Abstract
Purpose :
Two-photon (2P) imaging has become a powerful tool for retina research. While many exogeneous biosensors enable the investigation of retinal physiology in ex-vivo tissues, numerous endogenous fluorophores also reveal retinal functions, promising great potential for noninvasive application. Here, we present a multiphoton imaging platform for autofluorescence in retinal tissue with future extension to the living eye.
Methods :
The platform was developed from a custom 2P adaptive optics scanning light ophthalmoscope (2P-AOSLO) for in-vivo high-resolution imaging in the human eye. The optical path was extended and coupled with an objective for ex-vivo imaging. An ultra-short pulse laser (710 – 920 nm, <70 fs, 80 MHz) is used to illuminate the retina and to excite endogenous fluorophores (e.g. all-trans-retinol in photoreceptors, lipofuscin and melanin in retinal pigment epithelia (RPEs)). Currently, three channels can be recorded simultaneously: reflectance, dark-field, and 2P autofluorescence. Adult mouse tissues were examined as freshly isolated retinal flat mounts, eyecups, or intact eyeballs. All tissues were bathed in oxygenized saline. The laser power was kept at around 2 mW.
Results :
Photoreceptors were recorded after light exposure (590 nm). The most well-organized outer segments (~1.5-2 μm) showed improved visibility in 2P autofluorescence compared to reflectance images, however not all cells appeared to be functional. The typical RPE mosaic was clearly visible as a single layer of hexagonal cells (~15-20 μm/cell) with fluorescence evenly distributed within the cell except for the nuclei. Meanwhile, the reflectance image showed smaller structures, presumably microvilli, and the cell wall was apparent in the dark-field image. In intact eyeballs, the nerve fiber layer was the layer that provided the highest level of autofluorescence.
Conclusions :
Our system is based on an optimized all-reflective design, which allows us to minimize the laser power 2P laser for excitation and record weak fluorescence. Moreover, adaptive optics and a state-of-the-art retinal tracking system can compensate for aberrations and eye motion. Currently, ex-vivo results prove the feasibility of autofluorescence recording in the living eye. Through these advanced techniques, we set the stage for high-resolution 2P imaging of the retina in-vivo.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.