2.1. The fluorescence AOSLO customized for the mouse eye
A custom fluorescence AOSLO was built, combining wavefront sensing methodology previously developed for the mouse eye and design principles of broadband AOSLOs for the human eye [15
]. A schematic layout of the system is shown in
. Note that the actual optical system was folded in 3D, and was flattened in for illustration.
Fig. 1 Schematic of the mouse eye fluorescence AOSLO. LD: fiber coupled laser diode. SLD: fiber coupled Super Luminescent Diode. PMT: photomultiplier tube. SHWS: Shack-Hartmann wavefront sensor. F: band pass filter. 90/10: 90/10 beam splitter. HS: horizontal (more ...)
Three light sources were used for wavefront sensing and imaging after being coupled to single mode optical fibers and mounted on translation stages allowing for independent focusing. All source focuses could be adjusted by changing the distance between the source fibers and their collimating lenses. An 843 nm laser diode (LD) from Qphotonics (Ann Arbor, Michigan, USA) was used as the wavefront sensing source. A 789 nm superluminescent diode (SLD) with 11.5 nm bandwidth (InPhenix, Livermore, California, USA) was used as the reflectance imaging light source. An air-cooled Argon laser (CVI Melles Griot, Albuquerque, New Mexico, USA) provided multiple spectral lines between 457 and 514 nm for fluorescence imaging.
The optical path consists of five, 4-f, afocal telescope pairs that relay image the eye’s pupil onto several system pupil planes. All mirrors are commercially available with protective silver coating. The achromatic lens is a 400 mm effective focal length, 100 mm diameter achromat with broadband anti-reflection coating for 400 to 900 nm (Ross Optical, El Paso, Texas, USA). Note that the flatter side of the achromat faces the eye to minimize spherical aberrations for all angles required to form the imaging raster. The system pupil planes include the deformable mirror for wavefront correction, two scanners for raster scanning of the retina, and the SHWS lenslet array. The deformable mirror used was a large stroke hi-speed DM97 from ALPAO S.A.S. (Biviers, Grenoble, France). The deformable mirror controls both the monochromatic aberrations and focus adjustment, and the mouse is never moved, once it is positioned in the instrument. A resonant scanner (Electro-Optical Products Corp, Glendale, New York USA) line-scanned the retina horizontally at 15 kHz, and a slow galvometric scanner (GSI Group Corp, Massachusetts, USA) scanned the vertical direction at 25.5 Hz, forming a rectangular imaging raster. The wavefront sensor consisted of a lenslet array (Adaptive Optics Associates, Cambridge, Massachusetts, USA) with 7.8 mm focal length and 203 μm lenslet pitch, and a Rolera XR camera from QImaging (Surrey, British Columbia, Canada).
Light from the retina was split by a dichroic mirror into a reflectance channel and a fluorescence channel, before detection using photomultiplier tubes (PMT) H7422-40 and −50 from Hamamatsu Corporation (Hamamatsu, Shizuoka-Ken, Japan). The light reaching the PMTs was filtered spectrally by band-pass interferometric filters and spatially by confocal pinholes attached to the front of the PMTs. The confocal pinholes, used on the imaging channel, were between 1 and 3.9 Airy disks in diameter. The PMT output current was amplified and converted to voltage using transimpedance amplifiers (Femto, Berlin, Germany), inverted with in-house electronics, and digitized using a Matrox Odyssey eA framegrabber (Matrox International Corporation, Quebec, Canada).
The first order optical design follows basic design guidelines for a similar instrument built for the human eye [21
]. The deformable mirror is placed at the pupil conjugate closest to the eye, to minimize the area used on the optics. The other pupil plane elements are ordered to achieve a gradual change of magnification. Because of the large magnification factor between the deformable mirror plane and the pupil of the eye (approximately 7:1), an additional afocal telescope was added to gradually demagnify the pupil. In order to reduce the astigmatism due to using the spherical mirrors off-axis, spherical mirrors with long focal lengths (ranging from 375 mm to 1000 mm) were selected.
Due to the short focal length of the mouse eye, every degree in visual angle subtends only ~31-34 µm on the retina [28
], which is an order of magnitude smaller than in the human eye. Therefore, the AOSLO used in for this work was designed to use a small 3° FOV (field of view) with diffraction limited performance, which can be continuously increased up to 10° by changing the scanning angles on both scanners for finding areas of interest. To accommodate a large FOV using all reflective parts, the system performance was heavily driven by the large angle of incidence needed on the spherical mirror closest to the eye, and an achromatic lens had to be used as the last optical element with power to achieve diffraction-limited performance.
