range of energies, were delivered to the retina of Macaca mulatto,. Clinical examination and fluorescein angiography were performed at 1 hour in all eyes and ...
Articles Pathology of Macular Lesions From Subnanosecond Pulses of Visible Laser Energy Cynthia A. Toth,* Drew G. Narayan* Clarence P. Cain,~f Gary D. Noojin,-f Katrina P. Winter* Benjamin A. Rockwell,% and W. P. Roach%
Purpose. To demonstrate how current theories regarding ultrashort laser pulse effects may apply to ocular tissue, a prospective clinicopathologic study of macular lesions from ultrashort laser pulses compared the pathologic effects with the clinical and fluorescein angiographic appearance of the laser lesions. Methods. Ninety-femtosecond, 3-picosecond, and 60-picosecond laser pulses, throughout a range of energies, were delivered to the retina of Macaca mulatto,. Clinical examination and fluorescein angiography were performed at 1 hour in all eyes and 24 hours after exposure in selected eyes. Eyes were enucleated at 1 or 24 hours after lesion placement. The structure and extent of retinal lesions were scored for comparison with the clinical findings. Results. Focal retinal pathologic appearance correlated well with a clinically visible lesion observed 24 hours after laser delivery. Retinal lesions were small foci of retinal pigment epithelium (RPE) and retinal disruption, without choriocapillaris involvement. Lesions that contained focal RPE vacuoles or lifting of the RPE also demonstrated leakage, in fluorescein angiographic studies. Supratlireshold laser delivery frequendy caused focal columns of retinal injury and intraretinal hemorrhages from retinal vessel bleeding, with no rupture of choroidal blood vessels. Conclusions. The retinal response to ultrashort laser pulses at moderate energy followed a pattern of focal damage from laser-induced breakdown without significant thermal spread. Invest Ophthalmol Vis Sci. 1997;38:2204-2213.
Hivaluation of a new laser system for medical application, in diagnosis or in treatment, has two components: the safety of the laser for the proposed application and the potential for the laser to improve medical care in a cost-effective manner. Satisfaction of these requirements depends on the interaction of the laser with biologic tissues and the consequent information
or effect achieved. Laser tissue effects can be interpreted as a hazard in one context and as a treatment goal in another. Thus, determining how laser effects on tissue depend on the laser's parameters (pulsewidth or energy) is essential to assuring safe application and to evaluating the laser for use in the diagnosis or treatment of patients. Laser systems capable of producing pulses of less than 1 nanosecond (ns) in duration, have proliferated during the past 10 years. In previous studies of ocular From the *Duke University Eye Center, Durham, North Carolina; -\TASC, San Antonio, and ^Armstrong Laboratory, Brooks Air Force Base, Texas; and the §Air effects from subnanosecond pulses of laser energy, the Force Office of Scientific Research, Boiling Air Force Base, Washington, DC. threshold laser energy required to produce a visible Supported in part by Armstrong Laboratory (Brooks AFB, Texas), and by the Air Force Office of Scientific Research (Boiling AFB, Washington, DC) (grants F49620retinal lesion or a hemorrhagic lesion at different 95-1-0226 and 2312AA-92AL014 and Contract F33615-92-C-0017). The U.S. pulsewidths, in the rabbit or primate, have been idengovernment is authorized to reproduce and distribute reprints for governmental purposes notwitlistanding any copyright notation thereon. tified.1"7 Some investigators reported morphologic The views and conclusions contained in this document are those of the authors and characteristics of the laser lesions, although many used should not be interpreted as necessarily representing the official policies or endorsements, either express or implied, of the Air Force Office of Scientific Research a clinical endpoint for lesion threshold. Several findor the United States government. ings from these reports are of particular interest. First, Submitted for publication December 23, 1996; revised May S, 1997; accepted June hemorrhages were infrequently seen in 80- and 1003, 1997. Proprietary interest category: N. femtosecond (fs) pulse studies in the rabbit5'7 but were Reprint requests: Cynthia A. Toth, Department of Ophthalmology, Duke University common in the primate.6 Second, the time to appearMedical Center, Box 3802, Enuin Road, Durham, NC 27710.
