reflections from the end surfaces of the medium. This medium must be of a specific length to introduce constructive superposition between the various reflected electromagnetic waves at the desired wavelength. T h e lasing medium and reflecting surfaces (or external reflectors) serve as a resonant cavity, of quality factor Q. T h e resonant c a v i t y thus formed provides positive feedback, resulting in an increased interaction of photons with the excited atoms. A n increased photon density is p r o duced, at a photon energy E12 associated with reversion of the atoms from the excited to the ground state. A pulse of high peak power at a wavelength A = ch/E12 (where c is the velocity of light) is consequently produced. Since stimulated emission is initially triggered b y spontaneous radiative recombination from the metastable level, the system of active medium and reflecting surfaces can be considered as a light oscillator. This approach essentially neglects the energy input from the flash tube. F o r buildup to occur from stimulated emission for a particular resonant mode, the energy supplied b y the atoms during each pass of the beam through the laser cavity must be sufficient to overcome losses due to light transmission through the partially reflecting end surface, scattering at the reflecting surfaces and within the medium, and energy absorption without corresponding stimulated emission at the resonant frequency. T h e Q of the laser cavity is increased b y either suppressing these loss mechanisms or increasing the number of atoms excited to the metastable state per unit time. While transmission of light through the end surface is a loss mechanism insofar as the laser cavity is concerned, it is the means b y which useful laser radiation is obtained. This can be considered analogous to a useful loss mechanism where muscial sound is obtained when a string is plucked in air rather than in a v a c u u m . In an actual crystal, transitions from the metastable level occur not only at a discrete energy E12, but over a narrow band centered about E12. The wavelength of the stimulated emission is consequently not

156

SAMUEL FINE AND EDMUND KLEIN

at a single wavelength but over a band of frequencies. This band can be sufficiently broad to include several wavelengths which would resonate in the cavity. T h e predominant frequency in the laser beam will consequently be determined b y the distribution of photon energies within the band centered about 2 ? 12 and the Q of the laser cavity for that particular resonant wavelength. If the density of excited atoms is increased, oscillations occur for the modes with the least losses. A s stimulated emission occurs at these modes, fewer excited atoms are available for oscillation at modes with greater losses. Thus the relative energy and power densities would be concentrated in the major modes. B. Solid State

Lasers

Although the principle of stimulated emission has been recognized for over forty years, it appears that the first definitive proposals for utilization of this principle were made independently b y B a s o v and Prokhorov (8, 9, 10), T o w n e s et al. (73), and W e b e r (171). T h e first successful application of amplification b y stimulated emission was the ammonia maser developed b y Gordon, Zeiger, and T o w n e s in 1955 (74). This device operated at 24 gigacycles ( g c ) / s e c o n d , the inversion frequency of ammonia. In 1958, Schawlow and T o w n e s (156) proposed the extension of this principle to the visible region. In 1960, M a i m a n obtained light oscilla+ tions in ruby ( C r + + A l 2 0 3 ) (121, 122, 123). This was achieved b y p u m p ing an end-coated ruby rod with light from a flash tube, to a sufficient degree to obtain population inversion. T h e light output due to stimulated emission consisted of relatively coherent light at 6943 A emitted as

\

\ Ruby Pump lamp \ (flash tube) Dielectric (or silvered) 1 0 0 % reflecting coating \

Reflecting cavity '

\ \ . , Partial reflecting coating

FIG. 1. Simplified diagram of solid state laser.

BIOLOGICAL EFFECTS OF LASER RADIATION

157

FIG. 2. Output from ruby laser obtained with photodetector showing spiking. Sweep speed 0.5 msec per cm. Pulse duration 1 msec.

a pulse lasting about 1 msec in a beam of relatively small divergence. T h e essential components of this type of laser are shown in Fig. 1. T h e output from ruby is not continuous; each pulse consists of a number of spikes, each about 0.5 /xsec in duration (Fig. 2 ) . Although radiation within the spike is relatively coherent, there is generally no specific coherency for the radiation emitted between successive spikes. There also is considerable variation in spatial distribution across the ruby face during lasing, which affects the degree of coherency. T h e laser beam, although of narrow band width, is consequently not m o n o chromatic. It possesses a high degree of spatial and temporal coherency at high peak p o w e r density in comparison with other sources at optical wavelengths, but it not completely coherent. Adequate mathematical discussions of partial coherency have been developed (17). In a nondegenerate three-level system, represented b y the ruby (Fig. 3 ) , population inversion usually requires excitation of more than 5 0 % of the atoms. In a four-level system exemplified b y n e o d y m i u m - d o p e d glass, the terminal state for laser action is sufficiently a b o v e the ground state to form a relatively unpopulated state. Consequently, relatively few atoms need to be excited to result in population inversion. In crystal or glass lasers, the lasing element consists of a host crystal doped with various atoms. D o p i n g atoms are generally transition metals, rare earth atoms, or elements of the actinide series. In transitions of

158

SAMUEL FINE AND EDMUND KLEIN Absorption band

T

E2

_£t

_

Rapid non radiative transition • Metastable state Losing (or spontaneous) transition

- Ground state

B

A

FIG. 3. Simplified diagram of ( A ) three-level system; ( B ) four-level system.

electrons within the inner shells, shielding b y the outer electrons results in the occurrence of sharp spectra even when the atoms are in a host lattice. In some solid state systems, as the ruby, excitation is generally effected b y pumping with relatively broad band radiation. In other systems, with sharper transitions, pumping is effected with relatively narrow band radiation. Transition of the excited atoms to a metastable state is relatively rapid. Stimulated emission, due to buildup from initial spontaneous emission, occurs. Partial reflections occur at the reflecting mirrors and lasing action (or light oscillation) results. C. High Peak

Power

Pulses

Studies b y Hellwarth et al. (88, 115) resulted in the attainment of high peak power b y preventing oscillation until substantial population inversion was achieved. This can be produced by altering the reflectivity between one of the cavity reflectors and the cavity, thus affecting the quality factor (Q) of the cavity. Techniques for doing this include: 1. A rotating prism driven b y an electric or air-driven motor with critical positioning of the prism for production of oscillation at a time at which a high population inversion is present. 2. Interposition of a Kerr cell shutter which prevents the reflection of radiation until high population inversion has been achieved. 3. Interposition of thin films of organic dyes on glass plates, which alter irreversibly in transparency, allowing such units to be employed as "single-shot passive Q-switching" elements (127). 4. Interposition of metal phthalocyanines (163) dissolved in liquid organic solvents. These show enough bleachable absorber action when a certain intensity has been reached to serve as repeatable passive Q-switch elements for ruby lasers.

BIOLOGICAL EFFECTS OF LASER RADIATION

T h e advantage of passive Q-switching for biological studies

159 is its

low cost. It is unsuitable where pulse repetition frequency with an exact pulse-to-pulse interval is required. D.

Gaseous

Lasers

Lasing was first reported in a helium-neon gas mixture in 1961 (94). Since then, lasing, or stimulated emission in gaseous systems, has been reported at over 100 wavelengths, extending from the ultraviolet into the infrared (11, 12, 20, 56) to b e y o n d 30 microns. T h e basic design of the gas laser is relatively simple—a cylindrical discharge tube within an optical resonator, pumped in several w a y s including rf and dc. T h e length of the units in which lasing action has been obtained varies from a minimum of 2 inches to m a n y feet in length. A t some w a v e lengths, gas lasers can be operated continuously at power outputs exceeding 10 W and pulsed at peak power exceeding 100 W . Gaseous lasers can be made to exhibit a higher degree of coherence than either the solid state or semiconductor laser. T h e beam can therefore be focused to a small diffraction-limited spot size. T h e multitude of wavelengths and the high degree of coherence attainable should be of considerable interest for biological studies, particularly when coupled to a microscope system. E. Semiconductor

Lasers

Lasing in semiconductors was initially reported b y N a t h a n et al. Hall et al. (78), and Quist et al (146).

(186),

T h e semiconductor laser, whose operation depends on a p-n junction, consists of p and n doped compounds. Lasing was initially reported in gallium arsenide at 8400 A . Since then, lasing has been obtained in gallium arsenide phosphide alloys, indium phosphide, indium arsenide, indium gallium arsenide, indium stibnate, and indium phosphide arsenide. T h e wavelength ranges covered b y these lasers extends from 6600 to 51,000 A. A sharp line has been reported in silicon carbide p-n junctions at 4560 A . These, as well as other properties of semiconductor lasers, are discussed in reviews b y Burns and N a t h a n (22) and b y L a x (111). Continuous operation of G a A s lasers at levels in excess of 1 W has been attained at 2 ° K (21, 111); lasing has also been observed at r o o m temperature (91). Second harmonic radiation a c c o m p a n y i n g the 8400-A radiation of G a A s has been obtained. H o w e v e r , the output is small, 1.6 X 1 0 " 12 W , a c c o m p a n y i n g fundamental power of 5 X 10- 3 W (5,124). T h e potential attractiveness of the semiconductor laser for biological studies resides in its ease of lasing control b y electrical pumping and

160

SAMUEL FINE AND EDMUND KLEIN

its intrinsically small size. These factors m a y be particularly pertinent for intracavity and ophthalmic applications. F. Nonlinear

Optical

Effects

Nonlinear effects can be produced with lasers. This can result in a relatively coherent beam at a frequency which is a multiple of the primary laser frequency. Optical second harmonic generation, initially produced b y Franken et al. at the University of Michigan, using a ruby laser directed at a quartz crystal, resulted in a beam at a w a v e length of 3470 A (57). Efficiencies of the order of 1 part in 1 0 12 were obtained. Conversion efficiencies in second harmonic generation approaching 2 0 % have since been obtained in various laboratories, using index-matching techniques and Q-switched lasers (168, 169). Third harmonic generation has been produced. R a m a n laser action has been obtained b y W o o d b u r y et al. (37, 174). This involves absorption of incident laser radiation at a frequency j L , emission of a photon at fL ± fr, and a change in the vibrational energy of the molecule b y hfr. T h e theories concerning these effects have been discussed b y Terhune and Bloembergen (15,168). Pulsed laser radiation can be obtained at high p o w e r levels at w a v e lengths ranging from 2300 A to the infrared (168) b y use of frequency multiplication, R a m a n shift, and frequency mixing. T h e total energy within each pulse will, in general, be small. These techniques, however, m a y provide wavelengths, particularly in the ultraviolet, of significance for biological investigations at high peak power density. III.

P A R A M A G N E T I C R E S O N A N C E STUDIES OF L A S E R - I R R A D I A T E D BIOLOGICAL

SYSTEMS

Studies on the production of free radicals following laser irradiation m a y be of importance because of accidental exposure of laboratory workers. These studies m a y assist in understanding the physicochemical alterations associated with the potential production of new materials with radically changed characteristics produced b y high peak power density radiation. Free radicals have been suggested to be of importance in carcinogenesis and aging, and are accepted as factors in the primary mechanism of radiation damage to solids. Paramagnetic resonance has been observed in biological systems after heating and irradiation at X - r a y , ultraviolet, or visible wavelengths (16). Electron paramagnetic resonance studies of biological interest have been reviewed b y Sogo and T o l b e r t (161) and b y Smaller (160). C o m m o n e r and co-workers have studied free radicals in lyophilized nonirradiated biological systems (30). Free radi-

BIOLOGICAL EFFECTS OF LASER RADIATION

161

cals were found in a number of tissues including mouse liver; studies on skin were n o t reported. Studies on the production of free radicals in biological systems associated with laser irradiation were carried out b y Derr et al. (32, 83). Biological specimens irradiated at 6943 A included white and black mouse skin (in vivo), mouse liver, and preparations of fibrinolysin and collagenase. Signals were obtained in both nonirradiated and irradiated mouse liver. Resonances were not observed in white mouse skin and collagenase ( 2 : 1 signal to noise ratio for 1 0 14 spins). Resonances observed in black mouse skin and preparations of fibrinolysin, following irradiation, indicated with high probability that free radicals were generated in those biological systems b y laser irradiation. Hyperfine and line width determinations were not carried out. Further studies on pertinent in vitro systems, including proteins, chlorophyll, amino acids, and nucleic acids are warranted. Comparison with electron-spin resonance spectra which have been obtained following X - r a y or y-irradiation should be of interest. Electron-spin resonance studies during laser irradiation m a y provide information concerning intermediates occurring during irradiation. IV.

SPECTROSCOPIC U L T R A M I C R O A N A L Y S I S

A laser spectrograph has been developed (19, 137) and used for analysis of metallurgical faults. T h e operation of this equipment involves the focusing of a beam from a Q-switched ruby laser through a microscope on a selected small target. A sample from the preselected area is v a p o r ized b y the laser beam. B y means of carbon electrodes the temperature of the gases is then raised to spectral emissive levels. T h e resultant atomic spectra from the vaporized material are recorded photographically b y a spectrograph. This device has been used for spectroscopic analysis of tissues. T h e sample of tissue vaporized is about 50 microns wide and its weight is of the order of 10~7 gm (148). Ashing of the tissue is not necessary and analytical results are rapidly available (148). A. Soft

Tissues

With this type of unit, spectra have been obtained from sections of various normal tissues including kidney, adrenal, skin, pancreas, aorta, and brain, removed from the animal prior to analysis (63, 84, 120, 148). T h e elements determined in preliminary in vivo laser spectroscopy studies on the skin of normal intact b l a c k and white mice, and on melanomas in mice, include Fe, M g , Si, Cu, T i , A g , and Ca (54).

162

S A M U E L F I N E AND EDMUND K L E I N

B. Calcified

Tissues

Studies on in vitro undecalcified sections of canine cortical bone and on costochondral junctions were carried out b y Lithwick, H e a l y , and Cohen (112). Ca, P, M g , A l , and Si were consistently present. T i , Cu, Zi, and Y were present in lower concentration. T h e concentrations of M g , A l , and Si were found to be higher in y o u n g osteons than in the older osteons. P b was absent although it should be readily detectable by this method. Mechanical effects on bone were studied in association with spectroscopy. In old osteons, a stellate fracture resulted when the target included the Haversian canal, while an excavation was produced when the site of irradiation did not include a Haversian canal. T h e y o u n g osteons showed excavations or perforations limited b y the terminal cement line. Preliminary studies to determine the inorganic composition of calcified tissues were carried out b y H . M . Goldman, Ruben, and Sherman (63, 158) on supra- and subgingival calculi, mandibular cortical bone, and cervical and apical cementum. A variation in the concentration of P and M g between cortical and trabecular bone was noted in a study on the rat mandible. During wound healing, significant variations in Al content was noted on analysis of gingival tissues of the dog. Variations in M g and Si concentrations were observed in the osseous tissue. Further investigations are oriented toward quantitation of the method, and reduction of spectroscopic contamination of the specimen b y extraneous material. The technique m a y prove of significance in studies on localized changes in biological tissues, which are associated with variations in trace element concentrations. Use of the laser spectrograph in combination with the image converter high-speed camera operated in the streak mode has been considered for transient spectroscopy. V.

I N T E R A C T I O N S W I T H M A C R O M O L E C U L A R PREPARATIONS

Pulsed irradiation at relatively high energy density, of the order of 10 J (joules) per c m 2, has been studied in biological preparations using flash tube equipment. Although most flash photolysis studies were carried out in the gaseous phase, studies on solutions of macromolecules of biological origin have been reported b y Gibson and Ainsworth (62) and Grossweiner and M u l a c (77). In studies on pulsed photoexcitation of ovalbumin b y Grossweiner (76) and Grossweiner and M u l a c ( 7 7 ) , broad transient absorption spectra of both helium-saturated and airsaturated ovalbumin were determined. The transient absorption spectra of small molecules were studied in order to elucidate the sites of photon interaction with the macromolecule.

BIOLOGICAL EFFECTS OF LASER RADIATION

163

With the development of lasers, sources became available which provide high power densities at relatively narrow band widths as compared to those obtained from flash tube systems. T h e objectives of laser studies on macromolecular preparations include: (1) Determinations of changes in biological activity or in p h y s i c o chemical properties and elucidation of parameters affecting such alterations. (2) Exploration of effects of energy transfer agents on the induction or modification of the interaction. (3) Investigation of differential interaction with diverse biochemical preparations. A.

Enzymes

Optical density

Klein et al. (100) reported studies on aqueous lipase preparations containing methylene blue ( M b ) irradiated at 6943 A at energy levels ranging from 3 to 100 J per pulse. T h e number of exposures per sample varied from 1 to 20. The activity of lipase decreased (Fig. 4 ) as the total energy of the radiation was increased in the presence of M b ; in the absence of the dye, changes in lipase activity were not significant. T h e heat-labile c o m p o n e n t of pancreatic lipase was shown to have retained its activity, while the relatively heat-stable lipase cofactor was

° ' l 00

2

4

6

8

10 12

14

16 18 2 0 2 2 2 4 2 6 2 8 3 0

Time in minutes

FIG. 4. The effect of laser irradiation on the activity of pancreatic lipase in the presence of methylene blue and Janus green B. K E Y TO C U R V E S : 1, Irradiation of lipase without dye; 2, control lipase without dye; 3, irradiation of lipase with 0.005% methylene blue; 4, control lipase with 0.005% methylene blue; 5, irradiation of lipase with 0.005% Janus green B; 6, control lipase with 0.005% Janus green B.