After the spherical mirrors were chosen, the only degrees of freedom left were the folding angles or angles of incidence on the spherical mirrors. The optical setup was folded in a non-planar configuration to simultaneously minimize the monochromatic aberrations in retinal and pupil planes [21
]. The optical layout was designed using CodeV (Optical Research Associates, Pasadena, California, USA) and Zemax (Zemax Development Corporation, Bellevue, Washington, USA). The angles of incidence on the off-axis spherical mirrors were optimized on both the x and y dimensions, under constraints for ray clearance and mechanical mount clearance. The combined optical performance (RMS wavefront error) was optimized on the retinal plane for 9 points uniformly distributed in the scanning field and for three different vergences (−30 D, 0 D, 30 D).
The final system design had diffraction-limited performance for a vergence range of 60 D over a 3° × 3° FOV for high resolution AO imaging. The theoretical system optical performance is shown in
for the shortest wavelength reflected by the spherical mirror coating (450 nm). Note that all spot diagrams were calculated on the focal plane of the mouse eye, assuming a perfect mouse eye and a flat deformable mirror correction. The residual wavefront RMS at 450 nm was lower than λ/20 for all vergences. Because of the mostly reflective nature of the setup, the system performed better for longer wavelengths.
Fig. 2 Spot diagrams for 27 configurations evaluated at the retinal plane, over a 3° × 3° FOV for a vergence range of 60 D in the mouse AOSLO optical design. Configurations are grouped by vergences, and all configurations are diffraction (more ...)
The pupil plane performance was characterized as the image quality for pupil plane re-imaging. This was evaluated along the optical axis over the 3° × 3° scanning FOV, for a wavelength of 450 nm, using the same method as described previously [21
shows spot diagrams on the 4 pupil planes.
Fig. 3 Spot diagrams for the 4 pupil planes of the mouse AOSLO at 450 nm over a 3° × 3° FOV, for an on-axis point object at the SHWS pupil plane. Different scanning configurations are coded by color. Black circle represents the Airy disk. (more ...)
When the optical scanners move to form the imaging raster, if the aberrations are well corrected, the chief ray pivots around a stationary point in the center of the pupil planes. If this is not the case, then the chief ray moves or “wanders” in the pupil plane. This motion blurs the aberrations seen by the SHWS, thus degrading the AO correction. Therefore, we describe the optical performance of the AOSLO in the pupil planes in terms of both the wavefront RMS and the maximum displacement of the chief ray. This beam wander for a 3° × 3° FOV and 60 D of vergence range was ~1% of the pupil diameter (2.0 mm) for a wavelength of 450 nm, and ~4% for a combination of 450 nm, 650 nm and 850 nm light.
For an 8° × 8° FOV, the system is diffraction-limited over a vergence range of 40 D. The wavefront RMS for wavelength of 450 nm is better than are λ/14 for a −22 D to +18 D vergence range. Beam wander at the eye’s pupil is ~3% of the pupil diameter for 450 nm light, and ~7% for a combination of wavelengths from 450 nm to 850 nm.
2.2. Adaptive optics
Wave aberrations were measured and corrected at 15 Hz frame rate. Before any source focus compensation and AO correction, the SHWS spots often appear blurry, as shown in
. When the AO loop is closed, the spots become brighter and sharper, as shown in . It typically takes 0.2 seconds (i.e. three iterations) to reach a stable AO correction, with a residual wavefront RMS error at or below 0.05 µm. We always focused the beacon on the photoreceptors to maximize the quality of the SHWS spots. However, we often want to image other planes with light passing through the same deformable mirror. In that case, the change in deformable mirror focus required to image a different layer must be compensated in the wavefront sensing beacon by moving the source fiber tip, so that the beacon stays focused on the photoreceptors. The beacon was refocused by either maximizing subjective sharpness of the SHWS spots, or using a previous calibration done for similar layers.
Fig. 4 Typical SHWS spot patterns before the spots are focused on the wavefront sensing source or AO correction (a), and after AO correction (b). The spots are brighter and sharper after AO correction. These SHWS spot patterns are taken at a scanning field of (more ...)