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Pathology of Subnanosecond Pulse Laser Retinal Lesions ance of a minimum visible lesion (MVL) was delayed compared to that of longer pulse lesions.1"7 Third, fluorescein angiographic images did not improve the visualization of low energy, 90-fs pulsed laser lesions in the primate macula.6 In addition, there was an intriguing finding of bubble formation in the vitreous cavity of the rabbit eye, which occurred on delivery of suprathreshold 90-fs pulses of visible laser energy, when the focal plane was moved off the retina.8 In their report of a study of retinal damage from nanosecond laser pulses, Cleary and Hamrick speculated that acoustic transients from laser induced breakdown by nanosecond laser pulses in the mammalian eye could lead to damage.9 Marshall found evidence for thermal and mechanical damage in retinal lesions caused by a more than 20-nanosecond laser beam exposure that had a laser retinal image diameter of 100 to 800 //m.10 He found that in these lesions, all damage was associated with disturbance of the RPE, with no evidence of primary damage in the neural layers of the retina and with no evidence of cavitation. Zysset et al n and Vogel et al12 showed that shorter laser pulses (picoseconds) may induce optical breakdown with smaller foci of damage, which might be advantageous for intraocular applications.11"12 In parallel with the initial applications of ultrashort laser pulses to tissue, theoretical work and bench studies of ultrashort pulse laser effects on materials suggested that such nonlinear optical phenomena as laser-induced breakdown and self-focusing may have a significant role in the laser propagation and tissue effects in the eye.813"17 These results demonstrate that ultrashort pulse laser effects on tissue could be markedly different from damage previously seen from longer laser pulses. Here, sites of impact from plasmas and other nonlinear phenomena could have major effects on the tissue damage response from ultrashort pulses. The initial goal of the current study was to identify the pathologic correlates of clinically visible retinal lesions from ultrashort (picosecond and femtosecond) laser pulses in the primate macula. The second goal was to determine whether these pathologic characteristics clarified the clinical findings, and whether they supported or refuted the proposed nonlinear theories regarding this laser application. MATERIALS AND METHODS The treatment and procedures used in this study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with federal guidelines. Animals involved in this study were procured, maintained, and used in accordance with the recommendations of the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of
2205 Health Publication No. 85-23, revised 1985), the Office for Protection from Research Risks's Public Health Service Policy on Humane Care and Use of Laboratory Animals (revised 1986), and the Animal Welfare Act. Mature Macaca mulatto, weighing 3 to 4 kg were maintained under standard laboratory conditions, with monitoring and care during laser lesion placement, enucleation, and euthanasia as previously described.6 Single laser pulses with a range of energies from 0.08 to 12 fjj were delivered in a grid to the macular area of each eye at one of the three laser pulsewidths: 90-fs, 3-picosecond (ps) pulses or 60 ps, as previously described by Cain et al.6 The 90-fs and 3-ps laser pulses were 580 nm in wavelength, whereas the 60-ps pulses were 532 nm in wavelength. Four macular grids of three animals, with a total of 36 sites of laser delivery, were examined for tissue effects from 90-fs laser pulses. Four macular grids of three animals, with a total of 23 sites of laser delivery, were evaluated for tissue effects from 3-ps laser pulses. Two macular grids of two animals, with a total of 18 sites of laser delivery, were evaluated for tissue effects from 60-ps laser pulses. All laser energies reported in this study, were energies delivered to the cornea and measured as a percentage of beam delivered to a beam splitter. The pulsewidth was measured as previously described by Cain et al.6 The globes were obtained 1 or 24 hours after exposure, incised anterior to the equator, and immersed in a 3% glutaraldehyde and 0.1 M sodium cocodylate buffer immediately after enucleation. The posterior eye cup was cut from the anterior segment after 10 minutes and replaced in the fixative. The macular area was later dissected and embedded in Spurr's resin. One-micrometer sections were stained with methylene blue for examination. Sections in the region of laser delivery were obtained by aligning the first row of marker lesions and step sectioning at 20 //m through the length of each marker lesion for the three rows of the grid. Laser sites were examined using a Zeiss (Carl Zeiss, Oberkochen, Germany) light microscope. Digital images of the most extensive site of retinal effect for each lesion were captured using Scion Image on a Macintosh computer and manipulated to obtain comparable contrast and sharpness using Adobe Photoshop 3.0 (Adobe Systems, Mountain View, CA). The maximum width of each lesion was measured at the outer nuclear layer. During light microscopic examination, the extent of each laser lesion in sectioned retinal tissue was graded (Table 1) on the basis of the following criteria: A grade of 0 indicated that there was no retinal, RPE, or choriocapillaris abnormality at the site of presumed laser delivery, compared with the condition of the adjacent, normal retina. A grade of 1 indicated that only
Investigative Ophthalmology & Visual Science, October 1997, Vol. 38, No. 11
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TABLE l.