164

SAMUEL FINE AND EDMUND KLEIN

deactivated b y laser radiation. Proteolytic activity present in the lipase preparations was not altered b y laser radiation whether M b was present or not. These findings suggested that interactions other than thermal denaturation were associated with changes in biological activity. T h e effects on the enzyme system were produced only in the presence of an energy transfer agent, methylene blue. T h e enzymatic activities were differentially affected, since lipase activity was reduced, while proteolytic activity remained unchanged. The effects were not considered to be due to general heat effects, as indicated b y the finding that the heat-stable and not the heat-sensitive component of lipase was deactivated b y laser radiation. Although the temperature of the lipase suspension was raised from 2 ° C prior to irradiation to only 8 ° C following irradiation, transient localized heat effects cannot be excluded as causes of the differential reduction of lipase activity, particularly since lipase is considerably less heat-stable than pancreatic proteases. Thermal denaturation of lipase is usually accompanied b y changes in the electrophoretic and ultracentrifugal characteristics, which were not demonstrated following laser irradiation. Igelman et al. {92, 93) reported reduction in peroxidase activity, but found no changes in the activities of tyrosinase, trypsin, lysozyme, dehydrogenase, catalase, and amylase following laser irradiation. Rounds (149) reported 6 0 % reduction in the activity of lactic dehydrogenase following irradiation at 3471 A . Irradiation of D P N H or of the lactic dehydrogenase apoenzyme did not result in reduction of the activity, when the separately irradiated preparations were combined. The various parameters of plasmin activity were investigated (104) following laser irradiation at 6943 A (30-70 J / c m 2 ) to determine whether the activities of an enzyme preparation could be differentially altered b y laser irradiation with respect to the diverse substrates upon which it acts. In the absence of M b , the various parameters of plasmin activity remained unchanged following laser irradiation. In the presence of M b , the activities of the plasmin preparations were decreased b y laser irradiation. After 40 successive exposures, fibrinolytic activity was reduced to insignificant levels while appreciable levels of plasminogen activator activity, caseinolytic activity, and T A M e esterase activity remained. T h e observations on plasmin indicated that laser radiation m a y differentially affect the activities of an enzyme on its different substrates. This suggests that different activities of the same enzyme m a y be affected to different degrees or in different ways b y laser radiation. These effects m a y be due to differential primary interactions with the respective functional groups on the enzyme molecule, or to differential secondary effects following the primary interaction. T h e heterogeneity of the enzyme prep-

BIOLOGICAL EFFECTS OF LASER RADIATION

165

aration m a y also be involved, since the energy transformations associated with laser radiation m a y result in preferential interactions with some members of a population of closely related enzymes. A s the plasmin preparations studied were not pure, it is possible that one or more of the changes in activities were associated with components other than the fibrinolytic enzyme. T h e initial rates of plasmin activation were considerably lower than those of the corresponding control preparations. T h e rate of the second phase of activation, which represents a relative decrease in fibrinolytic activity, however, was not altered b y laser irradiation (103). This m a y be a further indication of a differential effect on enzyme activity at stages at which it is the limiting factor in the rate of the reaction as compared to stages at which other rate-limiting factors predominate. In order to determine whether these phenomena reflected effects on the plasminogen molecule or extended to the activator, urokinase was irradiated in parallel studies (103). In the presence of methylene blue as the energy transfer agent, the activity of urokinase in the activation of plasminogen was markedly reduced b y laser irradiation. B. Blood

Group

Substances

Studies on blood group substances were carried out in order to explore whether laser irradiation would alter the specific reaction of an antigen with a corresponding antibody (103). The activity of the blood group substances was assayed b y inhibition of hemoagglutination. Following laser irradiation, the activities of some preparations of the blood group substances were increased. T h e activity of blood group substance B was more markedly affected than that of blood group substance A . Heating of preparations of the blood group substances at 1 0 0 ° C for 10 minutes did not alter their activities. T h e inhibitory activity of irradiated (and control) specimens in the hemoagglutination test was not significantly altered b y dialysis. T h e electrophoretic mobilities and the boundaries observed on analytic ultracentrifugation did not reveal differences between irradiated and control specimens. T h e irradiation of blood group substances which resulted in an increase in immunochemical activity suggests that the number of functional groups required for the specific interaction of agglutinogen with isoagglutinin m a y have been increased. This could have occurred due to fragmentation of the molecule resulting in the liberation of active groups. Alternatively, the molecular conformation of the blood group substances m a y have been altered in such a manner that previously inaccessible functional groups were relocated in positions in which they were available for reaction. Interaction with laser radiation m a y also have

166

SAMUEL FINE AND EDMUND KLEIN

produced functional groups de n o v o or destroyed an inhibitory activity. Since dialysis of the irradiated specimens did not alter their activities, relatively large molecules appear to be associated with the increase in the agglutinogen activity. Failure to reveal differences between the electrophoretic and ultracentrifugal patterns of the controls and of the irradiated preparations does not exclude involvement of molecules which m a y have been too large to be dialyzable, but not large enough to be reflected b y changes in the moving boundaries. C.

y-Globulin

Studies on y-globulins were carried out to investigate the effects of laser radiation on proteins in regard to protein-protein interactions {103, 104). In the reaction of antiserum with preparations of human y-globulin, maximum precipitation occurred at lower concentrations of antigen (i.e., human y-globulin) when it had been irradiated than with the untreated controls. T h e curves for the reactions at serial dilutions of the irradiated specimens at constant levels of antibody were shifted to the r i g h t ' o f the control curves. T h e irradiated y-globulin preparations reacted less actively with rheumatoid serum than the untreated controls, while heated y-globulin showed a more intensive reaction. Upon gel diffusion and immunoelectrophoresis there were no apparent differences between irradiated and control y-globulin preparations in regard to the location of the precipitation arcs. Irradiation of preparations of y-globulin indicated differential effects on members of this molecular species. Irradiation resulted in loss of reactivity with serum of patients with rheumatoid arthritis, while energy of the same order of magnitude applied b y raising the temperature was without effect or increased the reactivity of the y-globulin preparation in this system. In the reaction with heterologous antibodies to human y-globulin, the irradiated antigen showed maximum reactivity at lower concentrations (of the antigen) than the untreated controls. Some of the irradiated specimens not only reacted maximally at lower concentrations, but also showed higher levels of maximum reactivity than the controls. Irradiation of heterologous antibodies to limulus serum appeared to increase the areas of the precipitin curve, quantitated photoelectrically, in comparison with control specimens. D.

Discussion

T h e changes in the immunochemical properties of the blood group substances and of the y-globulin preparations were produced b y laser radiation at 6943 A without the addition of an energy transfer agent such as methylene blue. A t the low power levels of standard spectro-

BIOLOGICAL EFFECTS OF LASER RADIATION

167

photometers, radiation at 6943 A is transmitted without detectable a b sorption b y these preparations. A t the relatively high power levels (10-100 k W range) of laser radiation, absorption of energy at 6943 A occurs, as shown b y the changes in biological activity. T h e fraction of transmitted energy does not appear to be a linear function of the energy of the incident radiation. Additional w o r k is needed to determine the relationship between incident and absorbed energy at a given w a v e length for these macromolecular preparations. If the lifetime of the excited state of a macromolecular population is of the same order of magnitude as the duration of the pulse, then significant interaction between excited molecules can occur. E v e n if the lifetime of the excited state is several orders of magnitude less than that of the pulse duration, a considerable number of molecules will still be in the excited state at any one time. Interactions, which are normally not detected, m a y become measurable b y end product analysis. M o l e c u l a r changes due to interactions between excited states m a y consequently be produced b e y o n d those which could be recognized if the equivalent energy were delivered at low power levels. Because of possible transient absorption states, the possibility of interaction of quanta with excited states of the atom also exists and m a y result in a detectable number of altered molecules which might not otherwise be measurable. VI.

MICROSCOPE-COUPLED LASER

RADIATION

Laser units have been coupled with microscopes to permit irradiation of biological specimens at high levels of magnification, particularly single cells and specific structures within cells. Optics applicable to the laser microscope are discussed in standard optics texts (34, 85, 14?), and have been reviewed b y T o w n e s (170) and M a l t (126) with particular relevance to laser-microscope systems. T w o factors of importance in laser-microscope systems with reference to laser irradiation are the spot size obtainable and limits of resolution between two successive irradiations. T h e almost parallel wave emitted coherently from the surface of a laser rod can, in the limit, have an angle of divergence primarily due to diffraction of about A / 7 ) , where A is the wavelength and D the diameter of the face of the rod. F o r D = 1 cm and A = 5,000 A, the angular divergence can be 1/20,000 rad (170). T h e angular divergence of crystal lasers has been much in excess of this, partially due to the lack of coherency across the emitting surface of the ruby. A n angular divergence less than 25 seconds of arc or less than 1/8000 rad has been achieved with gas lasers with plane parallel resonators. F o r a parallel, coherent beam passing through a small circular aperture

168

SAMUEL FINE AND EDMUND KLEIN

the image is diffraction-limited, resulting in an A i r y pattern consisting of a bright central disc surrounded b y a series of alternate bright and dark

rings

(34).

E i g h t y - f o u r percent of the light is concentrated

in

the central disc ( A i r y ' s d i s c ) . For

a lens of diameter

and

D,

focal

length / , the

central

bright

disc has a diameter d at the focal plane: (5)

d =

and, for small a, subtends an angle a = d/f T h e actual

at the lens

(147).

spot size achievable b y a good lens system is directly

related to the approximation of the actual divergence of the light to the theoretical diffraction limit k/D.

F o r a beam of the limiting divergence,

the spot size is limited b y diffraction to a linear dimension of the order of A / 2 . In the optical region this is about -g- micron and is a good representation of the minimal irradiation microscope system If

spot size achievable with a laser-

(170).

it is assumed that two points can be distinguished

as separate

when the center of one lies just outside the central A i r y disc of the other, then the limiting angle of resolution b y a lens is given b y E q . (6):

e

(6)

Therefore, m a x i m u m resolution between t w o irradiations

occurs with

short wavelengths of laser light and large diameter objectives. A second criterion, based on wavelength and numerical aperture

( N . A . ) , would

give a resolution d, approaching that of E q . ( 7 ) : (7)

d

The resolving power is therefore of the order of A / 2 for the standard microscope with an N . A . of about 0.95. Features which are desirable in laser-microscope systems include: 1. High, uniform intensity

over a small area, in the plane of the

object of interest. 2. L o w intensity in regions b e y o n d this area in this plane. 3. R a p i d beam divergence b e y o n d the plane of interest, in order to attempt to limit the high p o w e r and energy density to the plane of interest. 4. A diffraction-limited spot size in some cases. T h e laser b e a m is initially n o t parallel. Some aberration in the

microscope system. T h e o b j e c t under

is present

study is not located

at

BIOLOGICAL EFFECTS OF LASER RADIATION

169

the focal plane of the microscope where the spot size is smallest. H o w ever, marked differences in spot sizes have not been demonstrated, whether a film used as a test o b j e c t was placed at the principal focus of the objective or in the usual object plane. Spot sizes are usually determined b y the observable spot size on Polaroid film or a metallized film. A laser microscope developed b y the Technical Research Group e m ploying a laser with energy output in excess of 200 mJ at 6943 A, coupled to an upright microscope, can be focused to a spot size smaller than 5 microns. A similar type of unit has been developed b y Optics T e c h n o l o g y . A passive Q-switched laser coupled to an inverted microscope has been developed b y M a s e r Optics, Inc. A continuous gas laser operating at 6328 A has been focused through a trinocular microscope and a spot size of 2.5 microns obtained (125). Addition of a diaphragm t o these units should assist in obtaining a spot size approaching 1-2 microns. A.

Unicellular

Organisms

and Plant

Cells

Investigations on plant and animal cells have been carried out b y Saks et al., using a laser microscope (154,155, 178). T h e laser microscope facilitated destruction of a small section of the cell wall, to allow m i c r o needles and micropipettes operated b y a micromanipulator (108) to enter the cell readily (Fig. 5 ) . Sections of the cell wall and of cytoplasmic and nuclear constituents of the alga spirogyra were destroyed or altered at energy levels in the millijoule range. Methylene blue was used to provide vital staining for enhancing differential absorption of the radiation b y various cellular constituents. In further studies (155) sol-gel equilibria were found to be altered in amebae, producing modifications in ameboid movement. These alterations were both structural and functional, as indicated b y different rates of pseudopod formation and extension. T h e presence of green pigments (chlorophyll) or of blue and green dyes enhanced and localized the effects of irradiation. Cytoplasmic proteins became denatured, as indicated b y "pinching off" reactions in amebae and b y the production of spontaneous D e v a u x effects at the surface of oil drops which had been microinjected into the cytoplasm. R e c o v e r y of cells showing changes in structure or function of subcellular components were reported. Irradiated amebae had a significantly lower reproduction rate than nonirradiated controls. T h e growth rates of internodal cells of Nitella were reduced. M i c r o d r o p s of mineral oil or olive oil containing Nile blue sulfate were injected into the cytoplasm of amebae (178). W h e n the injected oil drops were irradiated through the 4 0 X objective, the microdrops

170

SAMUEL FINE AND EDMUND KLEIN

FIG. 5. A 6.4 micron opening produced in the cell wall of Nitella with a single pulse of 20 mJ incident at 6943 A X 200. (Courtesy of Dr. W . Saks and Dr. R. Zuzulo, New York University, New York.)

became distorted and were ejected. If the oil drops were repeatedlyirradiated with a beam minified 1450 X , accumulation of cytoplasmic debris occurred at the oil-cytoplasm interface without ejection of the oil drop. Essentially the same effect was produced in microdrops of olive oil or mineral oil containing Nile blue sulfate. If the amebae were stained with methylene blue, or if Nile blue sulfate was microinjected into the cytoplasm after the microinjection of the oil, the oil drops were ejected following irradiation. M u c h of the stained cytoplasmic debris was also ejected along with the oil. Saks et al. [155) concluded that injuries produced b y the smallest beam were specifically localized, and that even smaller beam sizes might produce highly localized changes in subcellular structures such as chromosomes or parts of chromosomes. B. Studies

on

Microcirculation

Studies on effects of laser radiation on microcirculation were carried out on skin flaps b y means of a laser coupled through a microscope (51). Flaps of abdominal skin and exteriorized mesentery with retention

BIOLOGICAL EFFECTS OF LASER RADIATION

171

of blood supply of anesthetized Swiss white and C 5 7 b l a c k mice were irradiated at 6943 A. T h e spot size obtainable at the focal plane was of the order of 5 microns. Emphasis was placed on the interaction with blood vessels. Some of the factors affecting the interaction were the energy and power density of the radiation at the site of impact, spot size, and diameter and shape of the blood vessel. A t low energy levels, constriction of the blood vessel occurred and persisted for several minutes. A t higher energy levels, blood flow within the vessel was arrested. This was accompanied b y formation of an intravascular clot. T h e structure of the wall of the blood vessel did not appear to be overtly altered. U p o n further increase in energy, aneurysmal dilatation occurred at the site of the clot formation. Interstitial extravasation of blood from smaller vessels and at sites of bifurcation was also produced (51, 104). Studies on small blood vessels were carried out b y K o c h e n and Baez (6, 106) with a T R G laser-microscope system. Exposure of muscular microvessels resulted in hyperresponsiveness to vasoactive agents and enhanced vasomotion of precapillary sphincters and metarterioles. A n increase in laser pulse intensity resulted in a distinct lesion on the inner surface of the irradiated vessel. Adherence of blood elements o c curred at that site. T h e cell aggregate either obstructed the vessel lumen or dislodged as an embolus. D i s l o d g m e n t was followed b y recurrence of agglutination. Obstruction, hemorrhage, and recanalization were o b served. Further studies included effects of laser radiation on acutely denervated rat mesoappendix perfused with mammalian Ringer solution, bovine albumin solution, and heparinized rat blood. C. Studies

on Formed

Blood

Elements

Studies on single cells irradiated at 6943 A using a ruby laser coupled through a microscope have been carried out at the French National Transfusion Center b y Bessis and Ter-Pogossian (14). T h e results of the interaction were observed through a closed-circuit television system. Breakdown of red cells appeared to occur at the site of impact. W h i t e blood cells and tissue cultures were also irradiated b y Bessis et al. (13, 14). Janus green was used as a vital stain for mitochondria in an attempt at selective irradiation of these cellular components. T i m e lapse photography was used to observe the effects on the irradiated cell and on adjacent cells to determine whether they were indifferent, attracted, or repelled. In parallel with in vivo studies b y Fine et al. on effects of laser radiation on vascular structures (51), studies were carried out with the lasercoupled microscope at 6943 A to determine the effects on separated

172

SAMUEL FINE AND EDMUND KLEIN

blood elements (104).

Studies on human red cells, white blood cells, and

platelets and on red and white cells of alligator blood were carried out. H u m a n and alligator red blood cells were damaged as the energy delivered was increased. T h e degree of damage varied with the energy of the radiation and the site of irradiation. H u m a n red cells usually contracted into an irregularly shaped b o d y of 2.5 microns in width in various planes; alligator red cells (elliptical, approximately 30 X 10 microns) contracted into multilobed structures, about one tenth to one third of the preirradiation size. W h e n a part of the cell not containing the nucleus was irradiated, the nonirradiated part remained relatively unchanged (Fig. 6 ) . R a d i a t i o n at

similar energy levels had no apparent

effect

on the

m o r p h o l o g y or ameboid m o v e m e n t of granulocytes. Addition of methylene blue (final concentration 0.0001%) ment, but the

altered

markedly the effects

site and energy of irradiation,

entirely

fragmented.

Ameboid

did not arrest ameboid m o v e of irradiation.

Depending on

the white cells were partially

m o v e m e n t was lost even when

or

struc-

tural integrity appeared to have been retained. Irradiation of platelets showed no apparent

effects unless methylene blue was added. In

the

presence of methylene blue, irradiation of platelets resulted in fragmentation with the release (or formation) of granular material.

FIG. 6. Alligator red cells exposed to microscope-coupled laser irradiation. The nonirradiated part of the cell, which contains the nucleus, remains unchanged (arrow).

BIOLOGICAL EFFECTS OF LASER RADIATION

VII.

173

T I S S U E AND C E L L C U L T U R E STUDIES

.1. Tissue

Culture

Studies

Tissue culture studies have been carried out b y R o u n d s et al. (11+9, 150-152). D o r s a l root ganglion cells of 7 - 1 0 - d a y chick embryos in plasma clot in the R o s e chamber were irradiated at 6943 A at 25 J per c m 2. Slight nuclear rotation of the neuron was observed as well as active pulsatile activity of the Schwann cells. I t was concluded that satellite cells in intimate association with the cytoplasm o f the neuron m a y be sensitive to laser radiation. Following laser irradiation of H e L a cells in tissue culture, chromosome abnormalities were observed, which morphologically resembled the nuclear changes following X-irradiation (151). Chromosomal abnormalities (Fig. 7) were induced in rabbit endothelial cells, which persisted through three subcultures following laser irradiation (151). Exposure to 25 J per c m 2 at 6943 A at a pulse duration of 1 msec resulted in immediate destruction of pigmented human skin, pigmented rabbit retinal epithelium (Fig. 8 ) , and a line of pigmented mouse melanoma. Similar but nonpigmented structures such as Caucasian skin, retinal epithelium from an albino rabbit, and a strain of mouse lung cells

FIG. 7. A chromosomal spread of a rabbit endothelial cell showing a dicentric chromosome (arrow) which persisted through 3 subcultures after exposure to 2 5 J per cm2 laser radiation. (Courtesy of Dr. D. Rounds, Pasadena Foundation for Medical Research, Pasadena, California.)

174

SAMUEL FINE AND EDMUND KLEIN

FIG. 8. (A) An image of pigmented retinal epithelial cells from a 12-day chick embryo prior to irradiation. The dark spheres constitute a cluster of pigmented cells which were not spread on the coverslip. The arrow draws attention to a metaphase figure containing pigment granules. (Courtesy of Dr. D . Rounds, Pasadena Foundation for Medical Research, Pasadena, California.) (B) The same area shown in Fig. 8A immediately following irradiation with 100 megawatts per cm 2 at a wavelength of 5300 A obtained through frequency doubling of a neodymium Q-switched laser. The cluster of pigmented cells and the mitotic figure (arrow) were disrupted. Other elements in the background were injured or killed, depending on the density and number of pigment granules per cell. (Courtesy of Dr. D . Rounds, Pasadena Foundation for Medical Research, Pasadena, California.)

showed no morphological changes for at least 24 hours under similar conditions of irradiation. Retinal epithelium cells containing more than eight pigment granules were destroyed, in contrast to cells with less then eight, when irradiated at 6943 A at 150 J per c m 2. Studies were carried out at 3471 A, 6943 A, and a combination of these wavelengths on pigmented and nonpigmented cell types. Cytotoxic effects leading to death within 1 hour occurred in nonpigmented rabbit endothelial cells irradiated at 3471 A , whereas 6943 A produced minimal injury to the same cell type (152). Deoxyribonucleic acid ( D N A ) metabolism was studied (153) in synchronized mammalian cells ( H e L a ) following laser irradiation. Cells maintained in tissue culture were synchronized with reference to D N A synthesis. Folic acid antagonists were employed to block D N A synthesis. Tritiated thymidine was added following a 16-hour block in the postmitotic growth period ( d ) and the cells allowed to proceed through D N A synthesis (S) and into the premitotic phase where they were fixed. T h e cultures were irradiated at 6943 A at a total energy density of 10 J per c m 2 during either the Ci or S phase. D N A metabolism,

BIOLOGICAL EFFECTS OF LASER RADIATION

175

as detected b y autoradiographic methods, following incubation in tritiated thymidine was measured. Differences in thymidine uptake in the Ci and S phase, respectively, were observed, which suggested the possibility of a b l o c k in D N A synthesis following laser irradiation. B. Studies

on

Microorganisms

Studies on the effects of laser radiation on microorganisms at 6943 A were carried out b y Klein, Fine, Neter, et al. (103, 104) a dn lethal effects on several species of cocci and coliform and chromogenic organisms observed. Surviving organisms of the Pseudomonas group showed reduction in growth and temporary decrease in pigment formation in subculture. Irradiation of a strain of Aspergillus niger in the presence and absence of methylene blue had no observable effects on this fungus. L . G o l d m a n et al. reported (68) destruction of cutaneous elements in Pityriasis versicolor following irradiation of cytological preparations in the presence of Evans blue. Changes were not observed in unstained preparations. V I I I . EMBRYOLOGICAL S T U D I E S

A. Effects

on Chick

Embryos

C h i c k e m b r y o s have been irradiated with a pulsed ruby laser (7). T h e embryos of unincubated fertilized chick eggs were irradiated in vivo through the shell, shell membrane, and several millimeters of albumin and then incubated for 22 days. Deformities included splay legs, club foot, and visceral protuberances. Increased mortality was reported. Abnormalities were also observed in embryos irradiated through a c e mented sterile coverslip placed over a small hole in the upper surface of the shell. B. Intrauterine

Irradiation

Laser irradiation at 6943 A of rat embryos and fetuses has been carried out in utero (38, 39). T h e feasibility was demonstrated of p r o ducing localized lesions of the rat fetus b y laser irradiation within the exposed uterus late in gestation. T h e intrauterine laser effect could be produced without rupture of the uterus or of the amniotic sac and without gross leakage of amniotic fluid. In other instances following irradiation late in gestation, a lesion was produced in a fetus in utero which appeared to be the same as a lesion observed in one of the litter mates on termination of pregnancy. I t appeared that lesions could be produced in structures deep to the fetal skin. Irradiation of the

176

SAMUEL FINE AND EDMUND KLEIN

placenta resulted in fetal resorption. C o m p a r a t i v e studies were carried out on neonatal animals. Histological examination showed dilatation of vascular channels with congestion and disruption, especially prominent at boundaries of surfaces between dissimilar tissues. Chronic inflammatory lesions including giant cell formation were observed. Laser radiation m a y offer a modality for investigation of the immediate reaction of intact fetal tissue and organs to localized injury in utero, provide information concerning reparative processes, and assist in the investigation of teratogenesis and other types of congenital defect. IX.

