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1From the Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; the 2Medical Laser Center Lübeck, Lübeck, Germany; the 3University Eye Hospital Regensburg, Regensburg, Germany; the 5Department of Biomedical Engineering, Tufts University, Medford, Massachusetts; and the 6University of Applied Sciences Hamburg, Hamburg, Germany.
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Abstract |
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METHODS. Acousto-optic deflectors repeatedly scanned the focus of a continuous wave (cw)-laser across the retina of Dutch belted rabbits, producing microsecond irradiation at each RPE cell. Two irradiation patterns forming separated lines (SEP) or interlaced lines (INT), different dwell times (2.5–75 µs), and repetition numbers (10 and 100 scans with 100-Hz repetition rate) were tested. Thresholds were evaluated by fundus imaging and angiography. Histology was performed for selected parameters.
RESULTS. Selective RPE cell damage was obtained with moderate laser power. The angiographic threshold power decreased with pulse duration, number of exposures, and applying the INT pattern. Ophthalmoscopic thresholds, indicating onset of thermal coagulation, were higher than twice the angiographic threshold for most tested parameters. Histology confirmed selective RPE cell damage for SEP irradiation with 7.5 and 15 µs; slower scan speeds or closed lines caused photoreceptor damage.
CONCLUSIONS. A cw-laser scanner can be set up as a highly compact and versatile device. Selective RPE damage is feasible with dwell times up to 15 µs. Greatest selectivity is achieved with short exposure times and separated scan lines. Interlaced lines and long exposure times facilitate heat conduction into photoreceptors. A scanner is an attractive alternative for pulsed selective targeting, because both selective targeting and thermal photocoagulation can be realized.
Many retinal diseases, such as retinal detachment and diabetic retinopathy, are treated successfully by conventional laser irradiation. However, the benefit for the patient has to be considered carefully when macular irradiation is performed, because of the resultant laser scotoma that can lead to severe loss of visual acuity. Furthermore, the thermal destruction of photoreceptors does not contribute to the therapeutic effect in macular diseases that are caused by dysfunction of the RPE cells, such as drusen in early age-related macular degeneration (AMD), diabetic macular edema, and central serous retinopathy. Selectively destroying RPE cells while preserving the photoreceptors may be the appropriate treatment for these diseases.5 The selective effect on RPE cells, that absorb approximately 50% of the incident green light,6 due to their high melanosome content, was first demonstrated using 5-µs argon laser pulses at 514 nm, with a repetition rate of 500 Hz in experimental rabbit eyes.5 Applying a train of brief, microsecond pulses that are on the order of the thermal relaxation time of the absorbing melanosomes (
1 µs) leads to high peak temperature or, depending on the exposure duration, to the formation of microbubbles7 8 9 10 11 12 13 around the melanosomes. Subsequently, the RPE cells are destroyed, whereas the background temperature increase remains at sublethal levels.14 Thus, collateral thermal damage is avoided, and photoreceptors remain intact as shown in various studies. Histology at different time points after irradiation demonstrated restoration of the blood–retinal barrier by proliferating and migrating RPE cells within 14 days after treatment.5 14 15 Clinical trials in which an Nd:YLF laser system was used with a pulse duration of 1.7 µs (up to 100 pulses, 100 and 500 Hz) demonstrated the clinical potential of this technique, confirming by microperimetry that central vision is not adversely affected after selective targeting (Roider J et al. IOVS 1998;39:ARVO Abstract 104).16
With these promising initial results, it is imperative that a compact, reliable, clinically viable laser system be developed for selective RPE treatment so that the therapeutic benefits of this approach can be tested and established in a larger patient population. Unfortunately, such a laser system has not been available so far; the pulse laser system used by Roider et al. in the earlier studies are bulky, cumbersome benchtop devices.15 16 17 Attempts to perform this treatment with a simple pulse diode laser source with 810 nm wavelength have been undertaken.18 19 20 However, the attempts were unsuccessful in creating selective RPE lesions reproducibly, most likely because the wavelength and pulse duration of the diode laser were both suboptimal,21 so that a temperature gradient between RPE and the adjacent tissue layers was not sufficiently established.22
