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D. Gray, W. H. Merigan, B. P. Gee, R. Wolfe, Y. Geng, B. Masella, D. Scoles, S. Luque, D. R. Williams; Adaptive Optics Enables Imaging of Axons and Dendrites of Rhodamine Labeled Macaque Retinal Ganglion Cells in vivo. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2853. doi: https://doi.org/.
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Although retrograde labeled ganglion cell somas can be imaged in vivo, fluorescence labeling does not completely fill the finer features of these cells: axons and dendrites. Additionally, conventional imaging systems do not have high enough resolution to resolve structures on the order of 2 microns. This study imaged these finer features using an in vivo analog of the fireworks that has been used to visualize retinal ganglion cells in vitro combined with adaptive optics fluorescence imaging.
Rhodamine dextran dye was injected into the LGN of macaque monkeys to label the retinal ganglion cells by retrograde transport. In vivo images were then acquired with a custom built, fluorescence adaptive optics scanning laser ophthalmoscope in the anesthetized monkey. An argon/krypton laser provided 530 nm excitation light for fluorescence imaging. Direct reflectance images were obtained simultaneously with light from an 830 nm laser diode and were used to register the fluorescence images. Photoactivation of the labeled cells (fireworks) was achieved by imaging for 10 to 15 minutes with a reduced field of view (1 x 1 deg) in order to increase the fluorescent signal.
Intense fluorescence from retinal ganglion cells was imaged at sufficiently high resolution that details of axonal and dendritic morphology were clear. Intensity profiles of filled axons show an average width of 1.6 microns compared to an expected width of 1.2 microns for a 0.5 microns wide axon convolved with a diffraction limited PSF for an eye of 0.23 numerical aperture. Direct comparison to confocal microscopy of the same cells showed that much of the same structure was visible in vivo.
Adaptive optics fluorescence imaging provides near diffraction limited resolution in vivo. When applied to labeled ganglion cells, fine dendritic structures are revealed, permitting classification of cell type. A quantification of the resolution of our imaging system shows that we are close to diffraction limited imaging.
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