Lesion Grade 0 1 2A
2V
3
4
H
Grading System for Retinal Lesion Histopathology
Extent of Retinal Damage
Choriocapillaris and Bruch's Membrane
Retinal Pigment Epithelium (RPE)
Photoreceptors
Inner Nuclear Layer and Plexiform Layer
Nerve Fiber Layer and Internal Limiting Membrane
No lesion at site of laser pulse deliver)' Only RPE cell effects Morphologic changes only in RPE cells and photoreceptor inner and outer segments and photoreceptor nuclei Grade 2A lesion plus RPE vacuoles or elevation of RPE cell from Bruch's membrane Lesion extends through RPE cells and outer and inner retinal layers but not into the nerve fiber layer Full thickness retinal lesion also involving RPE and sometimes including rupture of the internal limiting membrane Hemorrhage in or around laser lesion; only seen in grade 3 or 4 lesions
RPE cells were affected at the laser site, usually appearing as a single disrupted or lifted cell (Fig. 1). A grade of 2A indicated that morphologic changes could be found in RPE cells and photoreceptor inner and outer segments and photoreceptor nuclei, without inner retinal effects (Fig. 2). A grade of 2V indicated a lesion similar to grade 2A but with RPE vacuoles or separation of the RPE from underlying Bruch's membrane (Fig. 3). A grade of 3 indicated a lesion that extended through RPE cells and outer and inner retinal layers, but not into the nerve fiber layer (Fig. 4). A grade of 4 indicated a full-thickness retinal lesion, involving RPE through nerve fiber layer and sometimes including rupture of the internal limiting membrane (Figs. 5A, 5B). A grade of an additional H indicated that a hemorrhage was visible in the tissue sections (Figs. 6A, 6B). The term minimum visible lesion (MVL) designated a visible change in the fundus caused by laser insult, which was identified by at least two observers by ophthalmoscope. A fluorescein angiographic visible lesion (FAVL) denoted a focal change in fluorescence
at the macular laser site, which was visible in the early or late phase of the fluorescein angiogram. Comparison of the incidences of MVLs and FAVLs in grade 2A and 2V lesions was performed by chisquare (contingency table) analysis.
RESULTS The results of our study are summarized in two scattergrams that compare the grade of retinal pathology (Fig. 7) or the horizontal extent of the macular lesion (Fig. 8) with the laser energy delivered to the eye at each of three pulsewidths. Retinal pathology from the three laser pulsewidths occurred in the following patterns: • Nonlesion sites (grade 0) corresponded generally to sites at which the lowest energies had been delivered. The range of laser energies delivered for nonlesions overlapped with the range that produced lesions (Fig. 7). • There were no grade 1 lesions from 90-fs pulses.