We extensively tested the hypothesis that selective RPE damage can be created with a cw-laser in vivo, if the focused laser spot is rapidly scanned across the fundus so that each RPE cell just "sees" a microsecond pulse. A scanner, besides being a more compact alternative, has the ability to generate a variety of treatment parameters. By adjustment of scan speed (i.e., exposure time), repetition rate, and the shape of the scan pattern independently, treatment parameters can be optimized, and hypothetically the extent of selectivity can be controlled. The feasibility of scanning RPE cell targeting was demonstrated in preliminary ex vivo experiments employing a multimode-fiber–coupled Argon-ion laser scanner on sheets of porcine RPE.23 The feasibility of scanning selective targeting was further demonstrated, using a Gaussian scanning beam, on sheets of bovine RPE and for selected scanning parameters in vivo.24 25 In this study, we scrutinized the ability of a slit lamp–adapted laser scanner to create selective RPE lesions in the fundus of experimental rabbit eyes for a broad range of irradiation parameters. We evaluated the damage thresholds for various scan speeds, number of scans, and scan patterns by means of ophthalmoscopic and angiographic visibility. Furthermore considering the extent of selectivity by histologic examination, we identified several scanning parameters that are suitable for selective RPE targeting.
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Material and Methods |
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The scanner device was mounted on top of an ophthalmic slit lamp (SL-130; Carl Zeiss Meditec GmbH, Oberkochen, Germany). The focal plane of the scanner was carefully adjusted to coincide with the object plane of the slit lamp. Thus, the slit lamp served as the targeting device for both aiming within the fundus and focusing the treatment beam.
Animals
In total, 20 Dutch belted rabbits were used for the experiments. Rabbits were chosen because the density and location of light-absorbing pigments in the fundus are rather uniform and similar to that of the human eye.6 The rabbits were anesthetized with ketamine hydrochloride (35 mg/kg of body weight) and xylazine hydrochloride (5 mg/kg of body weight) and placed into a special holder system that allowed us to tilt and rotate the animal around its pupil in relation to the slit lamp. The experimental animals in this study were treated in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Experimental Design
The laser beam (Gaussian profile) was focused through a Goldmann contact lens onto the central fundus’ RPE in the rabbit eye by moving the retinal surface into the object plane of the slit lamp. The contact lens was locked to the animal holder to prevent unfavorable movements. The 1/e2-spot diameter in air was 27.5 µm. Calculations showed that the use of a planoconcave contact lens in cycloplegic emmetropic rabbit eyes leads to a magnification factor of 0.66.26 Thus, the spot size in the retina was calculated to be 18.1 µm.
In this study, irradiation was performed with two different scan patterns: The first scan pattern consisted of six separated lines (referred to as SEP) that were spaced approximately 60 µm apart (Fig. 1A) . The second pattern consisted of 21 interlaced lines without spacing (referred to as INT; Fig. 1B ). Both patterns illuminated a square field of approximately 300 µm length in the retina.
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ED50 Determination
Lesions were placed in the posterior retina by adjusting the slit lamp to allow the laser beam to enter the eye centrally. Visible suprathreshold marker lesions were used in all eyes to orient the particular nonvisible, selective laser scans. Marker lesions were placed by purposely causing coagulation of the neuroretina, using 100 scans at a 30-µs dwell time with 100-mW power. The test scans with various power levels were placed in the grid formed by these marker lesions. Approximately 30 test lesions were made in each eye (low 23, high 39), to determine the ED50. Ophthalmoscopic visibility, manifested by the whitening of the retina, suggests thermal denaturation. It also marks the ophthalmoscopic endpoint for this study. Ophthalmoscopic visibility was judged as positive or negative within moments after irradiation (Fig. 2B) . Thirty minutes after treatment, fluorescein angiography with injection of 10% fluorescein sodium into the ear vein was performed with fundus camera (Carl Zeiss Meditec GmbH, Oberkochen, Germany). If the RPE is damaged, the distal blood–ocular barrier, formed by RPE and Bruch’s membrane, will be compromised, and fluorescein can pool from the choriocapillaris into the subretinal space (Figs. 2A 2C) . Thus, fluorescein angiography was used to detect laser-mediated damage to the blood–ocular barrier, which defines the angiographic endpoint.
