Abstract
Purpose.:
The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. The authors have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo.
Methods.:
Enhanced green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague-Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy.
Results.:
Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics. Adaptive optics correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (measured at 904 nm, 1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was approximately 1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF.
Conclusions.:
With perfect aberration compensation, the in vivo resolution in the rat eye could be approximately 2× greater than that in the human eye because of its large numerical aperture (∼0.43). Although the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using adaptive optics to investigate the rodent eye in vivo with high resolution.
Naturally occurring, transgenic, and knockout rodent models are instrumental in the study of retinal disease mechanisms and in the development of treatments for human retinal dystrophies. To date, the majority of studies using rodent disease models rely on retinal histopathology to follow disease progression and the effect of candidate therapies. Histopathology yields high-resolution images and morphometric estimates of surviving retinal cells
1 ; however, the major drawback of this approach is that it does not allow longitudinal studies in the same animals.
In vivo imaging of the rodent retina offers the possibility of visualizing disease processes and progression in individual animals and of reducing the effects of animal-to-animal variation, background lighting, and genetic background. For example, it has been shown that in various neurodegenerative diseases, substantial modifications in the morphology of axons and dendrites can take place well before cell death.
2–8 These observations highlight the need to develop high-resolution in vivo imaging techniques capable of resolving subcellular structures (such as individual axons and dendrites) in rodent eyes.
The resolution of in vivo imaging is limited by the optical quality of the rodent eye. Compared with the human eye, rodent eyes have smaller axial lengths, higher optical powers, larger average refractive errors, and larger numerical apertures (NA;
Table 1 9–20 ). Rat eyes typically have a large hyperopic refractive error. Retinoscopy measurements have shown refractive errors in the range of +5 to +15 D in albino rats, with a strong dependence on the strain (Irving EL, et al.
IOVS 2005;46:ARVO E-Abstract 4334). A dilated rodent eye also has a larger numerical aperture than a dilated human eye. Thus, in theory, one could resolve smaller retinal features with a perfect correction of the eye's aberrations in a dilated rodent eye than in a dilated human eye.
Many studies have used single-photon fluorescence microscopy, fundus photography, two-photon microscopy, confocal microscopy, or scanning laser ophthalmoscopy (SLO) to image the living rodent retina, allowing the visualization of structures such as blood vessels, capillaries, nerve fiber bundles, photoreceptors, retinal ganglion cells (RGCs), retinal pigment epithelial (RPE) cells, and microglial cells.
21–39 Fluorescently labeled RGCs have been imaged in the rodent retina in vivo over a wide field,
21–28,36,38,39 with some studies showing the apparent loss of ganglion cells in diseased retina.
23,24,26,27,38,39 One report recently used a confocal laser scanning microscope to image ganglion cells and processes in vivo in transgenic mice that expressed yellow fluorescent protein (YFP) in a small subset of RGCs.
33 Resolution in all these studies could be improved by correcting the eye's aberrations with adaptive optics so that many fine features that could previously be resolved only in excised retina could now be imaged in vivo. Adaptive optics ophthalmoscopes have enabled near diffraction-limited imaging of cellular structures (such as individual photoreceptors, ganglion cells, and RPE cells) in living human and nonhuman primates,
40–44 and the resolution of subcellular features (such as ganglion cell axons and dendrites) in living nonhuman primates.
42,43 Recently, Biss et al.
31 fluorescently imaged mouse capillaries and microglia cells using an adaptive optics biomicroscope, showing that some of the benefits of adaptive optics found in primates can be realized in rodent eyes.
We describe here a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) for the rat eye. We show that cellular and subcellular features in the rat retina, such as fine capillaries and individual fluorescently labeled ganglion cell dendrites and axons, can be imaged. The in vivo resolution of our instrument was subsequently determined via confocal images of flatmounts of the enucleated retinas.
Rats were euthanatized after multiple in vivo imaging sessions. Retinas were removed and placed as wholemounts on slides with coverslips and covered in mounting medium (Vectashield; Vector Laboratories, Burlingame, CA). Wholemount images were acquired using a confocal microscope (Zeiss LSM510; Carl Zeiss, Inc., Oberkochen, Germany). Through-focus stacks were taken at the same locations as the in vivo images using a 10× (0.30 NA) and 40× (1.2 NA) microscope objective, and maximum intensity projection images were generated for each stack. The pinhole size in the confocal microscope was adjusted to match the in vivo imaging parameters. Ex vivo images acquired with the 1.2 NA objective were taken to represent the true size of the object.
The in vivo transverse resolution of the fAOSLO was determined by calculating the in vivo line spread function (LSF). First, average cross-sectional profiles were measured across the same individual dendrites in the in vivo and ex vivo images. Both in vivo and ex vivo profiles were normalized with regard to the background level. The in vivo LSF was calculated by deconvolving the ex vivo dendrite profile from the corresponding in vivo dendrite profile. The in vivo resolution of the fAOSLO was defined as the full width at half maximum (FWHM) of the in vivo LSF.
