Abstract
Purpose :
Calcium phosphate minerals are ubiquitous constituents of retinal drusen (PMID 25605911) and the presence of larger hydroxyapatite (HAP) deposits termed nodules is predictive of progression to advanced AMD within one year (odds ratio 6.4 : 1)( PMID 30404862). HAP can be imaged by fluorescence lifetime imaging microscope (FLIM) following staining with common tetracycline antibiotics (PMID 32319262). These results and others suggested that imaging HAP deposition in the retina might prove a useful biomarker for the early detection and monitoring progression of AMD and other diseases. However, some of the best performing tetracyclines are best excited at wavelengths near 400 nm, which raises safety issues and where background fluorescence particularly in the vitreous and lens is strongly excited. Since multiphoton excitation occurs only at the focal point, such imaging is effectively confocal and background fluorescence in the excitation path is minimized. In addition, there are minimal safety concerns with the excitation wavelength used by the two photon system (780 nm in this experiment) as it is similar to the wavelengths widely and safely used for OCT. Here we tested multiphoton excitation for imaging HAP in a FLIM using an ultrafast laser in the near-infrared for fluorescence excitation of HAP-bound tetracyclines.
Methods :
Donors provided consent under ethical standards. Cadaver eyes were dissected, flat-mounted, and stained with chlortetracycline or solutions essentially as previously described (PMID 32319262) and imaged in an ISS Alba FLIM with excitation at 780 nm, 90 fsec pulse duration, and 12 mW average power through a 20 X 0.7 NA objective. The instrument was calibrated with standards of known lifetimes.
Results :
Both HAP-bound doxycycline and chlortetracycline were efficiently excited by focused 780 nm pulses; measured average lifetime values were similar (3.6 and 1.9 nsec, respectively) to those measured previously for HAP-bound tetracyclines with one photon excitation (3.8 and 1.7 nsec, respectively); and both distinct from the typical average lifetime of the retinal background fluorescence (0.2 – 0.4 nsec; reviewed in PMID 28673870).
Conclusions :
Based on these results we propose that in vivo imaging of tetracycline-labelled HAP in the outer retina could become a useful tool to monitor mineralization using a fluorescence lifetime ophthalmoscope with multiphoton excitation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.