S T U D I E S ON N O R M A L

ANIMALS

Studies b y Fine, Klein, and co-workers (42-48, 51, 99, 104) were carried out at 6943 and 10,600 A , at energy levels ranging from 1" J to over 500 J per pulse, at peak power levels exceeding 100 megawatts, and at pulse repetition frequencies exceeding 1 pulse per second. Effects due to both unfocused radiation and radiation focused b y lens systems were investigated in hamsters, mice, rats, and m o n k e y s . T h e purpose of these experiments w a s : 1. T o determine the short-term and long-term effects of the interaction of laser radiation with skin and underlying normal and tumor tissue. 2. T o determine thresholds for gross and histological changes. 3. 4. 5. tion

T o determine the effect on biological pigments. T o evaluate the hazards associated with laser radiation. T o orient future studies of the biological interaction of laser radiawith biological systems in vivo and in vitro. A. Skin and Subcutaneous

Tissue

The initial studies b y Klein and Fine (99) were concerned with laser irradiation of the skin and mucous membranes, as well as with effects on deeper structures and tumor tissue in rodents including hamsters and mice (42-44^Emphasis was placed on the skin since it is a primary site of interaction in the intact organism. Observations on the skin, furthermore, could be carried out more readily than on less superficially located tissues. At energy levels of 3 0 - 5 0 J and pulse durations of the order of 1 msec, the interaction of the radiation with shaven skin was associated with a plume of back-scattered material and radiation from the area of impact (42). Skin lesions went through the stages of vesiculation with surrounding inflammation, exudation, crusting, and recovery. T h e majority of irradiated hamsters survived. In the animals which died

177

BIOLOGICAL EFFECTS OF LASER RADIATION

or were sacrificed, however, lesions were found deep to the skin lesion. T h e deeper lesions consisted of hemorrhage and edema in the abdominal wall and the viscera (i.e., intestine, s t o m a c h ) . Between the injured areas of the abdominal wall and the lesions in the underlying viscera, however, intervening layers of peritoneum appeared normal. L a c k of free blood in the

peritoneal

cavity

further

indicated

that some integrity

of the

peritoneal layers had been retained, despite extensive hemorrhages severe injury

to adjacent

substantiated

b y the

structures. These observations were

histological findings

and

further

of alternating injured

uninjured layers of tissues in the path of the radiation

and

(44)-

M i c r o s c o p i c findings include sharp delineation between nonviable and viable cells [43) > In the skin, the sharpness of the b o u n d a r y was indicated b y the close proximity o f depigmented hair follicles to pigmentcontaining follicles, and of pigment-free granules to pigment-containing granules. T h e blisters following laser radiation

were located within

the

epidermis. These findings suggested a degree of selectivity of the interaction of laser radiation with different biological materials or structures. T h e y suggested that the factors of importance might be related to the short duration and high peak p o w e r and peak power density, as well as optical and thermal properties of the tissue. Fine et al. (51) suggested that the energy could in some cases be considered as delivered in the form of an impulse, for purposes of analysis. T h e relative mildness of the superficial skin lesions did not correlate with the marked severity o f the associated deeper lesions. These observations were subsequently

confirmed and extended b y studies

energy and peak power levels repetition

frequencies

of the superficial

(46)-

(48, 51)

and

at relatively

A t higher total energy

(51),

at

higher

high the

pulse

severity

as well as of the deeper lesions was increased.

The

relation between severity of injury and energy delivered, however, did not appear to be linear. T h e disparity between the severity of the superficial skin lesions and the severity of the deeper lesions in the muscles and viscera remained

or became more marked. Thus relatively

minor

skin injuries were associated with extensive lesions of the internal strucin other studies

(4%)

at power levels exceeding 100 megawatts, interposition of uninjured

tures. E v e n

at

high energy

(over 500 J )

and

with

injured layers of tissues was present on gross observation. B. Abdominal In from

studies

on mice, at

a f-inch

and Pelvic

Regions

energy levels of 100 J

diameter ruby

laser

(51,

104),

the

per pulse

obtained

majority

survived

focused and unfocused irradiation of the anterior abdominal wall (Fig. 9 ) . In periodically sacrificed animals, lesions of the underlying viscera

178

SAMUEL FINE AND EDMUND KLEIN

FIG. 9. Appearance of superficial at energy level of 60 J.

skin

lesion following

unfocused

irradiation

(liver, intestine) were observed. Despite extensive hemorrhages and severe the

injuries peritoneal

to

adjacent

structures,

free

cavity. Between injured

blood

tissues,

was not

present

intervening

in

layers of

peritoneum appeared intact. Superficial skin lesions were well demarcated following

focused

or unfocused irradiation.

Underlying lesions of the

liver were well delineated following both focused and unfocused irradiation directed at the overlying intact skin

(Fig. 1 0 ) . Intestinal lesions

varied in severity and degree of delineation. M i c r o s c o p i c examination showed flattening

and elongation of nuclei

and cells, as well as vesiculation within the epidermis. Swelling and alteration the

hair

of staining

of collagen were present.

M e l a n i n granules

follicles were displaced or scattered. The gross

in

architecture

of the hair follicle was retained. Hemorrhage was present in the deeper tissue, particularly in the underlying muscle. T h e hemorrhages appeared to be related to gross vascular damage. In hepatic lesions a sharp transition from nonviable to living tissue was observed (Fig. 1 1 ) . In some cases damage extended along the hepatic vessels beyond this line of transition.

T h e general architecture was preserved. In the central part

of the hepatic lesion, flattening and elongation of cells and nuclei were present. In the sinusoids o f the outermost part of the lesion, intense congestion occurred immediately after irradiation, but frank hemorrhage was not observed. Necrosis of the intestinal wall usually involved a part of the circumference and was frequently associated with hemorrhage

BIOLOGICAL EFFECTS OF LASER RADIATION

179

FIG. 10. Lesion (necrosis) in the liver following laser irradiation of the intact animal. Ruby laser wavelength, 60 J.

FIG. 11. Microscopic appearance of hepatic lesion following laser irradiation of the intact mouse (single pulse, approximately 60 J).

180

SAMUEL FINE AND EDMUND KLEIN

into the wall and lumen. M i c r o s c o p i c lesions were occasionally seen in the pancreas, stomach, and other abdominal and pelvic organs. A t energy levels exceeding 300 J at 6943 A, directed at the intact abdominal surface of mice, the effects were more marked than in the 100 J range (49, 104). Survival time varied from immediate death to death within 2 days following irradiation. Free blood was present in the peritoneal cavity. Gross and microscopic lesions were present in the kidney, spleen, and pancreas as well as in the liver and intestine. At energy levels exceeding 500 J, at both 6934 and 10,600 A, similar although more severe effects were observed (51, 104) • A t these energy levels the relative mildness of the superficial lesions did not correlate with the marked severity of the associated deeper lesion. C. Irradiation

of the

Thorax

Radiation at energy levels below 100 J directed at the chest of mice, whether focused o r unfocused, did not result in death (51, 104). Energy levels exceeding 300 J at 6934 A resulted in immediate death or death within 2 days (51, 104). On autopsy, gross lesions were present in the lungs and heart. Sharp demarcation between hemorrhagic and relatively normal lung was evident on gross inspection. On microscopic examination, congestion of alveolar walls, frank hemorrhage into alveolar spaces, and almost complete loss of the staining properties of the cells of the alveolar wall were observed. Some of the vessels within the affected zone contained coagulated blood. A t energy levels exceeding 500 J, at both 6934 and 10,600 A, directed at the chest or abdomen of normal mice, time of death varied from immediate to 2 days postirradiation. T h e deeper lesions produced at these energy levels in the thoracic organs, as in the abdominal viscera, were more extensive than those produced at lower energy levels. D. Irradiation

of Exposed

Abdominal,

Pelvic,

and Thoracic

Organs

In view of the marked effects on viscera when irradiation was carried out through the intact skin, individual organs were exposed directly to laser radiation. Laser irradiation of exposed organs of white Swiss and black C57 mice was carried out at 6943 A at energy levels ranging from 4 to 75 J. Both unfocused and focused radiation was employed. T h e exposed organs included liver, spleen, pancreas, bladder, testis, kidney, stomach, lung, heart, and brain. Following irradiation the incision, through which the respective organ had been exposed, was closed. Irradiation of the stomach and intestine at energy levels of 75 J produced perforation and death, while irradiation of the liver produced a nonfatal

BIOLOGICAL EFFECTS OF LASER RADIATION

181

lesion. Unfocused irradiation resulted in a well-circumscribed dark red lesion in the liver. On sacrifice 2 days postirradiation, the lesions appeared well circumscribed, white, with a narrow red margin. Focused irradiation resulted in tissue volatilization, rupture of the capsule, and crater formation. Bleeding occurred in some cases, but ceased spontaneously within several minutes. W i t h unfocused irradiation at energy levels below 12 J (at 12 J per c m 2 ) , lesions were not evident on gross examination. A t energy levels a b o v e 18 J, lesions in the liver were evident. Irradiation of the spleen at over 50 J resulted in gross effects similar to those observed on irradiation of the liver. Bleeding was more severe than in the liver, but also stopped spontaneously. Unfocused and focused irradiation of the kidney resulted in interstitial and subcapsular hemorrhages without gross bleeding b e y o n d the confines of the kidney. Irradiation of the full bladder did not result in perforation or hemorrhage. Irradiation of the exposed heart resulted in instantaneous death. H e m o r rhagic lesions followed irradiation of the lungs. Following testicular and ovarian irradiation the animals were mated. Several generations of litters have so far shown no gross abnormalities (104) • M i c r o s c o p i c changes noted in the liver and spleen following direct irradiation were similar in type, but differed in degree from those found in these organs on irradiation through the intact skin. A typical mosaic pattern of vacuolization was seen within the liver parenchyma. This pattern was evident in the other directly irradiated organs. E. Irradiation

of the Head and

Brain

Studies of effects of pulsed unfocused and focused irradiation of the forehead in mice have been carried out b y Fine and Klein (47). The hair on the forehead was clipped and the area depilated. Of 41 animals irradiated at energy levels below 100 joules, 31 died within 24 hours of irradiation. Unfocused irradiation was followed b y death within 30 seconds in 10 of 23 animals. Neurological changes were noted in the animals which survived irradiation. Hemorrhages from the mouth, nose, auditory meatus, retro-orbital space, and orbit were observed. Although subperiosteal petechiae were present on the outer table on the cranial bones, gross damage to the underlying bone was not evident. Gross intracranial hemorrhages were observed in the meningeal spaces, the ventricles, and the conducting system, and within the substance of the brain distant from the site of impact (Fig. 1 2 ) . M i c r o s c o p i c a l l y , hemorrhages were seen in the underlying muscle in addition to cutaneous lesions. There were no apparent changes in the bone, but the cells of the bone marrow showed

182

SAMUEL FINE AND EDMUND KLEIN

FIG. 12. Cross section of brain following laser irradiation of the forehead of the mouse. Hemorrhages are evident in the meningeal spaces, the substance of the brain, and the conducting system.

elongated and condensed nuclei. Hemorrhages were present in the meninges and meningeal spaces, as well as within the cerebral and cerebellar cortex, within the ventricles, and at the base of the brain. Sharp demarcation of intracranial lesions was not observed grossly or microscopically. A t energy levels exceeding 100 J per pulse, similar results were o b tained on irradiation directed at the forehead. However, at energy levels in excess of 500 J, rupture of the skin overlying the skull with folding back of the edges at the site of the rupture was observed. Preliminary studies indicated that in the 50-100 J range, at 6934 A, of the order of 1 0 % of the incident radiation directly penetrates through the skin, muscle, and skull. In control studies the exposed brain was directly irradiated at energy levels of 10-12 J. Lesions produced in these animals were localized at the sites of irradiation and were compatible with survival. Studies were carried out by Earle et al. (35) on the effects of radiation, at 20 J per pulse at 6943 A, directed at the unshaven forehead of mice and rats. W i t h unfocused radiation, the hair and scalp of white mice were burned, but no immediate or late effects were found in the brain. With the beam focused to an area 2 m m in diameter on the scalp, a deep burn was produced in the skin, the cranium was intact, and acute subdural, subarachnoid, and slit-like intracerebral

BIOLOGICAL EFFECTS OF LASER RADIATION

183

hemorrhages were produced in the brains of some mice, but not of rats. W i t h the b e a m focused so that the focal point w o u l d be within the brain, if transmitted through scalp and cranium, acute ischemic necrosis and slit-like hemorrhages were produced in brains of rats and mice. T h e effects were fatal to mice within a few minutes. T h e rats appeared to be dazed, but were not killed. T w o rats were allowed to live for 11 days and the late effects were found to show features of healing contusions of the brain. In subsequent studies b y Earle and H a y e s (36), and H a y e s et al. (87), the unshaven heads of unanesthetized white mice were exposed to unfocused and focused laser radiation (into the brain) at 3 0 - 3 5 J per pulse at 6943 A. Thirty-seven animals following unfocused irradiation showed no ill effects, whereas all of the 16 animals exposed to a beam focused 2 m m into the brain showed immediate severe neurological s y m p t o m s which culminated in death. These results were considered to be predictable b y a thermal hypothesis, since 100 J per c m 2 incident on the skin of the head results in 10 J per c m 2 incident on the brain. If the absorption coefficient is 16 c m - 1 at 6943 A and the specific heat 1 c a l / g m / ° C , the outer 100 microns was computed to show a temperature increment of 4 6 ° C . If this energy were focused to 1 m m 2, however, the predicted thermal effect would be increased 100-fold and severe damage and death would occur. T h e brains of anesthetized cats were exposed through the dura to graded energy fluxes from a ruby laser. A 4 5 ° cone angle was utilized in an attempt to produce a trackless deep lesion. Immediately after exposure, the animals were injected with trypan blue and, after 1 hour, perfused. Graded effects (intradural and subdural hemorrhage) and increased permeability as evidenced b y the d y e were observed. A l l of the lesions appeared to be in continuity. D e e p lesions with sparing of superficial tissues were not observed at these energy and power density levels. F. Effects

of

Dyes

In v i e w of the differential interactions of laser radiation at 6943 A with melanin and hemoglobin, the effects of various dyes were explored (52). Applications of water black, methylene blue, and Janus green to depilated skin of mice increased the size and severity of the cutaneous lesions with variable effects on deep-seated visceral lesions as compared to nonstained controls following irradiation at 2 8 - 4 4 J per pulse at 6943 A . Phenol red application did not grossly alter the cutaneous lesion, but increased the severity of visceral lesions in some animals while picric acid produced no changes as compared to the controls.

184

SAMUEL FINE AND EDMUND KLEIN

G. Studies

at High Power

Densities

Fine, M a i m a n , Klein, and Scott (48) studied radiation effects at high peak power levels (exceeding 100 megawatts at energy levels of 3 J ) directed at the abdomen. D e e p as well as cutaneous lesions were produced. A t lower peak p o w e r levels (approximately 10 k W at 7-10 J ) , considerably less or no damage to the skin was observed. Focused radiation at the lower peak p o w e r levels at times resulted in injuries to the underlying subcutaneous and deeper structures. H o w e v e r , the deep lesions produced at high peak power were considerably more marked than at the lower peak p o w e r levels. These studies indicated that the power and power density of the radiation, as well as energy and energy density, are of importance in the interaction of electromagnetic radiation with biological systems. T h e threshold for a particular biological effect, at which power level becomes a parameter of consequence at a specific energy level, requires further study. H. Miscellaneous

Studies

Studies at pulse repetition frequencies exceeding one pulse per second were carried out b y Klein, Fine, and co-workers (46, 49, 51). The effects did not appear to be quantitatively similar when the same energy (of the order of 300-500 J ) is delivered at pulse repetition frequencies of 1 pulse per second (several joules per pulse) as compared to delivery of this energy as a single pulse of 1-msec duration. One reason m a y be that interaction of one pulse with tissue tends to alter the tissue in such a manner that absorption of radiation during the following pulse differs from that during the previous one. There is also time for dissipation of the energy between pulses. Studies of the effects of single pulses of laser radiation at 6943 A and energy levels up to 5 J (pulse duration approximately 1 msec) on skin have been reported b y L . G o l d m a n et al. (65, 66). These studies were carried out on the normal skin of rabbits. On skin and hair of albino rabbits no lesions were evident at low energy levels. In the black skin of the rabbit ear complete tissue destruction was produced. Sections of the ear taken 1 hour after exposure to the laser beam showed a deeply fissured area through epidermis and dermis, and into cartilage. T h e dermis showed compression and increased density of collagen with a moderately thick border of neutrophilic infiltration about the area. T h e adjacent dermis showed edema and diffuse infiltration of l y m p h o cytes and histiocytes. T h e epidermis adjacent to the impact area showed edema and loss of melanin. Klein, Fine, and co-workers (51, 104) investigated the effects of single

BIOLOGICAL EFFECTS OF LASER RADIATION

185

pulses, at 6943 A at energy levels ranging from approximately 30 m J to 100 J, on hemostasis and skin flaps in normal mice. Irradiation of skin flaps in which adequate circulation was maintained resulted in the production of intravascular thrombosis. T h e degree of injury to the blood vessel wall varied from nondetectable to frank interstitial hemorrhage. Factors affecting these results included direction of the beam, energy level, spot size, and size of the artery or vein. W i t h a laser unit focused through a microscope, thrombosis followed b y partial to complete obstruction was observed. Hemorrhage produced in the tail was studied at energy levels in the 50-J range [51). Defocused radiation produced hemostasis in heparinized animals, while unfocused radiation at these energy levels did not stop the bleeding. /. Comparative

Studies

In view of the relative mildness of superficial lesions in the presence of severe deep-seated visceral lesions following laser irradiation of the intact animal and in view of the discontinuity of the lesions along the direction in which injury had taken place, comparative studies on the direct application of heat b y means of a cautery needle were carried out (51). Lesions grossly equivalent to those produced b y laser radiation at 100 J per pulse at 6943 A were induced by electrocautery. T h e injury produced b y electrocautery was relatively uniform throughout the affected tissues, as represented b y a continuous coagulum along the path of the cautery needle. In contrast to animals in which abdominal lesions produced b y laser irradiation were compatible with normal survival, the animals in which abdominal lesions had been produced b y electrocautery died within 2 4 - 7 2 hours following perforation of the a b dominal wall. A group of animals were exposed to w h o l e - b o d y flash tube irradiation at input energy levels up to 5000 J per pulse. T h e animals survived these exposures without internal injuries. Regrowth of the hair was accompanied b y diffuse loss of pigmentation. J.