Pathology of Subnanosecond Pulse Laser Retinal Lesions
When 3- and 60-ps laser pulses were applied, grade 1 lesions were observed, associated with low-energy delivery. • The most frequently observed lesions were grade 2. These developed after laser delivery throughout a wide range of energies for all pulsewidths. • Grade 3 and 4 retinal laser lesions occurred at lower energies for 90-fs pulses than for 3- or 60-ps pulses. There were 13 sites with grade 0 pathologic appearance. Of these, 9 received laser energy below the ED50 for MVL,0 and thus had a less than 50% likelihood for formation of an MVL. At 6 of the 13 sites, clinical examiners reported an MVL. There were no FAVLs at the 13 grade 0 sites examined. The seven grade 1 lesions involved one to two RPE cells in maximal horizontal extent (Fig. 1). Effects on RPE included disruption of cellular elements, diminished staining of the cytoplasm, often with pyknotic nuclei but no obvious cell rupture. There was no underlying damage to Bruch's membrane or to the choriocapillaris. None of the seven, 1-hour-old, grade 1 lesions appeared as MVLs, whereas one of the two, 24-hour-old, grade 1 lesions was an MVL. With fluorescein angiography, one of three grade 1 lesions was visible at 1 hour, and two of two grade 1 lesions were visible at 24 hours. The FAVLs were focal faintly hyperfluorescent sites, with minimal late leakage. There were 39 grade 2 lesions, of which 28 lesions (72%) were less than 50 fj,m in horizontal extent; 35 lesions (90%) were less than 100 ^m across. The le-
mmmm, FIGURE i. Light micrograph of a grade 1 laser retinal lesion. The lesion was obtained 1 hour after delivery of a L32-/iJ, 60-ps pulse. There is a focal area of condensation of nuclear chromatin in one retinal pigment epithelium cell, with darkened cytoplasm and slight elevation from Bruch's membrane in both cells affected {arrows). This was not seen in adjacent normal retinal areas. The retina overlying the site of retina! pigment epithelium damage and the choriocapiltaris below and in adjacent sections appear unaffected.
FIGURE 2. Light micrograph of a grade 2A laser retinal lesion. The lesion was obtained 1 hour after delivery of a 1.39fij, 90-fs pulse. There is a focal site of loss of retinal pigment epithelium melanin pattern with disruption of the cytoplasm but no large vacuoles {open airows). The overlying photoreceptors demonstrate intracellular vacuoles in the inner segments and darkened cytoplasm of the outer segments. Pyknotic rod and cone nuclei, with immediately adjacent normal nuclei, are also visible {solid airmos). The remaining retinal layers appear unaffected.
sions involved RPE and photoreceptor inner and outer segments focally, with few pyknotic nuclei or nuclei with vacuoles. None showed any definite outer plexiform layer changes and all had normal inner retinal layers. The choriocapillaris was normal, and Bruch's membrane was intact in each of these lesions. Several 24-hour-old grade 2 lesions demonstrated broader areas of pyknotic photoreceptor nuclei than the 1-hour-old lesions of comparable energy (Fig. 8). In the 3- and 60-ps laser lesions, the horizontal extent of 1-hour lesions was greater and the broadening of lesions at 24 hours was greater than in the 90-fs laser lesions. There was a close correlation between a positive MVL score at 24 hours and presence of a pathologic lesion. At 24 hours all 19 grade 2 lesions were MVLs, whereas at 1 hour, only 29 of 41 (71%) grade 2 lesions were MVLs. The clinical and fluorescein angiographic appearance of grade 2 lesions related to the presence or absence of vacuoles in the RPE or elevation of the RPE from the underlying Bruch's membrane. Across all pulsewidths, 16 of 16 (100%) grade 2 lesions with RPE vacuoles, RPE elevation off Bruch's membrane, or both (grade 2V, Fig. 3) were MVLs at 1 hour. In
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FIGURE 3. Light micrograph of a grade 2V laser retinal lesion. The lesion was obtained 24 hours after delivery of a 2.44}j,\, 90-fs pulse to the macula. The central affected retinal pigment epithelial cell is elevated from Bruch's membrane. The central retinal pigment epithelial cell and the cell immediately adjacent have vacuoles in the cytoplasm. Note the intact choriocapillaris beneath the damaged retinal pigment epithelium.