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Histology
Selected parameters (10 and 100 repetitions of 7.5 and 15 µs SEP, 10 repetitions of 30 µs SEP, and 10 repetitions of 7.5 µs INT and 100 repetitions of 15 µs INT) were tested in two additional eyes after thresholds had been determined. Irradiation in each eye was systematically performed with four different doses based on previously measured angiographic thresholds (0.5x ED50, 1x ED50, 1.5x ED50, and 2x ED50). One parameter per eye was covered with 16 test scans (4 per dose), placed adjacent to the marker lesions. Resultant lesions were examined by slit lamp (ophthalmoscopic visibility), and fluorescein angiography (angiographic visibility). In addition, lesions were sectioned and the histology examined by light microscopy.
Eyes for histologic examination were enucleated in vivo in rabbits under deep anesthesia 1 hour after treatment. Immediately after enucleation, the globes were incised anterior to the equator and immersed in Karnovksy’s solution. Twenty-four hours later, the posterior eyecup was cut from the anterior segment and the globe immersed in fresh fixative overnight. The areas of interest (marker lesions and lased lesions of the retina) were dissected and placed in fresh fixative for another 24 hours. The lesions were then postfixed in osmium tetroxide, dehydrated in a series of alcohols, and embedded in resin (Epon 812). Thick (1 µm) serial sections were cut through the lesion with a microtome (Ultracut E; Reichert, Vienna, Austria) and stained with methylene blue and toluidine blue.
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Threshold Determination
The thresholds for tested parameters were evaluated based on the angiographic and ophthalmoscopic visibility of the lesions as shown in Figure 2 (10 repetitions of 30 µs, SEP). Figure 2B shows the fundus as viewed through the slit lamp 2 hours after irradiation. Six prominent grayish white marker lesions (labeled M) formed a grid. Test lesions were placed in the area outlined by the markers, with laser power increasing from left to right. Lesions are not visible in the left half of Figure 2B (0 for ophthalmoscopic ED50 calculation), but become increasingly ophthalmoscopically visible with increasing laser power toward the far right (1 for ophthalmoscopic ED50 calculation). Fluorescein angiography (Figs. 2A 2C) revealed where RPE cells were successfully damaged by the irradiation. Some lesions in the lower left of Figure 2A did not become visible (0 for angiographic ED50 calculation). In those, the applied dose was too low to cause any damage. In the same figure, other previously invisible test lesions appear (1 for angiographic ED50 calculation); those are selective lesions. In Figure 2C , lesions that were ophthalmoscopically visible in the slit lamp examination are also visible in fluorescein angiography. In the early phase of fluorescein angiography, lesions consistently appeared as separated hyperfluorescent lines that resembled the applied scan pattern. It is interesting to note that some lesions in Figure 2B —those lesions that are faintly visible between the reflections (r) and the top right marker—were not ophthalmoscopically visible immediately after irradiations. Those lesions were produced with a dose more than twice ED50 and began to appear only approximately 30 minutes after irradiation. The developing edema caused by irradiation leads to more significant visibility in this image (2 hours after irradiation).
Figure 3 shows the probability function for detecting positive lesions by fluorescein angiography and by ophthalmoscopy, using various scan speeds (indicated by the equivalent exposure time at the top of each panel), for 10 repetitions of the SEP scan pattern. For each exposure time, the damage probability is plotted as a function of increasing laser power. The top three panels (2.5-, 7.5-, and 15-µs exposure durations) show only the fluorescein angiographic results, because none of the lesions was ophthalmoscopically visible, even with maximum available laser power of 185 mW delivered to the eye. The bottom three panels (30, 45, and 75 µs) show both the angiographic and ophthalmoscopic results. The gap between the 15% probability for ophthalmoscopically and 85% probability for angiographically visible damage is the therapeutic window. Its width decreases with increasing exposure time.
