SignificanceAdaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE).AimTo quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO.ApproachAOFLIO was performed on six subjects at seven eccentricities. Four imaging channels (λex/λem) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter.ResultsA repeatability analysis revealed <11.8% change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters.ConclusionsMultispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.
Retinal ganglion cells (RGCs) are the layer of neurons in the retina that output directly to the brain. In primates, there are approximately 20 distinct types which each transmit an independent encoding of the external visual world in parallel to the brain.1 The wide variety of RGC types suggests that the retina undertakes a sophisticated set of computations that our used by higher brain areas to decode the visual world. Yet, little is known about the majority of the different types of RGCs present in the primate retina largely due to the rarity of many of them. Current understanding of RGCs largely derives from ex vivo electrophysiology experiments which are acute and require severing the optic nerve.2 This preparation removes the opportunity to study vision as an intact system and puts a time limit on how long a sample is viable for study before the tissue dies. Traditional electrophysiology also struggles to study the RGCs that serve the fragile fovea, the central high-resolution region of the retina. Adaptive optics (AO) ophthalmoscopy coupled with functional imaging and optogenetics provides a unique set of tools which allows the study of individual RGCs in situ.3,4 Here we describe a plan for a novel split field of view AO ophthalmoscope that will deliver visual stimuli precisely controlled in space, time, and color to the receptive fields of specific RGCs, while also simultaneously optically recording functional calcium responses from the same RGCs. The device also enables psychophysical experiments to study the perceptual impact of RGC types via optogenetics.
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