contrast to the number of grade 2V lesions that were MLVs at 1 hour, only 13 of 25 (52%) grade 2 lesions had no RPE vacuoles or RPE elevation off Bruch's membrane (grade 2A; Fig. 2) were MLVs at 1 hour (P > 0.01). All 12 grade 2A lesions that were not MVLs at 1 hour were from 90-fs laser delivery. Only 1 of 13 (8%) of grade 2A lesions was an FAVL at 1 hour, and only 1 of 8 (13%) of grade 2A lesions was an FAVL at 24 hours. In contrast to the number of grade 2A lesions that were FAVL, 13 of 15 (87%) of the grade 2V lesions were FAVL at 1 hour (P > 0.01) and 9 of 11 (82%) grade 2V lesions were FAVL at 24 hours (P > 0.01). All of the 90-fs laser-induced grade 3 and 4 lesions were excised and fixed at 1 hour after laser delivery. All sites of 90-fs laser delivery with more extensive retinal damage (grade 3 or 4) were narrow (21 to 65 fim) columns of retinal damage (Figs. 4, 5), except when bleeding occurred and blood extended beyoncb the column through inner retinal layers {Fig. 4, 6). In 90-fs laser grade 3 and 4 lesions, inner retinal layers demonstrated vacuoles and pyknotic nuclei in a narrow column, without a visible gradation of effect into surrounding tissue. There was a slight increase in width of the zone of outer nuclear layer damage, but the extent of RPE and photoreceptor injury was other-
wise similar to those of the grade 2 lesions. Bruch's membrane and the choriocapillaris demonstrated no injury at these sites. All of the grade 3 and 4 lesions were MVLs immediately and thereafter. All of the grade 3 and 4 lesions examined by fluorescein angiographic scan were FAVLs at 1 and 24 hours. There were five grade 3 and 4 lesions created by 3-ps laser pulses and one grade 3 lesion created by 60-ps laser pulses (Fig. 7). All of these lesions were produced by higher laser energies than was required to produce the 90-fs high-grade lesions (Fig. 7). The longer pulsewidth lesions demonstrated a pattern of tissue response similar to that of the 90-fs high-grade lesions, although one 24-hour-old lesion with an inner retinal hemorrhage had broader damage to the outer nuclear layer (151 (xm) at 24 hours when compared to damage in the other 1-hour lesions (Fig. 8) Hemorrhagic lesions (Fig. 4, 6) were produced by all pulsewidths. However, the energy levels for hemorrhage from 90-fs laser pulses were much lower than for 3- or 60-ps laser pulses (Fig. 7). There were no ruptures of Bruch's membrane, and there were no sites of bleeding into or from the choroidal circulation
4. Light micrograph of a grade 3H laser retinal lesion. The lesion was obtained 24 hours after delivery of a 6.58-juJ, 60-ps pulse. Note the focal area of retinal pigment epithelium and photoreceptor damage (open airows) with several scattered pyknotic nuclei in the outer nuclear layer adjacent to this site. The intraretinal hemorrhage from retinal vessels (solid arroivs) extends throughout the inner nuclear layer in this lesion. Despite the extensive retinal effect, the underlying choriocapillaris in this section and adjacent sections demonstrated no disruption or thrombosis.
