Separate lifetime signatures of macaque S cones, M/L cones, and rods observed with adaptive optics fluorescence lifetime ophthalmoscopy

Abstract

In the retina, several molecules involved in metabolism, the visual cycle, and other roles exhibit intrinsic fluorescence. The overall properties of retinal fluorescence depend on changes to the composition of these molecules and their environmental interactions due to transient functional shifts, especially in disease. This behooves the understanding of the origins and deviations of these properties within the multilayered retina at high lateral and axial resolution. Of particular interest is the fluorescence lifetime, a potential biomarker of function and disease independent of fluorescence intensity that can be measured in the retina with adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO). This work demonstrates the utility of the phasor method of analysis, an alternate approach to traditional multiexponential fitting, to evaluate photoreceptor two-photon excited AOFLIO data and separate them based on functional differences. Phasor analysis on fluorescence lifetime decay data allowed the repeatable segregation of S from M/L cones, likely from differences in functional or metabolic demands. Furthermore, it is possible to track the lifetime changes in S cones after photodamage. Phasor analysis increases the sensitivity of AOFLIO to functional differences between cells and has the potential to improve our understanding of pathways involved in normal and diseased conditions at the cellular scale throughout the retina.

Near Infrared Autofluorescence Lifetime Imaging of Human Retinal Pigment Epithelium Using Adaptive Optics Scanning Light Ophthalmoscopy

“…A similar effect was observed in another study in which S cones were distinguished in the phasor space while no such difference was observed in the mean lifetime space. 78 More specifically with our 8° temporal data set, the τ 1 and τ 2 values acquired from the eye with the hyperreflective spot were 94 ps and 707 ps, while that of the subject's other eye were 32 ps and 557 ps. As each of the individual data points changes in phasor space, the combined effect can sometimes be nonlinear.…”

Suppression of Subpixel Jitter in Resonant Scanning Systems With Phase-locked Sampling

“…Finally, real-time subpixel jitter suppression enables higher resolution imaging with resonant scanners, which may be particularly important for ophthalmic applications such as two photon imaging [ 17 ] the registration SLO images for retinal FLIM [ 18 ] where frames are usually averaged due to limited photon budgets. PSF widening up to 15% of the diffraction-limited resolution is avoided with jitter suppression.…”

“…RESONANT scanners are widely used in many different types of microscopy including confocal [ 1 ], [ 2 ] and two photon [ 3 ]–[ 6 ] microscopy as well as super-resolution imaging using stimulated emission depletion [ 7 ]–[ 10 ] microscopy. The combination of wide scan angle and very high imaging rate has driven recent advances in real-time live animal imaging [ 4 ], [ 11 ], [ 12 ] and neuroscience [ 3 ], [ 13 ], [ 14 ], enabled adaptive optics scanning laser ophthalmoscopy [ 15 ], [ 16 ], two photon excitation ophthalmoscopy [ 17 ] and fluorescent lifetime ophthalmoscopy[ 18 ] in awake patients as well as accelerated real-time diagnosis of human cancers [ 19 ]–[ 21 ]. Resonant scanning has also enabled in vivo super-resolution imaging of living animals[ 10 ], providing high speed imaging of the subcellular dynamics of live neurons.…”

Fluorescence Lifetime Imaging of Human Retinal Pigment Epithelium in Pentosan Polysulfate Toxicity Using Adaptive Optics Scanning Light Ophthalmoscopy

“… 30 Phasor analysis was performed in MATLAB, as previously described. 31 In short, data were binned at a kernel size of 17 × 17 pixels and a threshold was set for 50 photons. Previous studies suggest that lower thresholds are acceptable for phasor analysis and have used a threshold as low as 37 photons.…”