Dependence of RPE Damage Threshold on Irradiation Parameters
The probability curves of Figure 3 were calculated by the probit method. Thereby, the ED50 radiant exposures for angiographically and for ophthalmoscopically visible damage were determined. Figure 4 shows the ED50 radiant exposure plotted as a function of exposure time for 10 repetitive applications of the SEP pattern. With increasing dwell time (slower scan speed), the angiographic threshold power decreased while the threshold radiant exposure increased (Fig. 3 , Table 1 ). The angiographic ED50 ranged from 191 mJ/cm2 (157 mW) with 2.5 µs to 1279 mJ/cm2 (35 mW) with 75-µs exposure durations. The ophthalmoscopic threshold was not reached for 2.5-, 7.5-, and 15-µs dwell times with the maximum available laser power of 185 mW. For 30-, 45- and 75-µs dwell times, the ophthalmoscopic thresholds were 2046, 2302, 2557 mJ/cm2 (140, 105, and 70 mW), respectively. The ophthalmoscopic threshold was twice the angiographic threshold for 75-µs dwell time. This factor increased for shorter dwell times (up to 3.5- with 30-µs dwell time), indicating that for 10 scans of separated scan lines, RPE cells can be safely targeted without visible neurosensory retina coagulation. The results for 10 repetitions of the SEP scan pattern are summarized in Table 1 .
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Changing the scan pattern from separated to interlaced lines also caused a slight decrease in threshold, as shown in Table 3 . No ophthalmoscopically visible damage was observed with 10 repetitive applications of the INT scan pattern for 7.5-µs dwell time. With 100 repetitions of the INT pattern with 15-µs exposures, however, ophthalmoscopically visible lesions were observed at a factor of 1.5 above angiographic ED50. As described earlier, the same number of repetitions using the SEP pattern with 15-µs dwell time did not result in visible coagulation for radiant exposure of up to 3.1 times the angiographic ED50.
Probit analysis further yields the slope of the dose–response curve, defined as ED85/ED50 (Tables 1 2 and 3) . The slopes of individual probability functions were, with one exception, always smaller than 1.1 for both angiographically and ophthalmoscopically visible damage, indicating a steep, step-like response (see also Fig. 3 ). The steep slope of individual dose–response curves suggests stable experimental conditions (e.g., focusing) for all test lesions in the same eye. The slope of pooled probability curves, considering all lesions of the same parameter, was approximately 1.1, indicating little variation between the duplicate measurements.28
Assessment of Cell Damage
Besides the evaluation of the outcome of irradiation by slit lamp examination and fluorescein angiography, the extent of selectivity of selected parameters was also assessed by histology. Results for 7.5- and 15-µs exposures confirmed that RPE cells were selectively damaged when these parameters were used (Figs. 5 and 6) , consistent with the absence of visible coagulation for 10 and 100 repetitive applications of the SEP pattern. The damaged RPE cells were flat, with some of the debris lying on Bruch’s membrane, and the melanin granules lost their apical orientation. There was no observable difference in the damage from irradiation with 10 or 100 repetitions at radiant exposure of one or two times angiographic threshold. Likewise, no damage was observed throughout the neural retina. The outer segments of the photoreceptors that are in contact with the RPE remained unaffected, showing preserved photoreceptor structure and orientation. In all sections, Bruch’s membrane and choriocapillaris appeared intact.
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Similar results were obtained in histology after irradiation with 100 repetitions of the SEP pattern with 15-µs dwell times (Fig. 6C) . The cell damage was confined to the RPE cell layer and photoreceptors were preserved, even after irradiation with radiant exposure twice the ED50. However, damage to individual RPE cells was rarely found. Neighboring RPE cells were more frequently affected so that the origin of the initial laser impact often could not be determined.