Discussion

Discontinuty of the injuries to deep structures in the abdomen, thorax, or head was present (104). If the lesions observed had been produced in the manner of c o m m o n thermal injuries (as conductive heat) in which the energy is delivered over a longer period of time, the resulting damage would have been continuous and layers of normal (uninjured) tissues w o u l d not alternate with injured tissues. T h e sharp delineation between irradiated and adjacent nonirradiated tissues suggests that the

186

SAMUEL FINE AND EDMUND KLEIN

FIG. 13. Depigmentation of regrowing hair at site of irradiation.

short pulse duration of the laser beam and the thermal and optical properties of the tissue must be considered. Sharp delineation, however, was apparently limited to the sites of primary interaction. Subsequent secondary reactions (i.e., inflammatory exudates, hemorrhage, or reparative a c t i v i t y ) were n o t sharply demarcated. T h e secondary reactions, furthermore, did not occur along straight lines, but followed the distribution of the blood supply. Selective interaction of the radiation with pigments (51) (i.e., melanin, hemoglobin) and dyes (52), sharply limited to the site of interaction, further indicates that the absorption characteristics of the various tissue constituents must be considered. Another aspect of selectivity of the radiation is suggested b y the sequelae of the interaction with melanin. In the skin lesions produced b y laser irradiation, melanin is not apparent although the structure of the hair follicle and its ability to produce hair have been retained (42). Localized failure of melanogenesis occurred during the recovery period. Thus the hair over sharply circumscribed, circular areas was not pigmented as it grew b a c k at the sites of laser irradiation (Fig. 1 3 ) . T h e deep lesions present following irradiation of the head, the flattening and elongation of nuclei and cells observed microscopically, and the mosaic pattern of vacuolization observed following direct irradiation of exposed organs indicate that other factors than the usual thermal effects without change of phase must be considered in interpretation of the

BIOLOGICAL EFFECTS OF LASER RADIATION

187

observations. A s the energy is delivered in a short period of time, the circulation of the blood does n o t appear to participate significantly in its dissipation. Destruction of circulatory integrity is, however, of consequence in the response of the system. T h e data obtained so far indicate that hazards m a y be associated with laser radiation, particularly as energy and power output are increasing with advances in technology. Hazards of direct exposure to radiation in addition to the danger to the eyes (50) are suggested b y the presence of marked lesions deep to the surface in the presence of minor superficial lesions, late effects as indicated b y the loss of pigmentation (42)} and the persistence of chronic inflammatory reactions (46). Hazards of reflected or scattered radiation, charged particle p r o d u c tion, and possibly of secondary effects (at wavelengths other than that of the primary radiation) associated with the plume (46, 53) require c o n sideration since an appreciable level of energy appears to be involved, as indicated b y damage to the vidicon (of a closed-circuit television system) at 8 ft from the site of irradiation (Fig. 1 4 ) . These considerations warrant further investigation to determine limits of safety and to develop protective measures. Considerable additional information is required for adequate safeguards of personnel operating laser devices as well as for guiding clinical investigation of potential medical applications.

Fig. 14. Circular defect in vidicon seen on center of screen of closed-circuit television system. Defect was produced by back-scattered radiation, with vidicon placed at 8 feet behind irradiation site.

188

SAMUEL FINE AND EDMUND KLEIN

X.

EXPERIMENTAL TUMORS

Studies on the effects of laser radiation the

cheek pouch of the hamster

on tumors transplanted to

were carried

out as part of

initial

exploratory studies b y Fine et al. (42). Necrosis and resorption of transplanted

tumors were observed, while lesions produced in the

normal

cheek pouch were slight to moderate in severity. These studies suggested that some properties of tumors m a y be more suitable than normal structures for the investigation of the

interaction

of laser radiation with tissues. T h e relatively large cell population of tumors

is more readily

accessible in small

laboratory

animals

than

normal cell populations of comparable size. Inhibition of growth is an easily measurable tumor

parameter

of biological activity, since even a few

cells surviving exposure to radiation

can manifest

themselves

b y growing out to appreciable dimensions. Some components of tumors, such as pigment in the melanomas, m a y behave as an energy transfer agent and thus m o d i f y the overall interaction Subsequent studies to neoplastic tissues

(46, 101, 102)

(102).

indicated that extensive damage

could be produced b y laser radiation

in several

transplanted mouse tumors, as had been previously observed in tumors in the

hamster.

Investigations

b y Klein et al.

(102)

suggested that, depending on

the characteristics of the laser radiation and the properties of the experimental tumors, the effects of irradiation varied from complete regressions to accelerated deterioration of the tumor-bearing host. Fine et al. explored the

effects of laser

radiation

on Harding-Passey

(48)

melanoma

in Swiss mice at high peak power and high power density, and observed partial destruction and necrosis at relatively l o w energy levels. M c G u f f et al. (116) planted

to the

described studies on a number of tumors trans-

cheek pouch of the

hamster.

These authors

reported

regressions of several lines of experimental tumors of human and hamster origin following irradiation

at 6943 A. H u m a n tumors included adeno-

carcinoma of the thyroid and breast, and melanoma. T h e y subsequently reported (117)

controlled studies on tumors transplanted to both cheek

pouches. One of the tumors was irradiated while the tumor in the other cheek pouch served as a control. Complete regressions of the

irradiated

tumors followed exposures to a single pulse at 6943 A in the 100 J range. T h e nonirradiated

control tumors

continued to grow, indicating

regression was induced b y the radiation

that

and was n o t spontaneous or

due to immunological incompatibility. These authors also reported

(118)

regressions of experimental tumors following exposures for several hours

BIOLOGICAL EFFECTS OF LASER RADIATION

189

to continuous radiation in the milliwatt range at 6328 A obtained from a helium-neon gas laser. T h e sites at which tumor regression occurred following pulsed laser irradiation were free of tumor cells on microscopic examination. M c G u f f et al. (118) reported that resorption of tumors transplanted to the cheek pouch was not due to obstruction of the blood supply to the cheek pouch, although damage to the blood supply of the tumor following laser irradiation, as previously suggested b y Klein et al. (102), could not be excluded as a cause of tumor regression. McGuff et al. (119) compared the effects of laser irradiation (approximately 220 J at 6943 A ) with those of X-irradiation at 1000 R alone (a nontumoricidal dose) and in combination with laser radiation. The laser-irradiated tumors showed earlier regression than tumors exposed to the combination of X-irradiation and laser radiation. Tumors treated with X - r a y s alone did not regress, but continued to grow at a somewhat slower rate than the nonirradiated control tumors. M i n t o n and K e t c h a m (180, 181, 184) demonstrated regression of S91 Cloudman melanoma following irradiation at 6943 A ( r u b y ) and 10,600 A ( n e o d y m i u m ) at energy levels in excess of 1000 J per pulse per c m 2. These studies explored the possibility of correlating the predictability or probability of inducing regressions with the absorption characteristics of the tumor tissue (133) and the energy density of the radiation. A n attempt was made to predict the laser energy required to destroy a tumor implant of a specific size. In subsequent studies M i n t o n et al. (132) found that tumor size was an important factor in determining laser-induced regression, as had been previously indicated b y Klein et al. (102); the smaller the tumor the greater was the incidence of regression following laser irradiation at a given energy density. M i n t o n et al. (132) also suggested that the structural characteristics of the tumor, which affect its consistency, are of significance in the interaction with the radiation. Generation of pressure waves within the tissue, in addition to thermal effects per se, was considered to be of consequence in the interaction, in agreement with the observations and calculations b y Fine et al. (51). M o t i o n of an oscillating pendulum to which an anesthetized mouse was attached was measured b y M i n t o n et al. (132) following laser irradiation of the tumor. Variations in the amplitude of the oscillation were noted, depending on the characteristics of the tumor and overlying skin. In studies b y Klein et al. (102), regression in 7 5 % was observed following irradiation of Harding-Passey melanoma (average diameter 1 c m ) with single pulses at energy levels exceeding 300 J and energy density levels in excess of 150 J per c m 2 non-Q-switched. In some studies

SAMUEL FINE AND EDMUND KLEIN

190

carried out at 6943 A, regressions were also noted in tumors which had received radiation at similar total energy but in divided daily doses over several days. T h e initial

reaction of the tumor to laser irradiation

was similar,

whether or not it was followed b y regression. This reaction consisted of

a sharply

defined area o f discoloration at the site of

irradiation

with associated flattening. In tumors which regressed, the necrotic area remained stationary for 7 - 1 0 days and then decreased, while the palpable portion of the tumor started to undergo resorption. Healing, frequently associated with only slight scar formation, followed within 1-2 weeks thereafter

(Fig. 1 5 ) . In tumors which did not regress, the necrotic area

continued to increase. T h e residual viable part o f the tumor continued to grow at the periphery of the laser-induced necrotic area until death of the animal (Fig. 1 6 ) .

FIG. 15. Irradiation of Harding-Passey melanoma followed by complete regression of the tumor. Top—lateral view of tumor preirradiation. Middle—lateral view of site immediately following irradiation at 8 0 0 J at 10,600 A . Bottom—view of irradiation site 3 weeks following irradiation. No tumor observed grossly for 1 year postirradiation.

191

BIOLOGICAL EFFECTS OF LASER RADIATION

FIG. 1 6 . Irradiation of Harding-Passey melanoma followed by continued growth of tumor. Top—lateral view of tumor preirradiation. Middle—lateral view of site immediately following irradiation at 8 0 0 joules at 10,600 A. Bottom—continued growth of tumor 3 weeks postirradiation. Animal died 4 weeks postirradiation.

M i c r o s c o p i c a l l y , the

central

area of laser-induced necrosis differed

from the spontaneous necrosis in control tumors b y its smooth outlines, due to absence of perivascular extensions of surviving cells. Distorted cells surrounding small vesicular spaces were observed, which retained nuclear staining

characteristics longer than the deeper lying areas of

spontaneous necrosis. Biopsies, taken

after

6 months

from

irradiated

sites at which regressions had taken place, showed melanin-containing histiocytes but no residual tumor cells. This represented an indication of the previous presence of melanoma. In a number of animals in which melanomas

had

regressed

changes were present (46,

in the

following

laser

epidermal

irradiation,

cells of the

hyperplastic

skin

appendages

102).

In a group of animals to which R i d g w a y osteogenic sarcomas had been transplanted, regressions were observed following

irradiation

of

tumors up to 1 c m in diameter b y a single pulse at 300 J, 6943 A,

192

SAMUEL FINE AND EDMUND KLEIN

and energy density levels of 150 J per c m 2. A t higher total energy delivered in divided doses, regressions did not occur (102). Biopsies from sites at which regression of R i d g w a y osteogenic sarcoma had o c curred showed histiocytes containing hemosiderin. Lewis bladder carcinomas in C57 mice failed to regress following irradiation at 6943 A at energy levels in excess of 900 J delivered as single or multiple exposures. This m a y have been due to large tumor size on irradiation. Cloudman S91 melanoma (average diameter 1 c m ) failed to regress following irradiation with fractional doses at total energy levels of 150-250 J (102). In preliminary studies in animals which had two or more widely separated Harding-Passey melanoma nodules (1 c m in diameter), one tumor per animal was irradiated. In some of these animals both the irradiated and the nonirradiated tumors regressed. A group of six animals, in which regression of Harding-Passey melanoma had persisted for 9 months or longer, was reimplanted with Harding-Passey melanoma in order to explore the possibility of latent immunological incompatibility between the tumor and the host. T h e second transplant failed to grow in three of the six animals (105). Further studies are required to establish statistical significance. T h e reactions of neoplastic and of normal tissues to laser irradiation were similar in that early lesions were sharply demarcated from the noninvolved adjacent areas, the architecture and outlines of cellular components were partially retained, the area immediately affected was not determined b y the effects on the blood supply, the interaction with pigmented structures (i.e., melanin-containing tissue, muscle, liver, and highly vascular organs) was more marked than interaction with nonpigmented tissues (99, 102). The interactions of laser radiation with cell populations of normal and of neoplastic tissue were different in several respects (101, 102). In relatively homogeneous normal tissues such as liver, the initially induced lesions did not appear to increase in size and were not followed b y secondary necrosis in adjacent structures. Repair in normal tissues became apparent within 36 hours. Necrosis following irradiation of some tumors was progressive, proceeded usually to liquefaction, and involved tumor which had p r o b a b l y been outside the track of the primary radiation. It is difficult, however, to exclude the role of infection in this respect when dealing with ulcerated tumors and intestinal lesions (51, 102). The differences in the reactions of normal and of neoplastic cell p o p u lations were further indicated b y the several types of tumor under study. M e l a n o m a was selected in order to study the effects of interaction with

BIOLOGICAL EFFECTS OF LASER RADIATION

193

pigment over larger areas than was possible in normal animals. Cloudman S91 melanoma was included in order to study the effects of irradiation o n the rate and extent of metastatic involvement. Osteogenic sarcoma and bladder carcinoma were selected for studies of interactions with cell populations of connective tissue and epithelial origin, respectively. T h e data, using survival of tumor-bearing animals as a criterion, suggested differences in the interactions of the various types of tumor with laser radiation. T h e information available at present is insufficient to relate these differences to specific biological characteristics. Some relations of the parameters of laser radiation to the interaction with neoplastic cells are, however, apparent. T h e total energy of the radiation and the rate at which the energy is delivered are important factors. T h e effects of irradiation do not appear to be quantitatively similar when the same energy is delivered as a single pulse, at pulse repetition frequencies in excess of 1 pulse per second or in divided daily doses, respectively (46, 102). Averages of the survival time of irradiated tumor-bearing animals in which complete regressions were not observed indicated a reduction in the expected survival time (102). H o w e v e r , this observation must be qualified b y the difficulties of disassociating the effects of radiation on the general state of the animal from those on the tumor. Early fatalities usually followed laser irradiation of animals with large, far advanced tumors. T h e reaction to injury induced b y the radiation, particularly b y exposing considerable areas to infection, would be p o o r l y tolerated b y a debilitated animal. Irradiation effects on other organs, particularly the intestine, m a y have contributed to the increased death rate, although lesions of these internal organs to an extent similar t o those occurring in tumor-bearing animals were well tolerated b y normal mice (51). Furthermore, interaction with the tumor m a y result in dissemination of residual viable tumor cells b y direct extension or b y metastases through the blood and lymph. T h e continued chronic inflammation and epidermal hyperplasia in some animals, several months after the gross appearance had suggested complete healing, require consideration of the possibility of delayed effects of the irradiation (102). However, neoplastic changes attributable to laser irradiation have not so far been observed in normal or in tumor-bearing animals, when regressions permitted survival for protracted periods of observation. T h e information available at present on the interactions of laser radiation with tumors is largely qualitative. Establishment of statistically significant data, particularly in regard to correlating the several physical

194

SAMUEL FINE AND EDMUND KLEIN

and biological factors with the effects of interaction on tumors, will require considerable additional work. Further extensive studies will be required before information obtained from the interaction of laser radiation with neoplastic and normal tissues in animals can be extended to a consideration of clinical investigation. X I . CLINICAL STUDIES

Clinical investigations relating to ophthalmology are reviewed in Section X I I . The potential applications of laser technology to clinical medicine have been summarized b y Litwin and Glew (113)y w h o consider the use of fiber optics, the application of laser devices to gross and microsurgical procedures, and the exploitation of effects of specific w a v e lengths. M a l t and T o w n e s (125) have discussed clinical exploration of laser radiation. Fine, Klein, and Scott (46) have reviewed early experimental findings in normal and tumor-bearing animals as a basis for orienting eventual clinical studies and point out some of the possible hazards. T h e y further emphasize the need for study of long-term effects of laser radiation in animals, as well as for elucidating the mechanisms of interactions as prerequisites for clinical investigation. K e t c h a m and M i n t o n (98) have discussed the applicability of laser radiation in the therapy of malignant disease. Their studies on tumors in animals have been oriented toward establishing guidelines for determining the energy required to destroy tumors in patients. Devitalization of a tumor with several appropriately placed laser pulses, prior to removal of part of an organ which had been involved b y tumor, was suggested. R e m o v a l of a major amount of malignant tissue b y laser radiation as a palliative measure, when definitive cure is not possible, was considered. Studies in man have been v e r y limited. This is due to the short time in which laser devices have been available, the lack of background information from animal or in vitro studies, and the absence of apparent specific clinical indications. Such studies in man as have been reported were exploratory, could not be adequately controlled, and can therefore be regarded as being only at an early, preliminary stage. A. Studies

on Normal Skin, Benign Skin Lesions, Cytological Preparations

and

L. G o l d m a n et al. (65) reported a lack of effects or minimal lesions in normal Caucasian and pigmented human skin, respectively, at energy levels of 0.5 J per pulse obtained from a ruby laser. A t 5 J per pulse more extensive lesions were produced in pigmented human skin, but not in Caucasian skin. M i c r o s c o p i c examination revealed a superficial ulcer and

BIOLOGICAL EFFECTS OF LASER RADIATION

195

an inflammatory exudate. Histochemical studies failed to show changes in the sulfhydryl or disulfide groups of keratin. Cytological studies (64) on cutaneous cells indicated that colored components, whether of natural or extraneous origin, mediated the effects of pulsed laser radiation at 6943 A . Comparison of the effects of Evans blue, trypan blue, and eriochrome black on the type and degree of injury produced at the cellular level b y laser radiation (energy levels from 0.5 to 5 J per pulse at 6943 A ) revealed maximal destructive effects in the presence of Evans blue. Exfoliative preparations of melanoma cells showed extensive cytoplasmic changes with complete disappearance of melanin granules, but sparing of nuclear elements. Preparations of cells obtained from basal cell carcinoma, seborrheic keratoses, and verruca vulgaris revealed sharply demarcated areas o f destruction. In parallel studies (66) on normal human skin and benign skin lesions injuries induced b y laser radiation in colored skin were more pronounced than in white skin in regard to gross and microscopic appearance of the lesions. Acanthosis and bizarre nuclei resembling carcinoma in situ were reported in epidermal cells following laser irradiation (67). N o marked changes were found in psoriatic plaques following laser irradiation in the presence or absence of tar or dyes. Changes were observed in angiomas of infants and children, in port-wine angiomas, and in senile angiomas following laser irradiation at 6943 A. A n g i o m a s in children underwent resorption. Since these lesions have a high spontaneous incidence of regression, considerable additional studies on a controlled basis will be required before laser radiation can be evaluated as a potential therapeutic method and compared to the established p r o cedures in the management of this disease. B. Studies

on Basal Cell

Carcinoma

Superficial changes of basal cell carcinoma following laser irradiation at relatively low energy density, accompanied b y residual tumor cells and early recurrence, have been reported (68, 70). G o l d m a n and Wilson subsequently reported (71) laser irradiation of eight lesions in a patient with multiple nodular basal cell carcinomas at energy densities ranging from 20 to 18,000 J per c m 2. One of these lesions, irradiated with four successive pulses at 100 J per pulse, partially overlapping at a beam cross section of 0.9 c m 2, appeared to regress. T h e other seven lesions which had been irradiated showed partial destruction, but did not disappear during observation periods ranging from 1 to 5 months. In the skin adjacent to the site at which complete regression had been observed on excisional biopsy, a new basal cell carcinoma appeared within 6 months of irradiation.

