Abstract
Purpose :
Two-photon excited fluorescence (TPEF) of the eye’s fundus is a new imaging modality that has been recently demonstrated on humans. Using near-infrared fs laser pulses and two-photon excitation it is possible to probe retinal fluorophores with excitation spectra in the UV spectral range, impossible with one-photon imaging. This study was designed to verify the applicability of the newly developed instrument and imaging method with healthy volunteers without prior experience with this technique.
Methods :
The study was performed using a TPEF scanning laser ophthalmoscope schematically shown in Fig. 1a. Fluorescence was excited with laser light at 780 nm, 76 fs pulse duration, pulse repetition rate of 6 MHz, and average power of 0.3 mW. Imaging was performed in a dark room with no prior dark adaptation or pupil dilation. Typically, 90-120 frames were recorded per eye and averaged in post-processing. We investigated a group of 13 healthy subjects with no history of ocular diseases in two age groups, 20-30 (n = 9) and 50-53 (n = 3) years old.
Results :
In all cases, the recorded TPEF signal was significantly higher than the instrument noise and sufficient to reconstruct fundus images. With the first group of subjects, the mean fluorescence photon count per pixel was 45.8 x 10-3, SD = 7.4 x 10-3 (n = 9). With the second group, mean fluorescence photon count per pixel was 48.7 x 10-3, SD = 9.4 x 10-4 (n = 3). There was no significant difference between the two groups (Fig. 1b). Selected examples of images on naïve volunteers are shown (Fig. 1c). These results are comparable to those obtained on a trained eye (1 subject with substantial experience with this imaging modality, 10 sessions). Here, the mean photon count was slightly higher (89.9 x 10-3, SD = 5.8x10-3).
Conclusions :
The results show that our novel imaging method is applicable to imaging untrained subjects and could be used clinically. The TPEF signal was significantly higher than the instrument's noise, and of the same order of magnitude as for an experienced subject; sufficient to reconstruct the TPEF image.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.