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. 2012 Apr 1;3(4):715-34.
doi: 10.1364/BOE.3.000715. Epub 2012 Mar 15.

Adaptive optics retinal imaging in the living mouse eye

Ying Geng, Alfredo Dubra, Lu Yin, William H Merigan, Robin Sharma, Richard T Libby, David R Williams

Adaptive optics retinal imaging in the living mouse eye

Ying Geng et al. Biomed Opt Express. .

Abstract

Correction of the eye's monochromatic aberrations using adaptive optics (AO) can improve the resolution of in vivo mouse retinal images [Biss et al., Opt. Lett. 32(6), 659 (2007) and Alt et al., Proc. SPIE 7550, 755019 (2010)], but previous attempts have been limited by poor spot quality in the Shack-Hartmann wavefront sensor (SHWS). Recent advances in mouse eye wavefront sensing using an adjustable focus beacon with an annular beam profile have improved the wavefront sensor spot quality [Geng et al., Biomed. Opt. Express 2(4), 717 (2011)], and we have incorporated them into a fluorescence adaptive optics scanning laser ophthalmoscope (AOSLO). The performance of the instrument was tested on the living mouse eye, and images of multiple retinal structures, including the photoreceptor mosaic, nerve fiber bundles, fine capillaries and fluorescently labeled ganglion cells were obtained. The in vivo transverse and axial resolutions of the fluorescence channel of the AOSLO were estimated from the full width half maximum (FWHM) of the line and point spread functions (LSF and PSF), and were found to be better than 0.79 μm ± 0.03 μm (STD)(45% wider than the diffraction limit) and 10.8 μm ± 0.7 μm (STD)(two times the diffraction limit), respectively. The axial positional accuracy was estimated to be 0.36 μm. This resolution and positional accuracy has allowed us to classify many ganglion cell types, such as bistratified ganglion cells, in vivo.

Keywords: (110.1080) Active or adaptive optics; (170.4460) Ophthalmic optics and devices; (330.7324) Visual optics, comparative animal models.

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Figures

Fig. 1
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 scanner. VS: vertical scanner. M1-9: Concave spherical mirrors.
Fig. 2
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 limited for 450 nm of wavelength. The radius of Airy disk (Black circle) is 0.59 µm.
Fig. 3
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.
Fig. 4
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 5° × 5°. Each wavefront sensor spot is sampled by 16 × 16 pixels on the CCD camera. The width of both images is approximately 465 pixels.
Fig. 5
Fig. 5
In vivo reflectance image of the NFL close to the optic disk in the mouse eye. This image was an average of 100 frames. Confocal pinhole diameter was 2.1 Airy disks. Arrows: examples of nerve fiber bundles. Arrowhead: example of capillaries. Image was contrast stretched for display purposes. Scale bar: 20 µm.
Fig. 6
Fig. 6
In vivo reflectance image montage of the NFL in the mouse eye, showing a large blood vessel in the center, capillaries, and nerve fiber bundles. This location was over 15 degrees away from the optic disk. Size of this image was 553 µm × 230 µm, or 16.3° × 6.8°. Each individual image was an average of 50 frames. Confocal pinhole diameter was 2.1 Airy disks. Scale bar: 20 µm. Image was contrast stretched for display purposes only.
Fig. 7
Fig. 7
In vivo reflectance capillary images in the mouse retina. All images are taken at the same retinal location. (a), (b), (d) Capillary images at different depths. Each image is a registered average of 50 individual frames. (c) Standard deviation/motion contrast image corresponding to the depth of image (a). Arrows and arrowhead: dark regions and microscopic bright point structures within the intermediate capillary layer. Confocal pinhole diameter was 2.1 Airy disks. All images were contrast stretched identically for display purposes. Scale bar: 20 µm.
Fig. 8
Fig. 8
(a) Photoreceptors imaged in reflectance in the mouse eye. This image is an average of 120 frames. Scale bar: 10 µm. (b) Fourier spectrum of the photoreceptor image in (a), showing a concentration of energy at a fixed radius from the origin. The partial circle indicates the spatial frequency calculated using a 1.60 µm nearest neighbor distance. Confocal pinhole diameter was 2.1 Airy disks. Image (a) was contrast stretched for display purposes.
Fig. 9
Fig. 9
In vivo fluoresence images of a ganglion cell expressing YFP. (a-c) Individual images from three of the focuses, at focus steps of 11.6 μm. Each image at an individual focus step was a registered image average of 500 frames. (d) Maximum intensity projection image generated from 5 separate in vivo images taken at focus steps of 5.8 μm. Confocal pinhole diameter was 3.9 Airy disks. All images were contrast stretched identically to preserve their relative brightness. Scale bar: 20 µm.
Fig. 10
Fig. 10
In vivo imaging of the same fluorescent ganglion cells at times separated by one month. Image shown in (a) and (b) are taken one month apart. Both images are maximum intensity projection images generated from 10 separate in vivo images at individual focuses. Each image at an individual focus is a registered average of 750 frames. Confocal pinhole diameter was 1.9 Airy disks. Images were contrast stretched identically for display purposes. Scale bar: 20 µm.
Fig. 11
Fig. 11
Characterization of the in vivo resolution using an image stack of a fluorescent ganglion cell. (a) Maximum projection image for a focus stack. (b) One individual focus from the focus stack. Transverse cross section on a typical dendrite labeled in yellow is plotted as an example in (d). (c) An individual focus image 11.6 μm shallower than the focus in (b). Arrow indicates measurement position for a typical axial cross section shown in (e). (d) and (e) are the characterization of the in vivo transverse and axial resolution, respectively. Circle data points: in vivo measurement. Solid black line: spline fit to in vivo measurement data. Solid gray line: theoretical diffraction-limited axial PSF. Scale bar: 20 µm.
Fig. 12
Fig. 12
Direct comparison of in vivo and ex vivo mouse monostratified ganglion cell. In vivo image dimensions (degree to µm conversion calculated using paraxial eye model [28]) matched very well with ex vivo image dimensions. (a) Ex vivo histological image acquired using a 40x oil immersion confocal microscope with a 1.3 NA objective. (b) In vivo image in a mouse retina taken with AO correction over a 0.49 NA. The in vivo image was a maximum intensity projection image generated from 11 in vivo images taken at different depths. Ex vivo image was a maximum intensity projection image generated from an image stack of 51 images. Confocal pinhole size was 1 Airy disk for ex vivo, and 1.9 Airy disks for in vivo imaging. Scale bar: 20 μm.
Fig. 13
Fig. 13
Direct comparison of in vivo and ex vivo mouse bistratified ganglion cells. All imaging parameters used were the same as that used for the cell imaged in Fig. 12.
Fig. 14
Fig. 14
In vivo imaging of six more monostratified ganglion cells. Shadows from large blood vessels can be seen in images of cell 2, 4 and 6. Cells 5 and 6 are taken from transgenic mice with ganglion cells expressing YFP, and all other cells are labeled with retrograde viral vector. Images are contrast stretched for display purposes. Scale bar: 20 μm.
Fig. 15
Fig. 15
In vivo imaging of three more bistratified ganglion cells. The left column shows the maximum projection image of the cell; middle and right columns shows the dendrite stratifications at 2 different focuses. All images are contrast stretched for display purposes. Scale bar: 20 μm.
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