Abstract
Purpose :
To design adaptive optics enhanced fluorescence lifetime imaging ophthalmoscopy (AOFLIO) that can image the retinal structure and metabolic function at the cellular level and optimize AOFLIO photon detection.
Methods :
A picosecond diode laser (λ = 473 nm, pulse width 40 ps) was employed to excite the endogenous fluorophores in the retina and RPE in the living human eye. The autofluorescence lifetime was measured in a long spectral channel (LSC: 560 - 720 nm) and a short spectral channel (SSC: 500 - 560 nm) using the time-correlated single photon counting (TCSPC) method through an adaptive optics confocal scanning imaging system. A high-precision timing system generated the line and frame synchronization signals and the pixel clock by using a phase-locked loop to track the motion of the fast scanner, enabling the fluorescence lifetime to be measured across the 2D scanning field in a FIFO imaging mode. The retinal movement was calculated from the consecutive adaptive optics high-resolution confocal images of the same retinal area simultaneously acquired with the fluorescence lifetime measurement using a near-infrared light source (λ = 790 nm), ensuring precise registration of autofluorescence photons recorded in successive frames. The thresholds of the constant fraction discriminators and the time to amplitude converters of the photodetectors were optimized to ensure maximum photon detection. Under the safe light exposure limits, AOFLIO images acquired with laser pulse repetition rates at 80, 50, and 20 MHz were evaluated to assess the excitation efficiency.
Results :
Adaptive optics increased the photon number by 2.0 times in the LSC and 1.7 times in the SSC. AOFLIO produced high-resolution retinal autofluorescence lifetime images, rendering clear RPE cell structure, and indicating an inhomogeneous distribution of multiple fluorophores associated with specific metabolic states at the cellular level.
Conclusions :
High-resolution AOFLIO can precisely measure the retinal and RPE metabolic function and potentially characterize the distribution of different intrinsic fluorophores in the human retina and RPE at the cellular level. It can inform the interpretation of clinical FLIO imaging and improve the biological bases of retinal and RPE functions for next-generation imaging technology.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.