196

SAMUEL FINE AND EDMUND KLEIN

C. Studies

on Nevi

and

Melanoma

Studies on experimental melanoma in animals (102) had indicated partial destruction, which, depending on experimental conditions, was followed b y regression or accelerated deterioration of the host. Studies on the effects of laser radiation on malignant melanoma and other melanin-containing lesions in man were undertaken b y several investigators (71, 72, 89, 118). G o l d m a n et al. (70) found that irradiation of benign pigmented moles produced superficial coagulation necrosis, which was followed b y fibrosis involving tissues adjacent to the irradiated site. T h e fibrotic area was free of nevus cells and showed epidermal atrophy. In nevi, left in situ or excised, and in malignant melanoma excised following laser irradiation, differential effects on melanin granules, sparing of hair follicles, and sharp lines of demarcation between irradiated and adjacent nonirradiated tissue were observed, as shown b y previous studies in experimental animals (102). McGuff et al. (117) reported studies on laser irradiation at 6943 A of subcutaneous nodules of malignant melanoma in man. T h e degree of injury to the melanoma nodule increased as the energy density of the radiation was increased. Complete destruction of melanoma nodules, however, did not occur and the irradiated lesions regrew within 2 - 4 weeks. Helsper et al. (89) also reported lack of regression of melanoma in man following laser irradiation at 6943 A . D. Exploration

of Fiber

Optics

The possible clinical use of fiber optics for the transmission of laser irradiation, suggested b y Litwin and Glew (113), has been discussed b y Siegmund (159), J. and L . G o l d m a n et al. (64, 69), and K a p a n y (96). Siegmund (159) discussed the problem of obtaining fibers of durability adequate to withstand high intensity laser radiation. J. Goldman et al. (64) explored the transmission of laser radiation at 6943 and at 10,600 A b y means of fiber optics for illumination and destruction of deep-seated lesions. These studies indicate that considerable refinement of such techniques will be necessary before they can be adequately utilized. D a m a g e to red cells in vitro and to tissues due to laser radiation transmitted through a fiber bundle was observed. K a p a n y (96) described studies on the development of endoscopic devices and the limitations of energy conduction through fiber optic systems set b y the laser radiation damage to the fibers. T h e reported studies in man have been included in this review primarily for the sake of completeness. M o s t investigators have directed their efforts to the study of animals and in vitro systems rather than

BIOLOGICAL EFFECTS OF LASER RADIATION

197

to clinical studies, because of the undefined nature and extent of the hazards of laser radiation and the lack of safeguards against such hazards. The absence of specific clinical indications, furthermore, has failed to provide an urgent basis for subjecting patients to laser irradiation. A t this early stage of the studies on the biological effects of laser radiation, information available from animal and in vitro studies has not accumulated to the extent at which clinical studies would be the only remaining unexplored area. H o w e v e r , the rapidly increasing scope and v o l u m e of the animal and biological in vitro studies in progress m a y provide an early basis for the assessment of hazards, and for the delineation of specific medical indications for the application of laser radiation. XII.

OPHTHALMOLOGICAL STUDIES

Studies of the interaction of laser radiation with the eye are being carried out for two main purposes: (1) T o develop methods for protecting the eye against laser radiation. (2) T o evaluate the laser as a clinical tool in ophthalmology. In order to pursue these studies, information from several areas is of importance. These include knowledge o f : (1) Properties of the normal eye (especially h u m a n ) , in both normal and pathological states. This includes some knowledge of physiological optics, normal histology, and physiology of the more important tissues involved (i.e., retina and c h o r o i d ) , together with some knowledge of the pathological responses of these tissues to photic insult. (2) Properties of the laser beam, including wavelength, homogeneity of the beam, divergence, energy, and power. Injury to the various ocular media is of importance in both respects and is dependent on energy absorption in the various layers. A. Absorption

by Ocular

Media

Attenuation of radiation by the ocular media has long been studied on animal and even freshly enucleated human eyes U ) . M e y e r Schwickerath calculated an absorption curve from previous experimental measurements and from his own experimental absorption values of a 2.28-cm layer of water (129). Studies on the transmission of light through the ocular media of enucleated eyes of normal mature chinchilla gray and N e w Zealand rabbits were carried out b y Wiesinger et al. (172), using a B e c k m a n D U spectrophotometer as a monochromatic light source and a K o d a k Ektron lead sulfide cell as a detector. Further studies on enucleated eyes of chinchilla gray rabbits were carried out

198

SAMUEL FINE AND EDMUND KLEIN

b y Geeraets et al. [60). Transmission properties of the sensory retina, anterior to the pigment epithelium, were also measured in rabbit eyes. Early studies on the transparency of the ocular media of freshly enucleated human eyes were carried out b y Ludvigh and M c C a r t h y {114). T w o human eyes, considered to be normal in regard to transmission measurements, were studied b y Geeraets et al. (60). Ninety percent of the radiation between 4000 and 9000 A is transmitted through the ocular media (129). A t both longer and shorter wavelengths, increased attenuation occurs. Radiations below 4000 A to about 3200 A are mainly absorbed b y the lens, which, with its increasing yellow coloration with age, m a y absorb more of the blue-violet portion of the visible spectrum. Radiations near 3200 A are limited b y the cornea and are superficially damaging, producing burns (keratoconjunctivitis), and those between 3100 and 2850 A, being absorbed readily b y proteins and nucleic acids, m a y severely damage the surface of the eye. A t longer wavelengths, absorption b y water begins around 10,000 A. Depending upon the energy involved, or the duration of exposure, serious damage m a y result (e.g., glass blower's cataract), generally limited (in lower energy ranges) to the anterior ocular layers. Lenticular damage is n o w believed to be a result of the localized heat production generated b y absorption of the infrared in the overlying dark pigment epithelium of the iris, rather than a direct action upon the lens as is the case with X - r a y s and y-rays (110). T h e wavelength of neodymium in glass in 10,600 A. Higher energy levels at this wavelength can reach the retina, producing destructive lesions (4). T h e aqueous and vitreous b o d y m a y be considered like water in their absorption and scattering of photic energy. The retina, except for its vascular supply and pigment epithelium, is transparent and probably transmits most of the photic energy. T h e absorption of light is considerable in the pigment epithelium and choroidal layers, which are heavily pigmented with melanin granules in the non-albino eye. This absorption, therefore, is greatly influenced b y the degree of pigmentation, the lightly pigmented fundus absorbing much less of the energy than the more darkly pigmented fundus. A t 6900 A, 8 0 % of the radiation incident on the fundus is absorbed in the darkly pigmented fundus, whereas there is less than 1 0 % absorption in the albino fundus. A t 10,600 A, less than 4 0 % of the radiation incident on the retina is absorbed b y darkly pigmented fundi compared with less than 1 0 % for the albino eye. Peak absorption b y the fundus occurs at 5750 A , at the same general wavelength as some of the visual pigment absorption peaks. Some absorption peaks are considered to be related to difference in the type of photoreceptor (i.e., rods or c o n e s ) ,

199

BIOLOGICAL EFFECTS OF LASER RADIATION

while others m a y be related to hemoglobin absorption (4). These factors should be considered in the design of laser coagulators as more w a v e lengths become available. B. Threshold Study for

of ocular damage has

injury

methods

to the for

ophthalmoscopic

included investigations

eye, particularly

determination examination,

Studies

of light

in the

of

thresholds

retina-choroid layer.

threshold

levels

microscopy,

have

The

included

electroretinography,

electron m i c r o s c o p y , and histochemical and enzymatic studies. Pulsed xenon and carbon arcs have been used as light sources b y H a m et al. (80,

81,

82).

Minimum

threshold

doses o f the

order

of

1 J

per

c m 2 for the retina of rabbits at pulse durations of the order of 1 msec were obtained

B y extrapolations of these curves, energy levels

(82).

approaching 0.45 J per c m 2 will result in threshold injury

for pulse

durations of 10 /xsec. D a t a b y H a m et al. (83) on power density, exposure time, and image size for production of extremely mild lesions in the rabbit retina are shown in Fig. 17, a log-log plot of power density in watts per square centimeter vs. exposure time. M o s t of the data for exposure times greater than 15 msec comes from early experiments on rabbits, utilizing a high density carbon arc source. T h e m a x i m u m power density obtainable on the rabbit retina with this source was approximately 300 W per c m 2. F o r exposure times less than 15 msec or for image sizes 0.2 m m or less in diameter, an observable lesion in the rabbit retina was not p r o -

d>

O

o

- = 0 . 8 0 mm x = 0 . 5 2 mm ° = 1.0 mm

O

Watts/cm2

O

O

O

duced at this power density. Exposure times in the range 15 msec down

O

O

I03r

I0"8

mill i i i mill i i i mill i i i mill I 0 " 7 I 0 " 6 I 0 -5 I 0 " 4

• 11•

I0"3

• • 1

IO" 2

I0"1

Time in seconds

FIG. 17. Log-log plot in watts per cm 2 vs. time for minimal visible injury (in vivo) to rabbit retina. [Courtesy of Dr. W . Ham et al, Medical College of Virginia and Acta Ophthalmologica (S3)].

200

SAMUEL FINE AND EDMUND KLEIN

to 175 /xsec were obtained subsequently either b y using a Zeiss light coagulator operating under steady-state conditions or b y means of an electronically pulsed xenon source. Exposure times for ruby laser pulses resulting in lesions ranged from approximately 1 msec down to 200 /Asec, thus overlapping the data obtained from white light sources for the short exposure times. T h e region 200 fisec to 25 nsec is being investigated currently b y H a m et al., using a Kerr cell as an optical shutter. Laser studies were carried out on chinchilla gray and D u t c h rabbit eyes b y Geeraets et al. (61). M i n i m a l lesions were observed in the eyes irradiated with a pulsed ruby laser of 200 /xsec duration at a retinal dose of 0.72 J per c m 2. W i t h a Q-switched ruby laser at exposure times of 28.5 nsec, the retinal dose for a minimal lesion was 0.07 J per c m 2 at a power density of 2.3 megawatts per c m 2. A t power densities of about 20-28 megawatts per c m 2, discharge of pigment clumps occurred. T h e retinal image measured 0.76 m m in diameter at these power levels. Funduscopic examination, histological studies, and histochemical methods such as D P N diaphorase activity showed that minimal lesions produced with both Q-switched and non-Q-switched lasers are non-uniform due to hot spots in the laser beam (61). T h e threshold lesions were small. A s the energy was increased b y removal of attenuation filters, the lesions became more and more irregular until several small lesions could be recognized within the irradiated field. Further increase in energy resulted in lesions larger than the estimated size of the laser beam on the retina. Choroidal and retinal hemorrhages occurred in some cases, but were usually restricted to the center of the lesion. In threshold studies with a ruby laser, Campbell et al. obtained preliminary results indicating that the energy for a threshold lesion obtained with a laser beam is consistent with H a m ' s xenon arc data (166). Further studies on determining the minimal threshold dose are being conducted b y B . S. Fine and Geeraets, using electron micrographic techniques (Figs. 18, 19, 2 0 ) . T h e lesion shown in Fig. 19 was produced with a ruby laser. T h e pulse duration was 300 /xsec. T h e beam diameter at the retina was calculated to be 0.8 m m ; the retinal dose was 0.7 J per c m 2. Since the intensity across the irradiated field is n o t uniform, not all portions of the field received this calculated dosage. T h e lesion shown in Fig. 20 was produced in a pigmented rabbit eye. T h e time interval between photic insult and fixation of the tissue was approximately 5 hours. This lesion would be clinically visible and due to a dosage somewhat greater than calculated. T h e energy required to obtain a threshold lesion is consequently dependent on the method used to determine the threshold, and the pig-

BIOLOGICAL EFFECTS OF LASER RADIATION

201

ill

FIG. 18. Light micrograph of retina and choroid from a pigmented rabbit eye. Two retinal "hot spot" lesions limited to the outer retinal layers can be seen within the irradiated area. The inner layers are morphologically normal. Specimen was obtained 5 hours after photic insult at ruby laser wavelength. (Courtesy Dr. Ben S. Fine, Ophthalmic Pathology Branch, Armed Forces Institute of Pathology, Washington, D. C , and Dr. Walter J. Geeraets, Department of Ophthalmology, Medical College of Virginia, Richmond, Virginia, 41.)

mentation present. Preliminary studies indicate that less energy is required at high peak power with Q-switched lasers than at lower peak power for production of a lesion. P o w e r is consequently a parameter of importance in interaction of laser radiation and biological systems. C. Studies

on Ocular

Lesions

Other studies on the interaction of laser radiation with the eye have been directed toward determining the effects at energy levels in excess of those required to produce threshold injury. Studies of ocular lesions produced b y a ruby laser were initiated b y Zaret et al. (175, 176). A ruby laser (0.1 J per pulse, 0.5 msec pulse duration) was used. T h e pupils of adult pigmented rabbits were maximally dilated. W h e n the beam was directed at a region containing medullated nerve fibers proximal to the optic nerve head, a site where energy absorption is least efficient, the visible lesion was minimal, but bubbles were produced in the vitreous. W h e n directed toward pigmented regions, the lesion consisted of blanched coagulated retina, elevated in crater fashion and containing a small, centrally placed hemorrhage. F i v e days later, the appearance of the lesion was that of a flat white scar with pigment

202

SAMUEL FINE AND EDMUND KLEIN

Fig. 19. Electron micrograph of a region of normal photoreceptors (P) and pigment epithelium (PE) in a pigmented rabbit retina near the area of laser photocoagulation (see inset, Fig. 20). The typical lamellas of the photoreceptor

BIOLOGICAL EFFECTS OF LASER RADIATION

203

clumping in and around the area. Following multiple exposures, the hemorrhage extended into the vitreous. W i t h the beam directed toward the pigmented rabbit iris b y means of a short-focus lens, the lesion was characteristically dark brown and irregularly shaped. Several days later the pupil constricted in a grossly eccentric fashion. I n further studies on eyes of brown and chinchilla gray rabbits (176), a central zone containing a b l a c k deposit, together with a chorioretinal hole, o c casionally obscured b y a small gas bubble or hemorrhage extending into the vitreous, was observed. A n annular region of blanched coagulated retina began to evolve 1-3 seconds after exposure. A region of retinal edema which had the appearance of a halo surrounded the coagulated zone. T h e lesion in the brown and in the gray rabbits was similar. Reduction of the b e a m energy resulted in smaller areas of coagulation. In light m i c r o s c o p y studies a firm bond between retina and choroid was observed in the coagulated area. A l o n g the peripheral margins of the lesion, the external limiting membrane remained adherent to the surrounding pigment epithelium. However, the retina here was thin, and its normal architecture disrupted. Leucocytes and histiocytes were scattered throughout the matrix of cellular debris. T h e vasculature of the underlying choroid, which was of normal thickness, was markedly diminished and replaced b y fibrous tissue. T h e resultant cicatricial chorioretinal adhesion was similar to that produced b y other light sources. Initial experimental studies b y Koester, Snitzer, Campbell, and Rittler were reported, using a 0.04 J, 0.8 msec ruby unit (107). Further studies carried out b y C a m p b e l l et al. included a large series on eyes of adult pigmented rabbits using a laser and xenon arc photocoagulator (23). In these animal studies, the lesions produced b y the laser appeared to be at a more external level of the retina but were qualitatively similar to those produced b y the xenon arc unit. On microscopic examination, pigmentary changes appeared to occur earlier after laser irradiation. T h e lesion produced b y the laser consequently develops over a period of days. T h e occurrrence of vitreous bubbles m a y be of importance outer segments (P) are clearly seen, as is the mucinous interreceptor material (MPS). The pigment epithelial cell possesses delicate apical microvilli ( M V ) , a number of apically placed pigment (melanin) granules (PG), as well as a large dense "lipoidal" body. CH, choriocapillaris containing a red blood cell ( R B C ) ; BM, basement membrane of pigment epithelium; N, nucleus of pigment epithelial cell; NU, nucleolus; M, mitochondria which are basally located. Reduced from X 16,500. (Courtesy of Dr. Ben S. Fine, Ophthalmic Pathology Branch, Armed Forces Institute of Pathology, Washington, D. C , and Dr. Walter J. Geeraets, Department of Ophthalmology, Medical College of Virginia, Richmond, Virginia, 1^1)

204

SAMUEL FINE AND EDMUND KLEIN

FIG. 20. The inset, a light micrograph of a thin (1-1.5 microns) section to show a "hot spot" area and the demarcation from adjacent normal photoreceptors (see Fig. 19). The electron micrograph shows a region similar to Fig. 19, but

205

BIOLOGICAL EFFECTS OF LASER RADIATION

in considering the laser for a clinical tool. Follow-up of the lesion over a period of years is desirable. D. Temperature

Measurements

in Experimental

Retinal

Burns

Temperature rise and heat conduction at various distances from the irradiation center of the retina following exposure to a modified M e y e r Schwickerath light coagulator were determined b y N a j a c ct al. (135). Measurements were made on 21 eyes using one or two copper-constantan thermocouples in each eye. T h e severity of the lesion created varied from minimal to actual explosion of the retina. F o r threshold lesions, in which there occurs a reddish coloration comparable to an erythema with a small amount of coagulation in the center, the temperature rise at the site varied from 12° to 2 0 ° C . Lesions associated with dense white coagulation of the retina and choroid were associated with a temperature rise exceeding 3 7 ° C above the baseline temperature. Thermocouples placed 1 m m distant from the center of the burn recorded about 1.4°C, and 2 m m distant 0.2°C, for a threshold lesion. T h e temperature rise, therefore, appeared to be well localized at the site of the lesion, with rapid attenuation beyond the lesion. Campbell, N o y o r i , Rittler, and Koester studied intraocular temperature changes following laser and xenon arc radiation (25). BaldwinL i m a - H a m i l t o n microminiature chromel-alumel thermocouples, with an insulated sheath and a time response of 40 msec for the thermocouple, were used. T h e total energy output of the ruby laser was 0.065 J. Laser pulse duration was 0.5 msec, xenon arc duration 50 msec. T h e thermocouple was oriented parallel to the axis of the coagulating beam, with the heat-sensitive tip flush with the inner surface of the retina. taken from within the lesion. The photoreceptor outer segments, although sectioned more obliquely here, are grossly disrupted. In some areas the lamellas have disintegrated into tubular fragments ( T ) . Much of the mucinous interreceptor substance (MPS) is still present, and a peripheral cleft (F) typical of normal photoreceptor cross sections is still clearly seen. The darker photoreceptor (C) represents a degenerating cone cell outer segment. The "broken" appearance of the pigment granules within the pigment cell is considered to be mostly artifactitious. The cytoplasm and the microvilli of the pigment epithelial cell show a "granulation" effect, present also within the degenerating photoreceptor outer segments (free arrows). This is considered a true early response to the photic insult. These degenerative changes, here a result of photic insult, are in general nonspecific changes which may also occur with other forms of trauma. The choriocapillaris (CH) is shown. Reduced from X 16,500. Wavelength 6943 A, pulse duration 300 ,usec, and retinal dose 0.7 J per cm2. (Courtesy of Dr. Ben S. Fine, Ophthalmic Pathology Branch, Armed Forces Institute of Pathology, Washington, D . C , and Dr. Walter J. Geeraets, Department of Ophthalmology, Medical College of Virginia, Richmond, Virginia, 41-)