In Vivo fAOSLO Imaging of Fluorescently Labeled Ganglion Cells, Axons, and Dendrites
It is challenging to achieve diffraction-limited imaging for the rat eye because of its small size, high optical power, and large refractive error. Given the high optical power of the rodent eye, a large defocus value is needed for sectioning the retina. The total optical power for the human eye is ∼60 D and for the rat eye it is ∼300 D, yet the rodent retina is comparable in average thickness to that of human (typically 170 μm vs. 250 μm, respectively).
16,51 Whereas only ∼0.66 diopter is required to shift the focal plane in the human from the anterior to the posterior retina, an ∼11.5 diopter change in optical power is required in the rat. Considering the different pupil sizes inherent in different species, the amount of Zernike defocus (in micrometers) needed to section the whole retina is less than 1 μm for the human eye and 5 μm for the rat eye. The large refractive error of the rodent eye poses another challenge for in vivo cellular imaging. We used trial lenses and contact lenses to compensate for spherocylindrical errors, but using a larger stroke deformable mirror for refractive correction would provide a faster solution. Another issue to be considered when imaging with multiple wavelengths is the increased amount of chromatic aberration in the rat eye compared with that of the human eye; the chromatic aberration between 475 nm and 650 nm in the rat eye is reported to be 5.8 D.
52 Last, but not least, wavefront sensing using reflective infrared light (which returns primarily from the photoreceptors) can cause additional uncertainties for effective adaptive optics correction when imaging retinal layers away from the photoreceptor layer. For example, in the rat, the ∼11.5 D separation between the ganglion cell and photoreceptor layers could require modifications of other aberrations, such as spherical aberration, for best correction. Measurements of any variation in the wave aberration with different focal planes throughout the retina have yet to be made.
Careful animal handling is also important to achieve successful images. For fAOSLO imaging of the rat retina, we have found that isoflurane provides a better, more stable plane of anesthesia than ketamine and xylazine. The morbidity rates after anesthesia are lower, fewer eye movements are observed in the high-magnification images, and the duration of the anesthesia can be longer (more than 3 hours vs. 30 minutes) when using isoflurane. A heating pad helps to keep the animal warm and prevent transient, temperature-related cataract formation for imaging sessions longer than 1 hour. Using contact lenses to keep the eye hydrated eliminates the need to manually blink the eye or constantly hydrate the eye with drops and avoids distortions in the wavefront caused by degradations in the tear film or nonuniformities in the distribution of the hydrating drops. A contact lens also helps to reduce the lower order aberrations of the eye. We found that the spherical refractive power and corneal curvature of our Sprague-Dawley rat eyes changed dramatically with age. Their spherical refractive power decreased from 10 to 20 D to approximately 5 D as they matured. Therefore, it is important to carefully select and match the base curve, diameter, and power of the contact lens to the eye being imaged. Poor-fitting contact lenses may degrade the spots in the wavefront sensor images, resulting in poor adaptive optics corrections and image quality.
The ability to resolve subcellular features such as individual ganglion cell axons and dendrites in rodents in vivo provides us the opportunity to detect early changes in the retina on a microscopic level in disease models and to investigate fundamental questions about retinal disease pathophysiology. For example, adaptive optics imaging may help to determine whether remodeling occurs during glaucoma, whether axonal degeneration precedes dendrite degeneration, what the pattern of cell death is, and whether vascular degeneration precedes RGC death. Although we have concentrated on ganglion cell imaging in this study, this level of resolution indicates that it may be possible to use the fAOSLO to image individual Müller glial cells, RPE cells, and rod and cone photoreceptors that are fluorescently labeled in transgenic rodents
54,55 or labeled using cell-specific adeno-associated virus (AAV) and lentiviral vectors.
46,56–58
The mouse is the most widely used animal model for human retinal degeneration. A similar imaging method might also be applied to image multiple cells types in normal and transgenic mice. With the current powerful molecular techniques that exist for engineering the mouse, fAOSLO imaging of mouse models has potential for investigating disease mechanisms and evaluating the efficacy of drug therapies. Finally, it may also be possible to combine fAOSLO imaging with optical methods used to image the functional activity of cells, allowing optical recordings of the activity of single retinal cells or ensembles of retinal cells in vivo.
Supported by National Institutes of Health Grant EY014375, Foundation Fighting Blindness, Texas Advanced Research Program Grant G096152, and the National Science Foundation Science and Technology Center for Adaptive Optics (managed by the University of California at Santa Cruz, cooperative agreement no. AST-9876783). Alfredo Dubra holds a Career Award at the Scientific Interface from the Burroughs Welcome Fund.
Disclosure:
Y. Geng, None;
K.P. Greenberg, None;
R. Wolfe, None;
D.C. Gray, Optos (E, P);
J.J. Hunter, None;
A. Dubra, None;
J.G. Flannery, None;
D.R. Williams, Optos (C, P);
J. Porter, None
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.
The authors thank Benjamin Masella, William H. Merigan, Andrew Winterborn, Wendy Bates, Wanli Chi, Mina Chung, Richard T. Libby, Stephen Burns, Joseph Stamm, Terry Schaefer, Lu Yin, and Lana Nagy for assistance.