FIGURE
Pathology of Subnanosecond Pulse Laser Retinal Lesions
FIGURE 5. (A) Light micrograph of a grade 4 laser retinal lesion. The lesion was obtained 1 hour after delivery of a 3.37-//J, 90-fs pulse. The retinal damage extends from the retinal pigment epithelium (solid arrows), where there is mild disruption of the cytoplasm and displacement of melanin granules, to the nerve fiber layer, where large focal vacuoles and clefts are present (open arrows). All along the column of damage, pyknotic nuclei and focal vacuoles are present, with very minimal adjacent tissue effect. (B) Light micrograph of a montage from two adjacent tissue sections demonstrating a single grade 4 laser retinal lesion. The lesion was obtained 1 hour after delivery of a 4.88-^/J, 3-ps pulse. The pattern of effect is similar to that described in panel A.
in any of the tissue sections that included or were adjacent to the lesions. We found focal retinal vascular damage within all hemorrhagic lesions (Fig. 6B). The hemorrhage in all lesions extended laterally into the retina into either the nerve fiber layer, the inner plexiform layer, or the outer plexiform layer. Infrequently, the hemorrhage also spread along the column of presumed laser damage. Clinically, vitreous blood was not seen over any retinal hemorrhagic laser lesion at the 1- or 24-hour observation time point. DISCUSSION In our results, we demonstrate a reproducible pattern of retinal pathologic response to laser pulses from 90 fs to 60 ps. With 3- and 60-ps pulsewidths, the result of low energy laser delivery was often damage to a single RPE cell. With 90-fs laser pulses, however, the minimal injury was usually a narrow lesion involving the RPE and photoreceptors rather than the RPE alone. As energy increased, horizontal lesion size changed minimally for the 90-fs lesions, although vertical extent often changed dramatically (Fig. 7). At longer pulsewidths, there was less vertical increase in
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Actual Energy Delivered to Cornea (pJ) 7. The vertical extent of retinal lesions from ultrashort laser pulses delivered to the macula is compared with the laser energy that created the lesion. Laser lesions produced by three pulsewidths (90 fs, 3 ps, and 60 ps) and of energies ranging from 0.31 to 11.45 /xj were examined using a light microscope. The laser lesions were graded according to the vertical extent of the lesion through retinal layers, as described in Table 1. FIGURE
D60psat 1hr • 60 ps at 24hr A 3 ps at 1 hr A 3 ps at 24hr o90fsat 1hr • 90 fs at 24hr
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Actual Energy (uj) Delivered to Cornea FIGURE 8. The horizontal extent of retinal pathologic response from ultrashort laser pulses delivered to the macula is compared with the energy that created each lesion. The maximum horizontal extent of 1- and 24-hour-old grade 2, 3, and 4 laser lesions was measured at the outer nuclear layer for each lesion, using light micrographs.
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Investigative Ophthalmology & Visual Science, October 1997, Vol. 38, No. 11
pulses, in that laser-induced breakdown thresholds have been demonstrated to be lower at 90 fs than at 3 ps or 60 ps.8 The decrease in energy transmitted beyond the site of laser-induced breakdown could also explain the notable lack of choroidal pathologic response to the ultrashort laser pulses. The current results also demonstrate the importance of the examination at 24 hours in clinical threshold studies of ultrashort laser pulses. Only 52% of grade 2A lesions were clinically visible at 1 hour (MVL), whereas 100% were clinically visible at 24 hours. Despite the improved clinical visualization of 90-fs grade 2A lesions at 24 hours, we found only a small increase in the extent of damaged tissue when comparing 1- and 24-hour laser lesions. The 90-fs pulse laser effects on tissue at 24 hours after delivery were different from those reported in studies of nanosecond or longer laser pulses or continuous wave laser. In the latter, edema at the margins of the lesion was a prominent finding at 24 hours, and the broadest area of injury was often at the level of the RPE. Perhaps the changes