Fluorescence adaptive optics scanning laser ophthalmoscope (FAOSLO)

We have constructed a new adaptive optics instrument (Gray et al., 2006) equipped with adaptive optics, providing <2 micron transverse resolution and providing video-rate (27 Hz) acquisition of 512x512 fluorescence images of the retina. The FAOSLO has a confocal pinhole that improves contrast through increased optical sectioning (115 microns). Its adaptive optics system has a 904 nm laser beacon, a Shack-Hartmann wavefront sensor (WFS), and a 144 actuator Boston Micromachines MEMS deformable mirror (DM). The scanning system (VS: vertical scanner, HS: horizontal scanner) has an adjustable 0.5 - 2.89 degree field of view. Fluorescence imaging is achieved with an AR/KR tunable laser source and a photomultiplier tube (PMT) for fluorescence light detection. Infrared reflectance imaging of the retina is achieved with a 788 nm super luminescent diode source and an avalanche photodiode (APD) for light detection. The two imaging modalities have independent focus control and can be used simultaneously. For example, it is possible to image the cone mosaic in the infrared and the RPE mosaic with autofluorescence (AF) in the visible. Figure 2 shows FAOSLO images obtained in vivo: a) Using 488 nm excitation, a 495 nm long pass dichroic and 35 nm band pass filter centered at 520 nm, the complete capillary bed surrounding the avascular zone is visible (Scale bar = 150 microns) from Gray et al (2006) b) in vivo images of ganglion cell somas, axons, and dendrites labeled with rhodamine dextran retrogradely transported from an LGN injection (Scale bar = 50 microns) from Gray et al (2008). Imaging RPE Cells In Vivo with Autofluorescence The FAOSLO has provided the first in vivo images of the RPE cell mosaic in the normal primate eye (Gray et al, 2006; Morgan et al, 2008; Morgan et al, in press), taking advantage of the resolution provided by adaptive optics and the AF of lipofuscin inside RPE cells. We use a 568 nm laser line for excitation because it maximizes the signal at the detector, given the additional spectral constraints imposed by the ANSI maximum permissible exposure and the lipofuscin excitation spectrum. The fluorescent emission is collected over a 40 nm bandwidth centered at 624 nm. In a typical frame, the signal measured by the PMT corresponds to only 0.2 photons/pixel, so over a thousand frames are typically averaged to generate a single fluorescence image. Eye motion between successive frames requires image registration before averaging, but the fluorescence images are too dim to register with cross-correlation. To overcome this problem, the FAOSLO simultaneously records a high SNR movie of the photoreceptors using reflectance imaging in the near infrared and a low SNR fluorescence movie of the RPE in the visible. Since the two movies share the same retinal motion, cross-correlation of cone frames can be used to compute the eye motion correction for the dimmer RPE frames. Figure 3 shows the cone mosaic (a) and RPE cell mosaic (b) in exactly the same retinal location imaged simultaneously at 6.4 deg eccentricity. Cone density is 22,692 cells/mm2 while RPE cell density is 4,184 cells/mm2, corresponding to 5.4 cones/RPE cell. Discrete RPE cells can be seen because the cell nucleus does not contain lipofuscin and appears dark, whereas the cytoplasm surrounding the nucleus appears bright due to lipofuscin AF. Figure 3 c and d show a Voronoi analysis (Galli-Resta et al. 1999; Baraas et al., 2007) of the cone and RPE mosaics, respectively. This analysis provides information about the number of nearest neighbors surrounding each cell (color coded in the figure) that can be used to provide quantitative measures of the density and regularity of each mosaic. Light-induced changes in RPE Autofluorescence We acquired images of the RPE and cone mosaics at a given retinal location before and after a 15 minute exposure to an intense light delivered to a smaller, square patch of retina (0.5 deg) within the imaging field of view (2 deg). During the exposure, eye motion was monitored with the FAOSLO and the retina was manually stabilized. The ratio of the mean AF intensity inside and outside the exposure location was calculated for each pre- and post-exposure image. Each post-exposure ratio was divided by the pre-exposure ratio, which normalizes for any pre-existing local differences in AF intensity inside and outside the exposure site. We call this value the AF-ratio. A value of one means the light exposure had no effect on AF intensity, a value less than 1 indicates that the light exposure caused a reduction in AF, and a value greater than 1 would indicate that the light exposure caused an increase in AF. Figure 4 shows a series of RPE images of the same retinal location before and at various times following exposure to 586 nm light at a retinal radiant exposure of 788 J/cm2, illustrating the sequence of light-induced changes in RPE AF that we observed. Immediately post-exposure, a decrease in AF was observed at the site of the exposure. The AF-ratio dropped from a value of 1 to 0.66 immediately post-exposure and then recovered partially to 0.76, 1.5 hours post-exposure. Throughout the AF reduction and recovery, the RPE mosaic remains intact. However, 11 days post-exposure, we observed a disruption in the RPE mosaic at the exposure site that persisted as long as 165 days post-exposure. This was observed for radiant exposures ≥ 247 J/cm2. What is especially striking is that both AF reduction and RPE disruption occur at light levels below the maximum permissible exposure (MPE) specified by the American National Standards Institute's standard for the safe use of lasers (ANSI Z136.1, 2007). These retinal changes are all the more alarming given that the MPE is designed to be 10 times below the damage threshold (ANSI Z136.1, 2007). Recently, we have also observed AF reduction and RPE disruption with 488 nm exposures below previously published damage thresholds (Ham et al, 1979; Lund et al, 2006). Figure 5 shows the dependence of AF reduction and RPE disruption on the energy used in the light exposure over 3 log unit range. Figure 6 shows the effects of light exposure 6 days after exposures at two different intensities, 150 µW and 55 µW, both at 568 nm light for 15 minutes. For both the 150 µW and 55 µW cases, there is a clear disruption of the RPE cell mosaic (Scale bar = 50 microns). For the 150 µW case, the reflectance image also shows damage in the photoreceptor layer, no photoreceptors are observed at the site of the exposure. However, for the 55 µW case shown here, the photoreceptor layer does not show damage. This suggests that our dual imaging method may allow us to further chart the separate effects of light exposure on these two intimately related cell mosaics. AF reduction is photochemical in nature as evidence by our observations of reciprocity for exposure power and duration and that neither adaptive optics nor laser scanning exacerbated AF reduction and RPE disruption. The mechanism responsible for AF reduction and that for RPE disruption are integrating the total power delivered to the retina. Recent evidence with 488 nm light exposures also suggests that AF reduction involves multiple lipofuscin fluorophores. The AF-ratio was also measured for a total of 6 exposures with 1.6 mW of 830 nm light for 15 minutes. During the 15 minute exposure the 568 nm light was turned off, but was used to acquire the pre- and post-exposure RPE images. No changes in AF were observed with near-infrared light. This result makes it possible to image human retina safely in the IR with AOSLO technology, since 1.6 mW provides more than adequate power for high resolution retinal imaging.