206

SAMUEL FINE AND EDMUND KLEIN

T h e temperature change in the plane of the retina was sharply defined and focal in nature with both xenon arc and laser photocoagulation. A t the site of coagulation, the temperature rise was about 3 0 ° C . Temperature changes were extremely transitory, indicating that, with repeated coagulations, thermal summation could not occur intraocularly at the usual coagulation rate. A t 1 m m from the site of coagulation the temperature rise was less than 1 ° C , indicating narrow confinement of the site of coagulation. In the middle of the vitreous b o d y , the temperature rise was less than 1 ° C . An exposure time of 3 seconds to the xenon arc coagulator resulted in a C h o r o i d a l explosion.' 7 T h e temperature elevation exceeded 7 0 ° C in the center of the lesion and exceeded 2 2 ° C in the middle of the viterous. B,eturn of the temperature to normal exceeded 15 seconds. Attempts at focusing the xenon arc and laser beam on the vitreous indicated that heat absorption b y the vitreous m a y be less at the ruby wavelength than at the integrated zenon arc wavelengths, although other explanations were considered. T h e return of temperature to normal with the ruby coagulator was more rapid than with the xenon arc coagulator. Thermal summation was consequently considered as less likely to occur with the laser coagulator than with the xenon arc coagulator. In the temperature measurements discussed, modification of the actual temperature b y the thermocouple must be considered. Some attention has been directed to this problem b y the various authors. E. Protection

of the Eye from Laser

Radiation

Studies on protection of the human eye from laser radiation have been carried out b y Straub (164, 165). M e t h o d s considered include the automatic high-speed shutter technique triggered b y the arriving pulse, and colored absorption filters or dielectric reflection filters. Kerr cell shutters were disqualified because of the small angle of acceptance and insufficient opacity in the shut state. Phototropic niters of adequate response to far red with appropriately short response time (10~ 8 second) and of sufficient opacity were not available. The energy which could result in a lesion of the retina was calculated as 1.6 X 10~ 6 J based on studies b y H a m et al. in which an energy density of 0.88 J per c m 2 resulted in mild irreversible damage, assuming a minimal irradiation area of 2 X 10~6 c m 2. Allowing a safety factor of 100, the permissible safe energy entering the pupil was estimated to be 2 X 10~8 J. For a darkadapted eye in proximity to a 2J, 0.6 cm diameter, parallel laser beam, an attenuation of 80 dB would be required (164) • B G - 1 8 glass was determined as possessing an optical density of 2 or higher at wavelength ranges between 6943 A to 1-3 microns for 1-mm

BIOLOGICAL EFFECTS OF LASER RADIATION

207

thickness (2.36 for 1.08 m m at 6943 A ) . A n optical attenuation of 100 dB therefore required a thickness exceeding 4 m m . Filters incorporating B G - 1 8 glass were designed with sufficient transmission in the visible range and with sufficient attenuation at 6943 and 10,600 A (164, 165). Endurance tests on these filters included exposure to 20 firings at 0.2 J, exposure to 2 J non-Q-switched focused to 1 m m 2, and exposure to 0.4 J Q-switched focused t o 1 m m 2. N o gross deleterious effects were observed. W h e n exposed to 10,600 A at 32 J per c m 2 (64 J t o t a l ) , the filter cracked at 25 c m from the laser. A t longer distances spalling, including delayed spalling, occurred. T w o techniques were used to decrease mechanical strain. One involved placing 2 m m of B G - 3 8 glass in front of the B G - 1 8 glass, thus reducing the energy incident on the B G - 1 8 glass. T h e other consisted of coating the B G - 1 8 glass with a dichroic mirror that reflected 8 0 % of the energy at wavelengths ranging from 6943 to 10,600 A, and transmitting the visible light. Further studies on eye protection against laser radiation have been discussed b y Swope and Koester (166). F o r one m m N d doped glass laser fiber at 10,600 A , the laser energy for retinal damage is less than 1.5 X 10~ 3 J, assuming a threshold of 0.45 J per c m 2. F o r a ruby laser at 6943 A, with 0.5° beam spread, calculations indicated that 1 0 4 J output will cause damage to the eye. These calculations assume a beam spread, those by Straub (164) assume a parallel beam. T h e design of glasses incorporating reflectors with high reflectance at all angles of incidence was not considered practical. Studies were consequently carried out on B G - 1 8 and B G - 3 8 glass. B G - 1 8 glass crazed at 5.5 J delivered in an area 5 m m in diameter; B G - 3 8 crazed at 9.5 J in an area 5 m m in diameter. Combination of B G - 3 8 and B G - 1 8 resulting in an optical density exceeding 10, irradiated with 740 J, did not result in damage to the underlying B G - 1 8 glass. A filter consisting of an outer layer of B G - 3 8 glass, a center plate of B G - 1 8 glass, and an inner plate of clear, tempered glass has been designed b y the American Optical C o m p a n y . A unit consisting of B G - 1 8 glass of density 9 with a dichroic coating of density 1 in welders' e y e shields, considered effective against 6943 and 10,600 A, has been made available b y the Fish-Schurman C o m p a n y . Other units incorporating safety glass and B G - 1 8 have been designed b y T h e Technical Research Group, I n c . F. Retinal

Coagnlator

Calculations of retinal energy density or intensity incident on the retina from a ruby laser are given b y Solon, Aronson, and Gould (162).

208

SAMUEL FINE AND EDMUND KLEIN

The optimum divergence angle (
\/DL

where DL is the diameter of the beam, and A is the laser light wavelength. T h e m i n i m u m diameter, of any retinal image spot, hmin, is therefore: Amin ^ 2.44

f\/DE

where DE is the pupil aperture, and / is the focal length of the eye. (The theoretical basis for these equations is equivalent to that of Section V I . ) T h e far-field case, in which the eye can see light from all parts of the laser but the retinal image spot size is limited b y the Fraunhofer diffraction pattern formed b y the eye, the near-field case, in which the laser beam cannot be considered as coming from a point source, and the case where the beam m a y be smaller than the pupil as it enters the eye, under which conditions the spot size will be enlarged, are discussed. Studies on the laser directed to its use as a retinal coagulator have been carried out b y Zaret and Grosof et al. (75, 177), Campbell et al. (25, 26, 139), Schepens et al. (58, 141-1U, 157), K a p a n y , Zweng, Flocks et al. (55, 95, 179,) and Tengroth et al. (167). In his initial studies, Zaret indicated the possibility of using the laser in ocular photocoagulation therapy and pointed out some of the advantages (17). Problems associated with the use of the laser in clinical ophthalmology were further discussed b y Zaret (177). These include a peripheral enlarging zone of edema and coagulation beyond the discrete focal lesion to produce a firm resultant chorioretinal adhesion, the production of tissue carbonization, retinal holes, retinal implosion vapor bubbles, and hemorrhage, the necessity of the retina being in apposition with the pigment epithelium for photocoagulation of detached retina, and the difficulties associated with "welding" the transparent portion of the retina to the pigment epithelium where separation has occurred internal to the pigment epithelium. Studies b y Freeman, Pomerantzeff, and Schepens (58) have been carried out, using an indirect ophthalmoscopic unit. This system uses closed-circuit water cooling of a 3 inch X i inch ruby pumped b y a 1000 W xenon flash lamp to provide a more uniform output. T h e coagulating beam is deflected toward the eye b y a dichroic mirror through which observation is performed b y means of an indirect ophthalmoscope. Illumination of the fundus is provided for b y the ordinary light source of the indirect ophthalmoscope. Over 9000 coagulations have been carried

209

BIOLOGICAL EFFECTS OF LASER RADIATION

out on pigmented rabbits and a limited number of patients. Laser coagulation has been combined with diathermy

and xenon arc coagulation

in treating retinal breaks in the normally positioned retina and uveal malignant melanomas prior to enucleation. T o prevent

overtreatment,

irradiation is initiated at threshold levels and then increased to produce the required energy. Retinal images of about 0.15 m m could be produced in the emmetropic eye. T h e lesion produced was larger. Vitreous hemorrhages occurred at 10 times threshold energy. A t higher energy levels, gas bubble formation occurred. B y increasing beam divergence, the incidence of vitreous hemorrhage was reduced and a deep central hole was not produced. M a n a g e m e n t of intraocular tumors was explored b y surrounding

the

tumor base with an array of coagulations followed b y direct coagulation of the tumor apex. Retrobulbar anesthesia was not needed. A n intense miosis refractory to cycloplegics, occurring in several patients with m a lignant

melanoma, was reduced b y narrowing the

beam with a

dia-

phragm. T h e risk of dissemination of tumor cells during laser irradiation was

considered as p r o b a b l y no greater

than

during

the

enucleation

itself. Clinical studies on laser photocoagulation, following studies on coagulation in rabbits, were reported b y Koester et al

(107).

T h e unit had

an output of 0.04 J obtained from a 2.5 X 0.25 inch ruby pumped b y an

F T - 5 2 4 flash

tube. Further studies

b y Campbell et

al

(25,

26)

were carried out, using indirect ophthalmoscopic laser units with energy outputs

of 0.25-0.50 J at 6943 A at pulse durations

some patients, therapeutic

over 2 0 0 - 3 0 0 irradiations

intensity

were

not

of 0.5 msec. In

were carried out. Lesions of

associated

with

profound

intraocular

damage. Necrosis and scar formation were confined to the site of coagulation. In

the

quently

a

standard clinical lesion, pigment accumulation and

subretinal

gas

bubble were observed. N o changes in

frethe

vitreous b o d y were noted. T w e n t y percent of patients developed punctate corneal staining which disappeared within 24 hours. Lens opacities were not

produced

or

aggravated

in

patients

who

had

opacities

before

treatment. Further studies b y Campbell et al

(27) included the use of a lasing

fiber bundle. T h e laser emission emerges from the t o p of a small fiber bundle, thus permitting

the glass probe to act as a heating

element

at its tip. E x p l o r a t o r y clinical investigations have been carried out with this type of unit. Animal and clinical studies b y Zweng and F l o c k s (179)

have been

carried out, using a hand held direct ophthalmoscope unit, incorporating a 3 X i inch ruby p u m p e d b y two linear flash tubes with a laser output

210

SAMUEL FINE AND EDMUND KLEIN

of 0.25 J. T h e target is viewed through a direct ophthalmoscope head. The operator looks through a clear area. T o lase, the fully aluminized portion of the mirror is slid in front of the viewing aperture before the pumping light is activated. Studies on animals have included 60 rabbit eyes, 8 cat eyes, and 14 m o n k e y eyes. Ophthalmoscopic, slit lamp, and histological examination and fundus photography have been carried out. Bubbles produced in the retina and vitreous at energy levels in excess of 0.08 J, resulting in lesions measuring 0.8 m m in diameter ( 1 0 - 1 7 J per c m 2 ) , disappeared in approximately 15 minutes. A t higher energy levels retinal hemorrhages and holes developed. Retinal and iris photocoagulation was subsequently explored clinically. Retinal lesions varied from 0.1 to 0.25 m m . Retinal holes were treated with a cordon of lesions around the hole. Microaneurysms in diabetic retinopathy were irradiated without producing apparent changes. Electroretinographic studies in conjunction with laser-produced lesions in the human eye have been made b y Tengroth et al. (167). T h e technique used consisted of application of one electrode to the cornea temporally near the limbus with the reference electrode on the forehead, similar to the method of Karpe (97). The electroretinogram was recorded during irradiation b y four successive laser pulses directed at the retina of a subject in w h o m a carcinoma of the left orbit had been diagnosed and who was scheduled for exenteration of the orbit. Control studies using white light photostimulation of 150-msec duration were done. Electroretinography was carried out during laser and white light irradiation in a second patient. Unlike the white light stimuli, the laser beam was not dazzling. A small, central, circular, green after-image persisted for 30 minutes in the first patient. The electroretinography in response to laser radiation was considered as a response from the whole retina, possibly due to light scattering, since it was inferred that the focused laser pulse strikes too small a portion of the retina to evoke a measurable response from that area alone. A s discussed b y the previously referenced authors, the possible advantages associated with laser radiation in clinical ophthalmology (dependent on laser characteristics, including exposure time, m o n o c h r o m a ticity, ability to focus to a fine spot, and wavelength) are: 1. Brief exposure time reduces the danger of eye movement, permitting accuracy in cautery placement, and minimal injury to jacent tissue. 2. The short flash duration m a y result in less heating of tissue rounding the area to be heated, with less transfer of energy to vitreous and other media.

thus adsurthe

211

BIOLOGICAL EFFECTS OF LASER RADIATION

3. M i n i m a l absorption b y the tissue and media anterior to the pigment epithelium occurs at 6943 A . 4. T h e effect of ocular chromatic aberration is decreased b y the m o n o chromaticity of the light. 5. There is less risk of damaging the cornea, lens, and vitreous because of lack of infrared radiation. 6. M o n o c h r o m a t i c radiation allows simultaneous coagulation and o b servation using a dichroic system. 7. T h e fine focus is especially of value in treating lesions close to the macula. 8. Wavelength can be varied as a parameter within limits. 9. T h e larger p o w e r and energy densities available m a y be of value in complex problems of management, such as tumors with high reflectance in the visible spectrum and lesions in the periphery where

aberration

produces a marked image spread. 10. T h e laser can be coupled to fiber optic bundles to reach relatively inaccessible regions. T h e problems and disadvantages associated with the laser unit have been discussed b y the various authors. These include: 1. T h e

short

duration

of

the

laser

pulse

limits

control

during

exposure. 2. Spherical aberration occurs, induced b y the anatomic structures of the eye and the obliquity of the incident light rays for peripheral photocoagulation. 3. Diffraction b y the pupillary margin m a y affect the retinal image of the laser beam, thus preventing the retinal image from being in absolute focus on the retinal pigment epithelium and limiting the minimum spot size attainable. 4. Variation in pigment epithelium between different eyes and within the same eye affects absorption, and the total required energy. 5. Multiple coagulations m a y be accompanied b y reflex miosis. 6. Peripheral rays m a y strike the iris causing iris damage. Inadvertent iris irradiation m a y result in conduction of heat to the lens resulting in cataracts. 7. D i r e c t delayed cataractogenesis m a y occur. For example, R . F . cataracts develop after a latent period of m a n y years. 8. Ultraviolet radiation at the retina produced b y harmonic generation due to the crystalloid structures of the pigment granules within the retinal epithelium m a y affect the eye. Ultraviolet radiation is not normally present at the retina. 9. Factors associated with the laser can result in inconsistent coagula-

212

SAMUEL FINE AND EDMUND KLEIN

tions. These include: (a) heating of the crystal, which is decreased but not eliminated b y water cooling, ( b ) misalignment of the crystal, (c) alterations in reflectivity of the end coatings affecting beam intensity, intensity distribution, and divergency, ( d ) alterations in dichroic mirrors, flash lamp, and cavity, (e) precipitation of minerals in the cooling water. Further studies are consequently required to determine the applicability of the pulsed laser to clinical ophthalmology. Since relatively low power and energy levels are required, the gas laser and semiconductor laser m a y prove of significance for clinical ophthalmology. Dangers to the eye of the operator inherent in the solid state type of unit exist and should not be disregarded. Dangers from the semiconductor laser due to off-axis radiation and from the gas laser due to the high peak power densities possible on focusing on the retina must not be neglected. XIII.

MODES OF INTERACTION

A specific comprehensive theory 'to explain all the observations noted on interaction of laser radiation with physical and biological systems has not been achieved. This is partially due to the difficulties associated with elucidation of the energy transformations which occur, their relative importance, and the inherent complexity of biological systems. Attention has, in general, been directed toward an explanation of the specific phenomenon of interest during the experiment with relative neglect of other energy transformations which m a y occur. Hypotheses have been formulated regarding factors of major importance in interaction of high peak power (and energy) density laser radiation with in vitro and in vivo biological systems. Experiments have been conducted on physical models and biological systems to investigate the relevance of these hypotheses. T w o hypotheses have been advanced to account for the majority of the effects observed at the energy and power levels considered insofar as biological systems are concerned. These are: (1) The purely thermal hypothesis, based primarily on studies of tissue in vitro and in vivo in which the effects are considered as not differing significantly from those of a burn insofar as tissue is concerned, and not differing significantly from those due to temperature elevation per se. (2) A "multiple factors" hypothesis, based on in vitro and in vivo studies of various tissues and other biological systems, in which factors other than direct temperature elevation alone are of significance. In the multiple factors hypothesis, factors other than temperature

BIOLOGICAL EFFECTS OF LASER RADIATION

213

elevation per se must be considered in an evaluation of the effects of the interaction. Their particular importance is dependent on the energy (and energy density) and power (and power density) of the incident radiation, as well as on the properties of the specific biological systems irradiated. Interactions of high peak power radiation include initial effects which are a function of temperature distribution and its associated secondary effects, as well as photochemical reactions. In the study of the effects of the interaction, the following are among the factors which require consideration: 1. Physical properties of the media, including mechanical, optical, and thermal characteristics; 2. spatial and temporal energy distribution within the media; 3. energy transformations which a c c o m p a n y the interaction; 4 . effects of these transformations on thermal, mechanical, and biological properties both during and after the interaction; 5. type and degree of alteration of the biological system following this interaction. Parameters of importance in these respects are the properties of the radiation and ,the characteristics of the biological system. Parameters of the radiation are energy and energy density, power and power density, wavelength, and possibly the degree of coherency and polarization. Physical parameters of the biological systems include reflectivity, a b sorptivity, heat capacity or specific heat of the systems, latent heat of vaporization, specific heat of the vaporized material, heat c o n d u c tivity, volume alterations with and without phase transformation, and acoustic and mechanical properties. D i r e c t measurements of these parameters have not been made at high peak power levels. Measurements of some parameters, however, have been carried out at low power levels. Skin reflectivity measurements on some species have been made by D a v i s (31), H a r d y and Muschenheim (86), and Kuppenheim et al. (109). An estimate b y D a v i s of the linear absorption coefficient of fair skin is of the order of 10 c m - 1, between 6000 and 12,000 A (31). T h e absorption coefficient is greater for the superficial layers than for the deeper tissues. H e a t capacity determinations for skin ranged from 0.8 cal c m _ 3d e g _1 to 1.18 cal c m ~ 3d e g _1 (31, 90). Measurements of thermal conductivity have given values of the order of 5 X 10~4 cal c m _ 1d e g _ 1s e c _1 for tissues of various species, with considerable variation depending on the type of tissue (31, 90). A number of factors mitigate against the prediction of effects observed at high peak power (and energy) density levels from those observed

214

SAMUEL FINE AND EDMUND KLEIN

at low peak power (and energy) density levels. F o r some effects a threshold exists at specific levels of power ( a n d / o r energy) densities below which the effect does not occur. Examples include production of cavitation in liquids and occurrence of phase transformations in liquids and solids. Another factor precluding extrapolation is the suppression of detectability of a specific phenomenon b y concurrent events. Although a threshold m a y not exist for some effects, such as second harmonic generation and modes of recombination of excited atoms, the sensitivity and discrimination against other processes of the detection techniques are such that observation of the specific phenomenon is difficult at low power levels. Other nonlinear processes during and following the interaction m a y further accentuate the importance of specific parameters at high power and energy density levels, in comparison to their importance at low levels. A t low energy density and power density levels, the gross interactions are due to the usual increase in temperature per se, without change of phase of the system; however, as energy density and power density are increased, effects m a y be due to other than the usual thermal effects per se. For example, some effects m a y be related to production of pressure gradients within the system. Pressure effects can be produced in several w a y s . T h e y can be produced b y volatilization of material from the surface, with generation of a mechanical impulse proceeding into the biological system being irradiated, based on conservation of momentum. Although generation of pressure transients due to this type of interaction at the surface of biological systems does occur, it is p r o b a b l y not the mechanism of major importance in damage deep to the surface at high energy and power levels. A second mode of pressure wave generation is due to transient heating and associated thermal expansion of volume without change of phase. In studies on physical systems (28, 173), the generation of elastic waves has accompanied the absorption of radiation from high power light sources including electric arcs and ruby lasers, microwave radiators, and the incidence of a pulsed electron beam upon a solid target. F o l l o w ing localized heating, the temperature gradients produced, as a result of thermal expansion, strains in the b o d y leading to generation of stress waves which propagate a w a y from the heated surface. In biological systems, thermal expansion can occur either at the surface or internally due to internal absorption. This effect would be enhanced b y entrapment of radiation between reflecting surfaces in the absorbing region. A t high peak power levels it is possible that this second m o d e of pressure wave generation is overshadowed b y another m o d e of pressure generation.