Histology of irradiation at radiant exposure three times angiographic ED50 of 30-µs dwell time (10 repetitions, SEP) showed photoreceptors that appeared significantly altered in the area of laser impact (Fig. 7D) , although the therapeutic window was measured to be 3.3 at this laser setting. The damage extended through the outer segments of the photoreceptors into the nuclear layer of the photoreceptors but was not visible in slit lamp examination (Fig. 7A) ; however, edema was observed in these lesions after 30 minutes (Fig. 7C) . Reducing the radiant exposure to twice the ED50 reduced the extent of the photoreceptor disturbance. Here, damage was mostly confined to the RPE cell layer. However, surviving RPE cells became rare, and isolated pockets of photoreceptor damage were still found (Fig. 7E) . Many coagulated red blood cells were found in the choroid; however, bleeding through Bruch’s membrane was not observed.
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Discussion |
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We are currently developing a new technique for selectively targeting the RPE by scanning a focused cw-laser beam24 rapidly across the retina. The scanning paradigm creates short exposure durations without the need for a complex pulse laser source. By applying separated (SEP) scan lines on ex vivo bovine RPE flatmounts in a prior study, we obtained alternating lines of damaged and intact RPE cells that replicate the applied scan pattern, indicating damage confinement to the irradiated cells.24 We also showed, by means of fundus photography and fluorescein angiography in rabbits, that selective targeting is feasible in vivo using 7.5- and 15-µs exposure durations. However, fundus photography and fluorescein angiography alone were not sufficient to assess whether damage was strictly confined to the RPE layer. Recently, we presented preliminary histologic finding of selective RPE damage in vivo after exposure to a single scanning parameter (5 µs, SEP scan pattern). Selectively damaged RPE cells were seen next to intact photoreceptors for this treatment parameter.25
We have extended these studies to cover a much broader range of exposure parameters, extending from exposure times on the order of the thermal relaxation time of the absorbing structures (2.5 µs) to well beyond that regimen (up to 75 µs). These studies are important because in designing a scanning system for clinical applications, one has to take into account not only the requirement to achieve selectivity with a sufficiently large safety margin (i.e., therapeutic window), but also the need to have a reasonably compact and inexpensive instrument. As shown in Figure 3 , the therapeutic window clearly increases with decreasing exposure duration. However, the increase in therapeutic window comes at the expense of increasing laser power, with accompanying increase in system cost and bulkiness. As the advances in laser technology will inevitably drive down both size and cost, the comprehensive results presented herein will provide useful guidelines for future system design considerations with regard to available laser source and the achievable selectivity. Moreover, from a clinical perspective, it may be beneficial to have the ability to control the degree of selectivity during treatment with a single instrument. By adjusting the scan speed and/or scan pattern, it is possible to produce either selective RPE damage (Figs. 5C 6C) or nonselective (thermal) lesions that extend to the photoreceptors (Figs. 7D 8A) . This flexibility is a key feature of the scanning approach. For the highest degree of selectivity, damage to individual RPE cells can be achieved. Given this microscopic precision of the scanner, future laser treatment of central retinal diseases may be possible even in the fovea if a suitable feedback mechanism, such as the detection of microscopic intracellular bubbles, is used. In contrast, thermal coagulation can be realized with the same device by adjusting scanning parameters to facilitate heat diffusion into the neural retinal layers, for instance by slowing down the scan speed or by scanning without spacing between adjacent scan lines (the INT scan pattern). Thus, a laser scanner is a compact and reliable device for RPE targeting that can allow the laser treatment to be tailored to individual patients’ retinal disease.