that allow visualization of 90-fs lesions at 24 hours result from a change in the scattering of light from the area of injury, but the tissue change is not visible in light microscopic study. In the 3- and 60-ps laser lesions, there were several lesions with a wider area of outer nuclear layer involvement at 24 hours when compared with 1-hour lesions of similar energy. In comparison, previous studies of pathologic response to ultrashort pulses of laser energy to the primate retina used 5.9 to 35 picosecond pulses of 532 or 1060 to 1064 nm laser energy.1"4 Ham et al1 and Goldman et al2'3 found small foci of RPE and outer retinal damage from laser pulses or more than 25 ps. With increased energy, however, they demonstrated broadening of the lesions at the RPE and outer retinal layers and no columnar damage from the laser pulses. The other ultrashort pulse study,5 although using visible laser wavelengths (625 nm) and 85-fs pulses, maintained a fixed retinal spot size of 50 fxm rather than using the diffraction-limited focusing of the eye. That study used rabbit eyes, which have large areas of avascular retina, rather than primate eyes. Our study, in contrast with the previous femtosecond studies of Birngruber et al5 and of Ham et al,1 highlights the importance of spot size as a factor in the creation of retinal lesions and the importance of wavelength. The latter is not just a factor in energy absorption, but also a possible factor in the location of the focus of a laser beam in ocular media. The pathologic findings of this study support those in our previous ultrashort laser pulse clinical threshold study.6 In that study, the ED50 for MVL for 90-fs and 3-ps laser pulses at 580 nm wavelength and for 60-ps laser pulses at 532-nm wavelength were calcu-
lated to be 0.43 ii] and 0.58 fi], respectively.6 In this study, retinal damage from 90-fs, 3-ps, and 60-ps laser pulses was seen with laser energies as low as 0.31 fi], 0.7 fi], and 0.8 /i], respectively. Because the goal of this study was to identify the morphologic response to the laser effect in the retina rather than to examine extensive numbers of sites of threshold energy, in the 3-ps and 60-ps eyes, there were few retinal sites that received low-energy pulses that were close to the predicted threshold levels. Several features of our study may have influenced our analysis of retinal effects from the laser pulses. One cause for some of the grade 0 lesions could be loss of energy between laser system and the retina, whether from momentary corneal aberration from drying, or from an event (e.g., laser-induced breakdown) within the vitreous cavity. Our method of 20fim step-sectioning the macula for laser lesions is another important factor that may have allowed us to miss all or portions of the very small retinal lesions. Lesions may have received a lower grade than would have been found with serial sectioning, with which the intervening 20-fj.m between-step sections would not be skipped. Despite this limitation, we did not observe a pathologic lesion at only four sites that received laser energy at or above the ED50 for MVL. Finally, some of the lesions were obtained at 24 hours after the laser delivery and thus had time for biologic amplification of the injury with inflammatory or wound-healing response. This was demonstrated best by the increased extent of outer nuclear layer damage seen 24 hours after 3- and 60-ps pulsed laser delivery. The new data presented here include the first reports of narrow columnar damage from ultrashort laser pulses, supporting the theoretical work of the ultrashort laser group at Armstrong Laboratory, Brooks Air Force Base, Texas, which proposed laser-induced breakdown as a cause for the retinal effects from 90fs pulses at 580-nm wavelength.6 The present histopathologic findings also demonstrate intraretinal, not choroidal, hemorrhages in lesions from relatively lowenergy femtosecond pulses. With this histopathologic result, we have demonstrated that the mechanism of injury is clearly different from that of longer (nanosecond) pulsewidths. We have shown that laser-induced breakdown is a probable cause for most of the retinal lesions from 580-nm, 90-fs laser pulses. These histopathologic data are essential information for the laser applications community in the determination of safety standards for use of ultrashort lasers. The rapid onset of small intraretinal hemorrhages with only a moderate increase in energy, may suggest a potential for vision-threatening injury with these lasers, even at a few times the minimum visible lesion threshold exposure. In contrast, the small RPE "footprint" of these laser pulses—even at higher en-