2.3. Animals, and preparations for in vivo imaging
Adult wild type mice (C57BL/6J) or transgenic mice (B6.Thy1-YFPH) were imaged [32
]. All mice were from 3 to 10 months old and weighed between 20 and 30 grams. Mice were housed in standard mouse cages under 12 hour light/dark cycle. All animals were handled according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and to the guidelines of the University Committee on Animal Resources at the University of Rochester.
Mice were anesthetized with either Ketamine/Xylazine cocktail injections (100mg/kg and 10mg/kg) only or combined with isofluorane gas (1-2%) and placed on a heating pad prior to in vivo
imaging. Pupils were dilated with one drop of 2.5% phenylephrine (Neo-Synephrine) and one drop of 0.5% tropicamide. A 0 to +10 D rigid contact lens (Unicon Corporation, Osaka, Japan) with a base curve between 1.55 to 1.70 mm was placed on the eye to maintain corneal hydration during in vivo
imaging. Mice were stabilized on a bitebar stage with two rotational degrees of freedom as described previously (Bioptigen, Research Triangle Park, North Carolina, USA) [15
2.4. Power levels, image acquisition and analysis
The maximum permissible light exposure (MPE) for the mouse eye was calculated assuming that the mouse retina possessed the same susceptibility to light damage as the human retina. These calculations included a scaling factor that incorporated the differences in the spot size and retinal illuminance at the mouse retina due to its higher NA. For reflectance imaging, 40 µW and 250 µW were used for the 843 nm laser diode and 789 nm SLD, respectively. Even though the system was to be diffraction-limited for a 3° × 3° high resolution FOV ( and ), during imaging it was often found that this FOV was too small to cover areas of interest. In reality we often image with a larger FOV (e.g. 5° × 7°), at the cost of slightly larger system aberrations and the FOV is probably outside of the eye’s isoplanatic size. The combined powers were less than the MPE for a 20-minute exposure time for a 5° × 7° FOV according to the ANSI guide for the safe use of lasers [33
]. For fluorescence imaging, cells were imaged using 300 µW of 514 nm laser power measured at the pupil of the eye. This power level, combined with that from the other two sources, is 1.8 times the ANSI MPE for imaging the 5° × 7° FOV for 5 minutes [33
To convert from visual angle to dimensions on the retina, a paraxial model for 100 days old C57BL/6 mice was used [28
]. It was calculated that every degree of visual angle corresponds to approximately 34 µm for this model.
To image different layers of the retina using the two channels simultaneously (reflectance and fluorescence), source and detector focuses for both channels were adjusted separately prior to imaging. Sinusoidal frame distortion from the motion of the resonant scanner was compensated by estimating the distortion from imaging a grating target placed in a model eye, and resampling the images using equally spaced pixels. Single in vivo
fluorescence frames typically possessed very low signal to noise ratio (SNR). Therefore, multiple fluorescence frames were averaged to obtain an image with higher SNR. To account for eye movements between different frames, images were registered using the shifts calculated from simultaneously acquired reflectance images [35
2.5. Labeling of ganglion cells, in vivo ganglion cell classification, and ex vivo confocal imaging
Ganglion cells were fluorescently labeled with yellow fluorescence protein (YFP) by one of two methods. The first was a transgenic mouse line that expresses YFP in a small subset of retinal ganglion cells (B6.Thy1-YFPH). In the second method, retinal ganglion cells in an adult C57BL/6J mouse were sparsely labeled with YFP via transduction from retrograde viral vector (Equine infectious anemia virus carrying YFP gene; Vector courtesy of Drs. Edward M. Callaway and Ali H. Cetin at the Salk Institute of Biological Studies, California, USA).
Image stacks were taken from the somas and dendrites of the sparsely labeled ganglion cells for classification. Typical image stacks contained 10 slices (with 750 frames averaged for each focus), approximately 6 µm apart in depth and were collected in 5 minutes.
One mouse having retrograde viral vector labeling of ganglion cells was sacrificed after multiple in vivo imaging sessions. Retinas were isolated and placed as wholemounts on slides with coverslips and covered in mounting medium (Vectashield; Vector Laboratories, Burlingame, California, USA). To visualize boundaries of inner plexiform layer (IPL), cell nuclei in both the ganglion cell layer and the inner nuclear layer were labeled with DAPI overnight. Wholemount images were acquired using a confocal microscope (Olympus FV1000; Olympus, Center Valley, Philadelphia, USA). Image stacks were taken at the same locations as the in vivo images using a 40X (1.3 NA) oil immersion microscope objective, and maximum intensity projection images were generated for each stack.