In our scanning device, the radiation of the cw-laser is delivered to a slit lamp mounted scanning unit with an optical fiber. We scan a laser spot about the size of one RPE cell across the retinal surface with a speed such that every targeted cell will be irradiated with microsecond exposure. Selective cell damage was demonstrated with moderate laser power (on the order of 100 mW) by the appearance of lesions in fluorescein angiography with concurrent absence of ophthalmoscopically visible coagulation. For most of the parameters tested, photoreceptor coagulation was not achieved even with irradiation far above angiographic threshold. Histology of 7.5- to 15-µs exposure times with the SEP pattern confirmed microscopically the absence of damage to the photoreceptor layer; thus, selectivity was substantiated for these laser settings. Histology for 2.5-µs exposure was not performed; however, it is reasonable to expect the photoreceptors to be preserved by using a shorter exposure time (i.e., better thermal confinement to the RPE) with lower radiant exposure (Table 1) . Subtle damage to the photoreceptors may not be visible in light microscopy (Fig. 7) . However, it is known that restoration of the photoreceptor outer segments is possible as long as the cell nuclei remain intact. Roider et al.18 (Roider J et al. IOVS 1998;39:ARVO Abstract 104) have shown in trials in which microperimetry was used in patients that the full field of view recovered within 1 week to 3 months after initial detection of a small number of blind spots after selective RPE treatment.
Dependence of Cell Damage Thresholds on Irradiation Parameters
We observed in our experiments using 10 repetitive exposures with the SEP pattern that the angiographic ED50 radiant exposure increased with exposure time, whereas the angiographic ED50 power decreased (Table 1) . If laser energy is applied within the thermal relaxation time of the melanosomes (1 µs), heat diffusion is minimized, and the temperature is confined to the absorber. With increasing exposure times, heat diffusion becomes significant, and, thus, higher radiant exposure was necessary to replace the amount of heat energy that dissipated during the exposure.
The slight reduction of the RPE damage thresholds due to application of 100 repetitions of the SEP pattern (Table 2) can be explained by the fact that the 100-Hz repetition frequency may be too high to allow the tissue in the irradiated area to cool completely to body temperature. Thus, heat gradually accumulates over a large number of repetitions, leading to a lower measured damage threshold.
Irradiation with the INT pattern was markedly different from that with the SEP pattern. Lower angiographic thresholds were measured for both 7.5-µs (10 repetitions) and 15-µs exposures (100 repetitions; Table 3 ). At the 15-µs dwell time ophthalmoscopically visible coagulation of the neural retina was also observed. Applying the INT pattern (21 lines without spacing) versus the SEP pattern (six spaced lines) introduces two effects. First, the total energy applied to the tissue is higher as the duty cycle increases (for 15-µs dwell time, the duty cycle approached 55% in INT versus 15% in 15-µs SEP). Second, because adjacent lines in the INT pattern were produced with a time delay of approximately 3 ms, thermal energy that diffused away from the flying spot while the scan was in progress heated up adjacent target areas, yielding a lower threshold.
Dependence of Therapeutic Window on Irradiation Parameters
For selective laser treatment, a safety margin between the angiographic and the ophthalmoscopic threshold is crucial to the prevention of unintentional photoreceptor cell damage. To ensure that most targeted RPE cells are damaged, irradiation above angiographic ED50 is desirable, yet damage to photoreceptors is to be avoided. For this purpose, we define the safety margin as the ratio of ophthalmoscopic ED15 over angiographic ED85. This safety margin is commonly referred to as the therapeutic window (TW). Because of the intra- and interindividual variation in pigmentation, which can differ by a factor of two in humans,6 a therapeutic window as large as possible is desired in a clinical setting.
Varying only the scan speed while keeping other parameters constant, we were able to show that the therapeutic window increases for irradiation with decreasing exposure time for the SEP pattern from a factor of 1.6 (75-µs dwell time) to 3.3 (30-µs dwell time) (Figs. 3 4 ; Table 1 ). We expect the therapeutic window to be even wider at shorter dwell times because less energy is applied to the retina and there is less heat diffusion away from the RPE. The therapeutic window was not determined for scan speeds between 2.5 and 15 µs because the available laser power was not sufficient to create ophthalmoscopically visible lesions. According to Figure 3 , none of the angiographic 85% probability points overlapped with the ophthalmoscopic 15% points, even at exposure times that were significantly longer than the RPE thermal relaxation time. Angiographic ED100 was reached at approximately 30% above ED50 (Fig. 3) . As confirmed by histology, there was no damage to photoreceptors observed with 10 and 100 repetitions of the SEP pattern, with 7.5-µs exposures (on the order of the thermal relaxation time), or even with a 15-µs dwell time (longer than the thermal relaxation time) at doses of up to twice ED50 (Figs. 5 6) .