Pathology of Subnanosecond Pulse Laser Retinal Lesions ergy—and the relative lack of choroidal hemorrhage, raise the possibility of future clinical applications of these laser pulses in situations in which a retinal incision without underlying choroidal effect is needed. Overall, these data help enhance our understanding of the effects of such ultrashort laser pulses. Key Words femtosecond laser pulses, fluorescein angiography, hemorrhage, histopathology, laser injury, picosecond laser pulses, ultrashort laser pulses Acknowledgments The authors thank David Katz, PhD, for his assistance with statistical analysis and Ewa Worniallo for her meticulous support of the histopathology effort. References 1. Ham WT, Mueller HA, Goldman AI. Ocular hazard from picosecond pulses of Nd:YAG laser radiation. Science. 1974; 185:362-363. 2. Goldman AJ, Ham WT, Mueller HA. Mechanisms of retinal damage resulting from the exposure of rhesus monkeys to ultrashort laser pulses. Exp Eye Res. 1975;21:457-469. 3. Goldman AI, Ham WT, Mueller HA. Ocular damage thresholds and mechanisms for ultrashort pulses of both visible and infrared laser radiation in the rhesus monkey. Exp Eye Res. 1977; 24:45-56. 4. Taboada J, Gibbons WD. Retinal tissue damage induced by single ultrashort 1060 nm laser light pulses. Applied Optics 1978; 17:2871-2873. 5. Birngruber R, Puliafito CA, Gawande A, Lin WZ, Schoenlein RT, Fujimoto JG. Femtosecond laser-tissue interactions: Retinal injury studies. IEEE J Quantum Electron. 1987;QE-23:1836-1845. 6. Cain CP, Toth CA, DiCarlo CD, et al. Visible retinal lesions from ultrashort laser pulsed in the primate eye. Invest Ophthalmol Vis Sci. 1995; 36:879-888. 7. Toth CA, Cain CP, Stein CD, et al. Retinal effects of ultrashort laser pulses in the rabbit eye. Invest Ophthalmol Vis Sci. 1995; 36:1910-1917.
2213 8. Cain CP, DiCarlo CD, Rockwell BA, et al. Retinal damage and laser-induced breakdown produced by ultrashort-pulse lasers. Graefes Arch Clin Ophthalmol. 1996;234(suppl):S28-37. 9. Geary SF, Hamrick PE. Laser-induced acoustic transients in the mammalian eye. / Acoust Soc Am. 1969; 46:1037-1044. 10. Marshall J. Thermal and mechanical mechanisms in laser damage to the retina. Invest Ophthalmol Vis Sci. 1970;9(2):97-115. 11. Zysset B, Fujimoto JG, Deutsch TF. Time-resolved measurements of picosecond optical breakdown. Applied Physics B 1989;48:139-149. 12. Vogel A, Capon MR, Asiyo-Vogel MN, Birngruber R. Intraocular photodisruption with picosecond and nanosecond laser pulses: Tissue effects in cornea, lens, and retina. Invest Ophthalmol Vis Sci. 1994; 35:30323044. 13. Hammer DX, Thomas RJ, Noojin GD, Rockwell BA, Kennedy PK, Roach WP. Experimental investigation of ultrashort pulse laser-induced breakdown thresholds in aqueous media. IEEE J Quantum Electron. 1996;QE-32:670-678. 14. Kennedy PK. A first-order model for computation of laser-induced breakdown thresholds in ocular and aqueous media: Part I—theory. IEEE J Quantum Electron. 1995; QE-3L2241-2249. 15. Kennedy PK, Boppart SA, Hammer DX, Rockwell BA, Noojin GD, Roach WP. Afirst-ordermodel for computation of laser-induced breakdown thresholds in ocular and aqueous media: Part II—comparison to experiment. IEEE J Quantum Electron. 1995;QE-31:22502257. 16. Gerstman BS. Thompson CR, Jacques SL, Rogers ME. Laser-induced bubble formation in the retina. Lasers SurgMed. 1996; 18:10-21. 17. Thompson CR, Gerstman BS, Jacques SL, Rogers ME. Melanin granule model for laser-induced thermal damage in the retina. Bull of Math Biol. 1996; 58:513553. 18. Borland RG, Brennan DH, Marshall J, Viveash JP. The role of fluorescein angiography in the detection of laser-induced damage to the retina: A threshold study for Q-switched, neodymium and ruby lasers. Exp Eye Res. 1978; 27:471-93.