BIOLOGICAL EFFECTS OF LASER RADIATION

215

A third m o d e of pressure w a v e generation is due to transient heating with change of phase, especially to a gaseous phase. If this effect occurs internally and the gaseous or volatilized products are entrapped, an extremely high pressure transient can be produced. Radiation from Q-switched systems can produce a relatively rapid pressure increase in comparison with radiation from comparable non-Q-switched systems. W i t h the former, rapid pressure buildup can occur, resulting in transmission of a shock wave. These shock waves m a y travel with relatively little attenuation and consequently affect areas at some distance from their origin. High speed photography (51) has indicated that effects of this t y p e m a y be produced in tissue. Outward hemispherical distensions of the a b dominal walls of mice were observed when radiation at 6943 A at an energy level of 75 J, non-Q-switched, was directed at the abdominal surface. T h e millisecond duration of the distension was of the order of the duration of the laser pulse. Some effects observed distant from the site of impact in the intact animal, particularly within an enclosed cavity such as the cranium, m a y be due to pressure alterations with change of phase. Effects on in vitro systems, such as aqueous solutions of p r o teins, m a y be associated with phase transformation on m a c r o - or m i c r o levels. Studies on pressure w a v e formation and transmission have been carried out (51, 128). Pressure waves, primarily of acoustic v e l o c i t y , occurred within the gelatin model following irradiation b y Mendelson (128). with a Q-switched laser at an energy of 3 J (0.5 J per pulse) T h e pressure waves were rapidly attenuated with depth in the gelatin. T h e interfaces normally present, and produced between tissue c o m ponents in vivo and between a solution and its container or within the solution itself in in vitro systems, must be considered. Following absorption of radiation within a region, multiple reflections can occur from the interfaces on both a gross and microscopic level. This m a y result in trapped radiant energy within the specific m a c r o - or microscopic region, particularly if there are changes in wavelength and alterations in the m e d i u m during irradiation. Optical properties of tissue affect the initial distribution of energy. A considerable fraction of the radiation m a y consequently be absorbed deep to the surface with subsequent entrapment of the energy within a relatively small volume. A s previously discussed, studies on intact animals have indicated damage to some tissue while the intervening tissue layers are relatively undamaged. F o l lowing laser irradiation of gelatin models, bubbles were produced, particularly at the interfaces. These effects m a y be dependent on optical properties, on the presence of interfaces, on the presence of cavitation, and on mechanisms other than temperature elevation per se.

216

SAMUEL FINE AND EDMUND KLEIN

High electric fields m a y be of importance at extremely high

peak

power densities. T h e equations for the electric field in v a c u u m are readily obtained. T h e p e a k value of the electric field E for a plane w a v e p r o p a gating in a medium with permittivity e and permeability ^ is: E

=

V2W

VV/e

where W, the radiative flux density, is the time average of W(E

X

H),

the instantaneous power flow in rationalized M K S units, and the electrical

conductivity is small. F o r a v a c u u m , p/e = 367.7 o h m . F o r

a

flux density of 1 megawatt per c m 2, a peak electric field of 2.74 X 1 0 6 V per

meter

is

obtained

for

vacuum.

In

tissue,

however, the

effects

m a y be quite different because of the c o n d u c t i v i t y of the media. Studies in biological systems have failed to demonstrate that a high electric field is an important parameter in the interaction at the power and energy levels so far studied. A t peak power level in the gigawatt region this factor m a y become important. T h e production of sonic frequencies following irradiation of biological systems has been demonstrated Q-switched

irradiation

{52). H y p e r s o n i c frequencies

of solids and

Townes et al. (29, 59).

liquids

have

following

been observed b y

H a r m o n i c generation has been obtained

These energy transformations

should be considered in an

(168).

evaluation

of effects on biological systems. As indicated at the beginning of this section, there is as yet no satisfactory theory to explain the modes of interaction between laser radiation and biological systems. One hypothesis, the "thermal" hypothesis, m a y not have sufficient scope to explain all pertinent observations, particularly

at high energy and power density. T h e second hypothesis,

the

"multiple factors" hypothesis, is incomplete. Attention will have to be directed toward elucidation of the relative importance of the energy transformations

various

in a consideration of this second hypothesis.

X I V . SUMMARY AND CONCLUSIONS

Studies have been carried out on in vitro

systems, single cells, tissue

and cell cultures, individual organs (particularly the eye and the s k i n ) , intact animals,

and

tumors.

Clinical studies

have been conducted in

ophthalmology, on normal skin, and on benign and malignant lesions. Lasers coupled with microscopes and spectrographs have provided tools for the

investigation

of biological systems. T h e observations to date

provide a basis for orienting further, more definitive investigations. Control studies

of t w o types would be desirable. One type should

evaluate the biological effects of incoherent radiation at the same energy and power densities as are obtainable with lasers. H i g h power densities,

BIOLOGICAL EFFECTS OF LASER RADIATION

217

however, of the order of megawatts or gigawatts per square centimeter, are n o t attainable with incoherent sources. A second group of control studies should involve the utilization of incoherent sources which would provide energy densities equivalent to those produced b y lasers, but at lower peak power densities. It would be desirable to carry out this second group of studies at wavelengths similar to those of laser radiation, since wavelength (and polarization) m a y be of consequence. A n attempt at comparative studies at lower peak power levels, using mercury arc and flash tube sources, has been carried out b y Fine and Klein on both in vitro and in vivo systems. Other studies on biological systems with high intensity sources, such as carbon arcs (31), provide information of importance for comparison with the effects of laser radiation on biological systems. Investigations with the light coagulator in ophthalmology as discussed b y Meyer-Schwickerath (129) are pertinent. Although factors, such as the broad band characteristics of the radiation and the lower peak power densities attainable, render problematical the use of such studies for control purposes, further investigation of the effects of incoherent sources on biological in vitro and in vivo systems m a y , in themselves, prove significant. It is evident that power density is of importance in some cases. Irradiation of skin at 1000 J per c m 2, at a power density of 1 megawatt (irradiation delivered in 10~3 s e c o n d ) , will result in effects which differ from those obtained if irradiation is carried out at a power density of 1 m W (irradiation delivered in 1 0 6 seconds). T h e difference is partially dependent on heat flow from the irradiated region b y conduction, radiation, and convection, as well as on blood flow. Further studies at various energy and power levels are required to evaluate the importance of power as a parameter for the production of specific biological effects. Although some variation can be obtained b y Q-switching, continuous independent control of laser power and energy has not been achieved; studies are warranted, however, at such levels as are available. Coherency is of importance in that it permits focusing to a small spot size. Whether coherency per se is of consequence in biological studies will be difficult to assess, particularly since considerable scattering occurs in some systems. T h e advent of relatively high power gas lasers m a y assist in assessing the relative importance of both coherency and polarization, particularly insofar as in vitro systems are concerned. Further investigations of methods of measurement of various laser beam parameters, and of the interaction with biological systems, are required. Measurement of energy and power during the laser pulse necessitates the design of beam splitters which will sample a definite fraction of the beam independent of beam polarization. Calorimeters must be

218

SAMUEL FINE AND EDMUND KLEIN

developed which are independent of wavelength and peak power over the required range. Temperature- and pressure-sensing elements to measure the respective quantities over the required wavelength band, which are relatively insensitive to radiation, possess the necessary band width and sensitivity, and minimally perturb the system under investigation, require development {138). Design of suitable air-spaced microscope lenses for Q-switched laser microscope systems is required. T h e physical characteristics of various biological systems must be denned at high peak power levels. This includes determination of values for parameters, such as reflectivity, absorptivity, heat conductivity, degree of scattering of electromagnetic radiation, and acoustic and mechanical properties, as functions of various laser beam parameters. In this regard, a preliminary investigation of absorption of radiation b y the plume of back-scattered material in the irradiation of various materials has been carried out b y Fine, Hergenrother, Klein, et al. A more detailed study at various wavelengths, energy levels, and power levels is warranted. Although extrapolation of effects on small animals, at the energy and power levels studied, to effects at higher levels on large animals is difficult, some degree of extrapolation is necessary for assessment of short- and long-term hazards to man at high energy and power levels. Charged particle production and other factors in addition to temperature elevation per se m a y be of importance insofar as hazards are concerned. Their relative significance requires further study. Other areas of potential importance include disinfection and sterilization, particularly at ultraviolet wavelengths, under usual conditions or for special purposes, such as might arise in the space p r o g r a m ; synthesis of macromolecules of biological interest from smaller components; and possible existence of communication systems basic to communication between organisms of the same species, using modulated coherent electromagnetic radiation (4.6). Extension of the production of coherent parallel radiation to X - r a y wavelengths should prove of interest for radiological studies and biophysics. X V . ADDENDUM

A. Oral

Tissues

Laser spectroscopy studies on oral tissues have been discussed. Other studies on oral tissues have been carried out. Extracted human teeth have been irradiated at 6934 A, at 12-25 J per pulse, using unfocused, defocused, and focused beams b y L o b e n e and Fine {113a). A n area of enamel 4 - 8 m m in diameter with a fused glasslike appearance surrounded

BIOLOGICAL EFFECTS OF LASER RADIATION

219

each opening. Undecalcified sections revealed an amorphous zone of enamel immediately adjacent to the c a v i t y . R e d u c e d birefringence was observed in the amorphous zone and disrupted enamel rods adjacent to the c a v i t y on examination under polarized light. N o gross differences were noted on X - r a y diffraction studies of hydroxyapatite crystals from the irradiated region in comparison with those from a nonirradiated area. Other in vitro studies have been carried out b y Stern and Sognnaes (163a), and both in vitro and in vivo studies b y Kinersly et al. (98a), and L . G o l d m a n et al. (72a, 72b). A sensation of heat was reported b y Kinersly et al. following irradiation of the stained lip at 0.5 J. Herpes simplex of the lip was unaffected at that energy level. Fiber optics was used b y G o l d m a n et al. to irradiate a molar in vivo. Further in vitro investigations and in vivo animal studies are required to determine the applicability of laser radiation to materials in dentistry, and the desirability of initiating in vivo irradiation in man. T h e use of fiber optics coupled to high power gas laser systems, such as ionized argon units, warrants exploration. B. Microscopy

and

Holography

The advantage of using monochromatic, coherent light to obtain i m proved contrast in interference microscopy was proposed b y T o w n e s (170). Barnes (personal communication) has carried out studies using 6328 A illumination in conjunction with phase microscopy. Preliminary studies at 6328 A b y Fine and Klein (unpublished data, 1964) indicated that this wavelength is relatively unsatisfactory for direct visualization in microscopy. H o w e v e r , with the increasing number of wavelengths available, possible improvements in direct viewing m a y be obtained. T h e advent of laser beams possessing high temporal and spatial c o herency has given increasing impetus to holography studies, originally described b y G a b o r in 1949 (58a). T h e basis of this "lenseless photogr a p h y " is the reproduction of a three-dimensional image of the original object in two steps. T h e first stage is the formation of an interference pattern on a photographic film, due to the interference of reflected (or diffracted) rays from an object, illuminated b y a coherent source. In the case of reflected rays a reference beam is obtained from a mirror. In the second stage, when a coherent beam is passed through this p h o t o graphic film, and observed, an image of the scene similar to that viewed b y binocular vision is observed. Magnification can be measured in two w a y s . It can be obtained b y geometrical magnification. A portion of a fly's wing magnified b y geometrical techniques is shown b y Stroke (165a). A second method is b y irradiation of the scene at one wavelength, and then irradiating the inter-

220

SAMUEL FINE AND EDMUND KLEIN

ference pattern on the G a b o r (58a),

film

at a longer wavelength. In the studies b y

a mercury v a p o r lamp was used as a source, and holograms

were obtained at these frequencies. G a b o r intended to use the technique for improving the resolving power of electron microscopes. A diverging electron beam would be used to produce the diffraction pattern on

the

film. T h e film would then be viewed with visible light resulting in m a g nification. H e suggested its application to X - r a y microscopy, which

has

not been feasible in practice because of the unavailability of practical focusing systems at these wavelengths. The systems originally proposed using holography were, however, incapable of resolving points less than 10,000 A apart, since the two fringe patterns of the two points are t o o closely spaced for the X - r a y plate to resolve.

( X - r a y wavelengths

of the order of 1 A . ) Stroke and Falconer (165b,

165c)

are

suggested that

this could be overcome b y deflecting all the waves diffracted b y the object into a direction where they would m a k e a zero or v e r y small angle with the reference wave, and thus maintain separability of the various waves. Techniques for

achieving this effect,

cussed b y Stroke (165a).

and

initial experiments are

dis-

A nonmathematical presentation of principles

of holography is given b y Leith and Upatnieks (111a).

T h e technique

of holography applied in the X - r a y region m a y permit three-dimensional visualization of macromolecules to be achieved in much shorter time than heretofore possible. ACKNOWLEDGMENT

The authors gratefully acknowledge the many invaluable comments and suggestions by Dr. Ben Fine, George Washington University, Washington, D.C.; Dr. George Moore, Roswell Park Memorial Institute, Buffalo, New York; Dr. Welville Nowak, Dr. Martin Litwin, Dr. Ronald Scott, and Mr. W . Peter Hansen, Northeastern University, Boston, Massachusetts. REFERENCES 1. First Annual Conference, Biological Effects of Laser Radiation, April 30 to May 1, 1964, Washington, D.C., proceedings published in Federation Proc. Symposia Issue 24(1), Pt. 3, Suppl. 14 (Jan-Feb. 1965). 2. Conference of the Laser, New York Academy of Sciences, May 4-5, 1964, New York, proceedings to be published in Ann. N.Y. Acad. Sci. (hereafter cited as Proc. Conf. Laser—Ann. N.Y. Acad. Sci., 1964). 3. Third Boston Laser Conference, August 5-7, 1964, Boston, Massachusetts, Abstracts of Biological Sessions (hereafter cited as Abstr. 3rd Boston Laser Conf., 1964). 4. Adler, F. H., "Physiology of the Eye—Clinical Applications." Mosby, St. Louis, Missouri, 1959. 5. Armstrong, J. A., Nathan, M . I., and Smith, A. W., Appl. Phys. Letters 3, 69 (1963). 6. Baez, S., and Kochen, J. A., Proc. Conf. Lasers—N.Y. Acad. Sci., 1964-

BIOLOGICAL EFFECTS OF LASER RADIATION

221

7. Barnes, F. S., Lang, K. R., Daniel, J. C , and Maisel, J. C. Nature 201, 675 (1964). 8. Basov, N. G., and Prokhorov, A. M . , Zh. Eksperim. i Theor. Fiz. 27, 431 (1954). 9. Basov, N. G., and Prokhorov, A. M . , Zh. Eksperim. i Theor. Fiz. 28, 249 (1955). 10. Basov, N. G., and Prokhorov, A. M , Dokl. Akad. Nank SSSR 101, 47 (1955). 11. Becker, C. H., Cox, G. C , and McLennan, D . B., Proc. IEEE 51, 358 (1963). 12. Bennett, W . R., Jr., Appl. Opt. Suppl. 1. p. 24 (1962). 13. Bessis, M., Gires, F., Mayer, G., Normarski, G., Compt. Rend. 255, 1010 (1962). 14. Bessis, M., and Ter-Pogossian, M . , Proc. Conf. Laser—N.Y. Acad. Sci., 1964. 15. Bloembergen, N., Proc. IEEE 51, 124 (1963). 16. Blois, M . S., Brown, H . W., Lemmon, R. M., Lindblom, R. O., and Weissbluth, M . (eds.), "Free Radicals in Biological Systems," Proc. Symp. Stanford, California, 1960. Academic Press, New York, 1961. 17. Born, M., and Wolf, E., "Principles of Optics." Pergamon Press, Oxford, 1959. 18. Born, M., "Atomic Physics." Hafner, New York, 1964. 19. Brech, F., and Cross, L., Appl. Spectry. 16, 59 (1962). 20. Bridges, W . B., Proc. IEEE 52, 834 (1964). 21. Burns, G., and Nathan, M . I., IBM J. Res. Develop. 7, 72 (1963). 22. Burns, G., and Nathan, M . L, Proc. IEEE 52, 770 (1964). 23. Campbell, C. J., Rittler, M . C , and Koester, C. J., Trans. Am. Acad. Ophthalmol. Otolaryngol. 67, 58 (1963). 24. Campbell, C. J., Noyori, K. S., and Rittler, M . C , Acta Ophthalmol. Suppl. 76, 22. 25. Campbell, C. J., Noyori, K. S., Rittler, M . C , and Koester, C. J., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-71 (1965). 26. Campbell, C. J., Noyori, K. S., Rittler, M . C , and Koester, C. J., Proc. Conf. Laser—Ann. NY. Acad. Sci., 1964. 27. Campbell, C. J., Noyori, K. S., Rittler, M . C , Innis, L, and Koester, C. J., 118 A?in. Conf. Am. Med. Assoc. (Sect. Ophthalmol.), 1964. 28. Carome, E. F., Clark, N. A., and Moeller, C. E.. Appl. Phys. Letters 4, 95 (1964). 29. Chiao, R. Y., Townes, C. H., and Stoicheff, B. P., Phys. Rev. Letters 12, 592 (1964). 30. Commoner, B., Townsend, J., and Pake, G. E., Nature 174, 689 (1954). 31. Davis, T. P., in "Temperature: Its Measurement and Control in Science" Am. Inst. Phys., eds.), Vol. 3, Part 3, p. 149. Reinhold, New York, 1963. 32. Derr, V. E., Klein, E., and Fine, S., Appl. Opt. 3, 786 (1964). 33. Derr, V. E., Klein, E., and Fine, S., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-99 (1965). 34. Ditchburn, R. W., "Light," 2nd ed. Wiley (Interscience), New York, 1962. 35. Earle, K. M., Sterling, C , Roessmann, U., Ross, M . A., Hayes, J. R., and Zeitler, E. H., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-129 (1965). 36. Earle, K. M., and Hayes, J., Abstr. 3rd Boston Laser Conf., 1964. 37. Eckhardt, G., Hellwarth, R. W., McClung, F. J., Schwarz, S. E., Weiner, P., and Woodbury, E. J., Phys. Rev. Letters 9, 445 (1962). 38. Edlow, J., Fine, S., Vawter, G. F., Jockin, H., and Klein, E., Life Sciences. 4, 615 (1965). 39. Edlow, J., Farber, S., Fine, S., and Klein, E. Abstr. 3rd Boston Laser Conf., 1964.