It is interesting to note that 15-µs INT exposures had a much narrower therapeutic window (1.5) than did SEP exposures (Table 3) . Heat diffusion geometry introduced by irradiation with separated scan lines can be modeled as separated cylinders, whereas heat distribution with INT exposure approximates a disc. The latter has greater heat conduction into the photoreceptors.
As expected, thermal coagulation that is seen by histology to be confined to the photoreceptors (i.e., not extending through the entire thickness of the retina) may not be visible in slit lamp examination (Fig. 7 , 30-µs SEP). Therefore, the therapeutic window for 30-µs SEP exposure should be smaller than 3.3 indicated by the ophthalmoscopic and angiographic measurements. Furthermore, ophthalmoscopic lesions that are initially invisible can appear with time after irradiation, as has already been observed in the pulsed laser approach.17 This was more likely when energies higher than angiographic threshold were applied (Figs. 2 7 8) . Those lesions were not visible during the first 10 minutes after irradiation but became visible after 30 minutes. This late-onset visibility is attributed to damaged RPE that weakens the blood–retina barrier and leads to extracellular fluid leaking in those particular areas (i.e., edema). The change of the scattering properties of the neurosensory retina, due to the edema in the subretinal space, eventually leads to ophthalmoscopic visibility. In fact, leakage though the compromised blood–ocular barrier is the basis for fluorescein angiography, currently the standard method of detecting laser-mediated RPE cell damage. Just as lesions created with the INT pattern appear brighter in fluorescein angiography than those created with the SEP pattern, we observed the most significant edema in lesions that were created with the INT pattern above ED50 (Fig. 8) . The total area of compromised barrier was larger in INT than in SEP, yielding a higher fluid diffusion rate into the subretinal space. Consequently, edema developed more quickly in INT than in SEP lesions. It is therefore imperative to judge ophthalmoscopic visibility within moments after irradiation to avoid misjudging edema as coagulation, yielding thresholds that differ in dependence from the time of determination.
In a clinical setting, dosimetry in each patient becomes crucial. To determine a therapeutically useful dose and the therapeutic window in individual patients based on slit lamp examination and additional angiography 1 hour after treatment bears the risk of overtreatment or unsuccessful treatment because the operating physician will seek to avoid collateral damage. Successful yet safe treatment could be achieved reproducibly if the treatment outcome could be monitored during the laser energy application. Considering the cell damage mechanism, selective RPE destruction was originally thought to be achieved by thermal necrosis of the cell.5 14 However, recent investigations show that microbubble formation occurs around melanosomes in suspension with pulse durations within the nanosecond and microsecond regimen.7 8 9 Calculations show that rapid vaporization is initiated when the melanosomes’ surface temperature reaches approximately 150°C.9 Brinkmann et al.9 argued that cell death originates from microbubble formation, rather than thermal denaturation, after applying a train of microsecond pulses. We observed in a related project that the thresholds for cell damage and bubble formation correlate well at pulse durations of up to 10 µs.10 Bubble formation leads to a transient increase in backscattered light from the RPE that can be monitored using optical means to provide feedback as to whether RPE cells have been damaged 10 11 12 . Likewise, the onset of cavitation can be detected by acoustical means.13 Thus, detecting bubble formation during the exposure can serve as immediate feedback, indicating that the angiographic endpoint (i.e., damage of RPE cells) has been reached. The result of the feedback can be used by the physician to adjust dosimetry manually. It is likewise conceivable that a device can be developed that automatically interrupts the treatment once RPE cell damage has been measured.
Outlook
Our ED50 threshold for scanning with 2.5-µs dwell time is 191 mJ/cm2, similar to the threshold of 189 and 143 mJ/cm2 that Framme et al.17 measured in rabbits by using a spot diameter of approximately 100 µm with 5- and 1.7-µs pulses, respectively. Whether the scanning approach requires intrinsically higher radiant exposure compared with the pulse technique23 is a subject for further investigation. Comparison of threshold values aside, the scanner bears several other advantages over pulsed systems. The laser scanner can be set up as a compact device that is entirely adapted to a targeting device, such as a slit lamp. In this study, we coupled a commercially available cw-laser via single mode fiber to the scanner. We are currently setting up a new scanning device that incorporates a compact cw-laser of 1.2-W output power into the slit lamp–adapted device, eliminating the need for optimizing the fiber coupling efficiency and increasing the available power at the cornea from 185 mW to approximately 700 mW. Future experiments, therefore, will be designed to investigate the extent of selectivity and the width of the therapeutic window for shorter exposure times. Most important, a scanner is able to create a variety of irradiation parameters, as opposed to the pulsed approach that can cover a small range of pulse durations with a fixed pattern. Furthermore, by scanning a small laser spot to produce spaced lines, a heat-diffusion geometry is introduced that allows selective RPE damage, even at exposure times that are significantly longer than the thermal relaxation time of the absorbing structures (Tables 1 2) . In contrast, the large spot diameter of the pulsed approach (approximately 100 µm in diameter) may lead to an unnecessary temperature increase in the center of the lesion and to heat diffusion into the photoreceptors; this possible outcome is indicated by the smaller therapeutic window of 1.9 that Framme et al.17 determined in rabbits after 5-µs pulsed exposure. In fact, by changing the heat diffusion geometry introduced by different scan patterns, a scanner can control the extent of the selectivity. Selectivity is best achieved by irradiation with exposure times on the order of the thermal relaxation time and using separated lines (SEP). Thermal photocoagulation can also be realized using the same device, by slowing down the scan speed (i.e., increasing the dwell time) and using a scan pattern that facilitates heat conduction into the photoreceptors (INT). Thus, a scanner can serve both as a source for selective targeting and as a thermal photocoagulator.
Recovery of selective RPE lesion produced by the pulsed approach typically takes approximately 14 days.5 By creating different RPE damage patterns and lesion sizes, the scanner will enable us to investigate whether the recovery time of the RPE defect can be accelerated. However, it remains to be shown whether the smaller area of recovering RPE after scanning SEP treatment is actually capable of removing existing edema or drusen. Future work will incorporate in vivo follow-up examinations regarding the restoration of the RPE barrier in the animal model.
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Conclusions |
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We have shown that the scanner can be useful in both selectively targeting the RPE and thermal photocoagulation. Adjustment of the dwell time and the applied scan pattern allows control if the heat diffusion geometry and extent of selectivity. Irradiating generously spaced locations with dwell times on the order of the thermal relaxation time is ideal for selective targeting; precision of individual RPE cell damage can be achieved. Slowing down the speed of the scanner (i.e., increasing the exposure time) and selecting a scan pattern that facilitates heat conduction into the photoreceptors is beneficial for thermal coagulation. Thus, thermal coagulation can be performed with the same device simply by adjusting the scanning parameters. Future experimental setups will incorporate a compact cw-laser on the targeting device as well as an online feedback system that monitors cell death during irradiation. Pending further experiments and eventual clinical trials, the scanning method may allow optimization of treatment parameters to individual patient’s needs.
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Footnotes |
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Supported by National Eye Institute EY12106 and by Lumenis, Inc.
Submitted for publication July 12, 2006; revised November 30, 2006; accepted January 19, 2007.
Disclosure: C. Framme, None; C. Alt, Lumenis (F); S. Schnell, None; M. Sherwood, None; R. Brinkmann, None; C.P. Lin, Lumenis (R)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Charles P. Lin, Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, CPZN-8240, 185 Cambridge Street, Boston, MA, 02114; lin{at}helix.mgh.harvard.edu.
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