222 40. 41. 42. 43.

SAMUEL FINE AND EDMUND KLEIN

Einstein, A., Physik. Z. 18, 121 (1917). Fine, B. S., and Geeraets, W . J., Unpublished data (1964). Fine, S., Klein, E., Scott, R. E., and Seed, R., Skin 2, 43 (1963). Fine, S., Klein, E., Farber, S., Scott, R. E., Roy, A., and Seed, R. E. J. Invest. Dermatol. 40, 123 (1963). 44. Fine, S., Klein, E., Scott, R. E., Seed, R., and Roy, A., Life Sciences 1 , 30 (1963). 45. Fine, S., Klein, E., Scott, R. E., Aaronson, C., and Donoghue, J., Abstr. 2nd Boston Laser Conj., 1963. 46. Fine, S., Klein, E., and Scott, R. E., IEEE Spectrum 1, 81 (1964). 47. Fine, S., and Klein, E., Life Sciences 3, 199 (1964). 48. Fine, S., Maiman, T. H., Klein, E., and Scott, R. E., Life Sciences 3, 209 (1964). 49. Fine, S., Klein, E., Ambrus, J., Cohen, E., Ambrus, C , Derr, V. E., and Nowak, W., Federation Proc. 23, 442 (1964). 50. Fine, S., Klein, E., Hardway, G., Scott, R. E., King, W., and Aaronson, C , J. Invest. Dermatol. 42, 289 (1964). 51. Fine, S., Klein, E., Nowak, W., Scott, R. E., Laor, Y., Simpson, L., Crissey, J., Donoghue, J., and Derr, V. E., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-35 (1965). 52. Fine, S., Klein, E., Laor, Y., Abstr. 3rd Boston Laser Conf., 1964. 53. Fine, S., Klein, E., Nowak, W., Hansen, W., Hergenrother, K., Scott, R. E., and Donoghue, J., Northeast Electronics Research and Engineering Meeting (I.E.E.E.) Record p. 158 (1964). 54. Fine, S., Klein, E., Brech, F., and McNary, W . F., Unpublished data (1963). 55. Flocks, M., and Zweng, H. C , 113th Ann. Conf. Am. Med. Assoc. (Sect. Ophthalmol.), San Francisco, 1964. 56. Fowles, G. R., and Jensen, R. C , Proc. IEEE 52, 851 (1964). 57. Franken, P. A., Hill, A. E., Peters, C. W., and Weinreich, G., Phys. Rev. Letters 7, 118 (1961). 58. Freeman, H . M., Pomerantzeff, 0., and Schepens, C. L., Proc. Conf. Laser— N.Y. Acad. Sci., 1964. 58a. Gabor, D., Proc. Roy. Soc. (London) A197, 454 (1949). 59. Garmire, E., and Townes, C. H., Appl. Phys. Letters 5, 84 (1964). 60. Geeraets, W . J., Williams, R. C , Chan, G., Ham, W . T , Jr., Guerry, D., I l l , and Schmidt, F. H., Arch Ophthalmol. (Chicago) 64, 606 (1960). 61. Geeraets, W . J., Ham, W . T., Jr., Williams, R. C , Mueller, H. A., Burkhart, J., Guerry, D., I l l , and Vos, J. J., Federation Proc. 24(1), Pt. 3, Suppl. 14, 5-48 (1965). 62. Gibson, Q. H., and Ainsworth, S., Nature 180, 1416 (1957). 63. Goldman, H . M . , Ruben, M . P., and Sherman, D . B., Oral Surg., Oral Med., Oral Pathol. 17, 102 (1964). 64. Goldman, J., Hornby, P., and Long, C , J. Invest. Dermatol. 42, 231 (1964). 65. Goldman, L., Blaney, D. J., Kindel, D. J., and Franke, E. K., J. Invest. Dermatol. 40, 121 (1963). 66. Goldman, L., Blaney, D . J., Kindel, D . J., Richfield, D. F., and Franke, E. K., Nature 197, 912 (1963). 67. Goldman, L., Abstr. 2nd Boston Laser Conf., 1963. 68. Goldman, L., Blaney, D. J., Kindel, D. J., Richfield, D. F., Owens, P. and Homan, E. L., J. Invest. Dermatol. 42, 247 (1964).

BIOLOGICAL EFFECTS OF LASER RADIATION

223

69. Goldman, L., Blaney, D . J., Freemond, A., and Hornby, P., J. Am. Med. Assoc. 188, 230 (1964). 70. Goldman, L., Igelman, J. M . , and Richfield, D . F., Arch. Dermatol. (Chicago) 90, 71 (1964). 71. Goldman, L., and Wilson, R., J. Am. Med. Assoc. 189, 171 (1964). 72. Goldman, L., Proc. Conf. Laser—N.Y. Acad. Sci., 1964). 72a. Goldman, L. et al, J. Am. Dental Assoc. 70, 601 (1965). 72b. Goldman, L. et al, Nature 203, 417 (1964). 73. Gordon, J. P., Zeiger, H. J., and Townes, C. H., Phys. Rev. 95, 282 (1954). 74. Gordon, J. P , Zeiger, H. J., and Townes, C. H., Phys. Rev. 99, 1265 (1955). 75. Grosof, G., Proc. Conf. Laser—N.Y. Acad. Sci., 1964. 76. Grossweiner, L. D., J. Chem. Phys. 24, 1255 (1956). 77. Grossweiner, L. D., and Mulac, W . A., Radiation Res. 10, 515 (1959). 78. Hall, R. N., Fenner, G. E , Kingsley, V. D., Soltys, T. J., and Carlson, R. O., Phys. Rev. Letters 9, 366 (1962). 79. Hall, R. N., Solid-State Electron. 6, 405 (1963). 80. Ham, W . T., Jr., Weisinger, H., Guerry, D., I l l , Schmidt, F. H., Williams, R. C , Ruffin, R. S., and Shaffer, M . C , Am. J. Ophthalmol. 43, 711 (1957). 81. Ham, W . T., Jr., Weisinger, H , Schmidt, F. H., Williams, R. C , Ruffin, R. S., Schaffer, M . C , and Guerry, D., I l l , Am. J. Ophthalmol. 46, 700 (1958). 82. Ham, W . T., Jr., Williams, R. C , Geeraets, W . J., Ruffin, R. S., and Mueller, H. A., Acta Ophthalmol. Suppl. 76, 60 (1963). 83. Ham, W . T., Jr., Williams, R. C , Mueller, H . A., Ruffin, R. S., Schmidt, F. H., Clark, A. M . , and Geeraets, W . J., Acta Ophthalmol, submitted for publication. 84. Harding-Barlow, I., Personal communication (1964). 85. Hardy, A. C , and Perrin, F. H., "The Principles of Optics." McGraw-Hill, New York, 1932. 86. Hardy, J. D., and Muschenheim, D., / . Clin. Invest. 13, 817 (1934). 87. Hayes, J., Helwig, A., Earle, E., and Lele, A., Abstr. 3rd Boston Laser Conf., 1964. 88. Hellwarth, R. W., in "Advances in Quantum Electronics," 2nd Intern. Congr. Quantum Electronics (J. R. Singer, ed.), p. 334. Columbia Univ. Press, New York. 89. Helsper, J. T., Sharp, G. S., Williams, H. F., and Fister, H. W., Cancer 17, 1305 (1964). 90. Henriques, F. C , Jr., and Moritz, A. R., Am. J. Pathol. 23, 531, (1947). 91. Howard, W . E., Fang, F. F., Dill, F. H., Jr., and Nathan, M . I., IBM J. Res. Develop. 7, 74 (1963). 92. Igelman, J. M . , Federation Proc. 24(1), Pt. 3, Suppl. 14, 5-94 (1965). 93. Igelman, J. M . , Rotto, T. C , Scheckter, E., and Blaney, D . J., Proc. Conf. Laser—N.Y. Acad. Sci., 1964. 94. Javan, A., Bennett, W . R., Jr., and Herriot, D . R., Phys. Rev. Letters 6, 106 (1961). 95. Kapany, N. S., Peppers, N. A., Zweng, H. C , and Flocks, M . , Nature 199, 146 (1963). 96. Kapany, N . S., Proc. Conf. Laser—N.Y. Acad. Sci., 1964. 97. Karpe, G., Doc. Ophthalmol. 2, 268 (1948). 98. Ketcham, A. S., and Minton, J. P., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-159 (1965).

224

SAMUEL FINE AND EDMUND KLEIN

98a. Kinersly, T. et al, J. Am. Dental Assoc. 70, 593 (1965). 99. Klein, E., and Fine, S., Paper presented at Stephan Rothman Research Club, Am. Acad. Dermatol., 1962. Chicago, Illinois. 100. Klein, E., Fine, S., Cohen, E., Ambrus, J., Neter, E., Lyman, R., and Scott, R. E., Proc. Meeting Am. Coll. Physicians, Atlantic City, New Jersey, 1964. 101. Klein, E., Fine, S., Scott, R. E., and Farber, S., Proc. Am. Assoc. Cancer Res., (1964). 102. Klein, E., Fine, S., Laor, Y., Simpson, L., Ambrus, J., Richter, W., Smith, G. K , and Aaronson, C , Federation Proc. 24(1), Pt. 3, Suppl. 14, S-143 (1965). 103. Klein, E., Fine, S., Ambrus, J., Cohen, E., Ambrus, C , Neter, I., Bardos, T., and Lyman, R., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-104 (1965). 104. Klein, E., and Fine, S., Proc. Conf. Laser—Abstracts N.Y. Acad. Sci., 1964. 105. Klein, E., Fine, S., Laor, Y., Donoghue, J., and Simpson, L., J. Invest. Dermatol. 43, 565 (1964). 106. Kochen, J. A., and Baez, S., Proc. Conf. Laser—N.Y. Acad. Sci., 1964. 107. Koester, C. J., Snitzer, E., Campbell, C. J., and Rittler, M . C , J. Opt. Soc. Am. 52, 607 (1962). 108. Kopac, M . J., Science 113, 232 (1951). 109. Kuppenheim, H . F., Dimitroff, J. M., Malotti, P. H., Graham, I. C , and Swanson, D . W., J. Appl. Physiol. 9, 75 (1956). 110. Langley, R. K., Mortimer, C. B., and McCulloch, C , AM A Arch. Ophthalmol. 63, 473 (1960). 111. Lax, B., Solid-State Design 4, 26 (1963). 111a. Leith, E. N., and Upatnieks, J., Sci. Am. 212(6), 24 (1965). 112. Lithwick, N. H., Healy, M . K., and Cohen, J., Submitted for publication. 113. Litwin, M., and Glew, D., / . Am. Med. Assoc. 187, 842 (1964). 113a. Lobene, R. R., and Fine, S., "Interaction of Laser Radiation with Oral Hard Tissues," to be presented at the International Association for Dental Research, July 1965. Abstract to be published in J. Dent. Res. 114. Ludvigh, E., and McCarthy, E. F., AM A Arch. Ophthalmol. 20, 713 (1938). 115. McClung, F. J., and Hellwarth, R. W., Appl Phys. 33, 828 (1962). 116. McGuff, P. E., Bushnell, D., Soroff, H. S., and Deterling, R. A., Jr., Surg. Forum 14, 143 (1963). 117. McGuff, P. E., Deterling, R. A., Jr., Gottlieb, L. S., Fahimi, H. D., and Bushnell, D., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-150 (1965). 118. McGuff, P. E., Deterling, R. A., Jr., Gottlieb, L. S., Bushnell, D., and Roeber, F., Proc. Conf. Laser—N.Y. Acad. Sci., 1964. 119. McGuff, P. E., Deterling, R. A., Jr., Levy, C. K., Bushnell, D., and Roeber, F., Abstr. 3rd Boston Laser Conf., 1964. 120. McNary, W . F., Rosan, R. C , and Healy, M . K., Abstr. 3rd Boston Laser Conf., 1964. 121. Maiman, T. H., Phys. Rev. Letters 4, 564 (1960). 122. Maiman, T. H., Nature 187, 493 (1960). 123. Maiman, T. H., Phys. Rev. 123, 1145 (1961). 124. Malmstrom, L. D., Schlickman, J. J., and Kingston, R. H., J. Appl Phys. 35, 248 (1964). 125. Malt, R. A., and Townes, C. H., New Engl J. Med. 269, 1417 (1963). 126. Malt, R. A., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-122 (1965). 127. Masters, J. E., Ward, J., and Hartourni, E., Rev. Sci. Instr. 34, 365 (1963). 128. Mendelson, J., Abstr. 3rd Boston Laser Conf., 1964.

BIOLOGICAL EFFECTS OF LASER RADIATION

225

129. Meyer-Schwickerath, G., "Light Coagulation" (translated by S. M . Drance), p. 24. Mosby, St. Louis, Missouri. 130. Minton, J. P., Zelen, M . , and Ketcham, A. S., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-155 (1965). 131. Minton, J. P., Proc. Conf. Laser—Ann. N.Y. Acad. Sci., 1964. 132. Minton, J. P., and Ketcham, A., Abstr. 3rd Boston Laser Conf., 1964. 133. Minton, J. P., Life Sciences 3, 1007 (1964). 134. Minton, J. P., and Ketcham, A., Cancer 17, 1305 (1964). 135. Najac, H., Cooper, B., Jacobson, J. H., Shamos, M . , and Breitfeller, M., Invest. Ophthalmol. 2, 32 (1963). 136. Nathan, M . I., Dumke, W . P., Burns, G., Dill, F. H., Jr., Appl. Phys. Letters 1, 62 (1962). 137. Norris, J. A., Abstr. 3rd Boston Laser Conf., 1964. 138. Nowak, W . B., Fine, S., Klein, E., Hergenrother, K , and Hansen, W . P., Life Sciences, 3, 1475 (1964). 139. Noyori, K . S., Campbell, C. J., Rittler, C , and Koester, C. J., Arch. Ophthalmol. (Chicago) 72, 254 (1964). 140. "Optical Masers," Appl. Opt. Suppl. 1 (1962). 141. Pomerantzeff, O., Schepens, C. L., and Freeman, H . M., Brit. J. Ophthalmol. 48, 298 (1962). 142. Pomerantzeff, O., and Schepens, C. L., Brit. J. Ophthalmol. 48, 306 (1964). 143. Pomerantzeff, O., Brit. J. Ophthalmol. 48, 311 (1964). 144. Pomerantzeff, O., Brit. J. Ophthalmol. 48, 315 (1964). 145. Quantum Electronics Issue, Proc. IEEE 51, No. 1 (1963). 146. Quist, T. M., Rediker, R. H., Keyes, R. J., Krag, W . E., Lax, B., McWorter, A. L., and Zeiger, H. J., Appl. Phys. Letters 1, 91 (1962). 147. Robertson, J. K , "Introduction to Optics," 4th ed. Van Nostrand, Princeton, New Jersey, 1954. 148. Rosen, R. C , Healy, M . K , and McNary, W . F , Science 142, 236 (1963). 149. Rounds, D . E., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-116 (1965). 150. Rounds, D . E., Proc. Conf. Laser—N.Y. Acad. Sci., 1964. 151. Rounds, D . E., and Chamberlain, E. C , Abstr. 2nd Boston Laser Conf., 1963. 152. Rounds, D . E., Chamberlain, E. C , and Obigaki, T., Proc. Conf. Laser—Ann. N.Y. Acad. Sci., 1964. 153. Rounds, D . E., and Adams, J. E., Abstr. 3rd Boston Laser Conf., 1964. 154. Saks, N. M., and Roth, C. A., Science 141, 46 (1963). 155. Saks, N. M., Zuzolo, R. C , and Kopac, M . J., Proc. Conf. Laser—N.Y. Acad. Sci., 1964. 156. Schawlow, A. L., and Townes, C. IL, Phys. Rev. 112, 19 (1958). 157. Schepens, C. L., and Freeman, H., Paper presented at Med. Phys. Soc. Meeting, Boston, Massachusetts, 1963. 158. Sherman, D . B., Ruben, M . P., and Goldman, H. M., Abstr. 3rd Boston Laser Conf., 1964. 159. Siegmund, W . P., Proc. Conf. Laser—N.Y. Acad. Sci.. 1964. 160. Smaller, B., Advan. Biol. Med. Phys. 9, 225 (1963). 161. Sogo, P. B., and Tolbert, B. M., Advan. Biol. Med. Phys. 5, 1 (1957). 162. Solon, L. R., Aronson, R , and Gould, G., Science 134, 1506 (1961). 163. Sorokin, P. P., Luzzi, J. J., Lankard, J. R., Pettit, B. D., IBM J. Res. Develop. 8, 182 (1964). 163a. Stern. R. H.. and Sognnaes, R. F., J. Dental Res. 43, 873 (1964).

226

SAMUEL FINE AND EDMUND KLEIN

164. Straub, H. W., Protection of the Human Eye from Laser Radiation, Army Material Commands (Harry Diamond Labos.) Rept. No. TR-1153, p. 15 (1963). 165. Straub, H. W., Federation Proc. 24(1), Pt. 3, Suppl. 14, S-78 (1965). 165a. Stroke, G. W., Intern. Sci. and Technol. pp. 52-60 (May 1965). 165b. Stroke, G. W., and Falconer, D . G., in "Symposium on Optical and Electrooptical Information Processing Technology," Nov. 9-10, 1964 (J. T. Tippett et al., eds.). M.I.T. Press, Cambridge, Massachusetts. To be published. 165c. Stroke, G. W , and Falconer, D. G., Phys. Letters 13, 306 (1964). 166. Swope, C. H., and Koester, C. J., Appl. Optics, 4, 523 (1965). 167. Tengroth, B., Karlberg, B., Bergquist, T., and Adehed, T. Acta Ophthalmol. 41, 595 (1963). 168. Terhune, R. W., Solid-State Design 4, 38 (1963). 169. Terhune, R. W., Maker, P. D., and Savage, C. M., Appl Phys. Letters 2, 54 (1963). 170. Townes, C. H., Biophys. J. 2, No. 2, Part 2, 325 (1962). 171. Weber, J., IRE Trans., Prof. Group. Electron Devices 3, 15 (1963). 172. Wiesinger, H., Schmidt, F. H., Williams, R. C , Tiller, C. 0 . , Ruffin, R. S., Guerry, D., I l l , and Ham, W . T., Jr., Am. J. Ophthalmol. 42, 907 (1956). 173. White, R. M., J. Appl. Phys. 34, 3559 (1963). 174. Woodbury, E. J., and Ng, W . K , Proc. IRE 50, 2357 (1962). 175. Zaret, M . M . , Breinin, G. M . , Schmidt, H., Ripps, H., Siegel, I. M . , and Solon, L. R., Science, 134, 1525 (1961). 176. Zaret, M . M . , Ripps, H., Siegel, I. M . , and Breinin, G. M., Arch. Ophthalmol. (Chicago) 6 9 , (1963). 177. Zaret, M . M . , Federation Proc. 24(1), Pt. 3, Suppl. 14, S-62 (1965). 178. Zuzolo, R., Saks, N. M., and Kopac, M . J., Abstr. 3rd Boston Laser Conf., 1964. 179. Zweng, H. C , and Flocks, M., Federation Proc. 24(1), Pt, 3, Suppl. 14, S-65 (1965).

Quadro Moderno 5 pz. cm 150x90 Stampa su Tela Arredamento Casa Arredo Design | Murphy Brown | IMDb: 7 S2E11 Deutschland 83 - Season 2