Imaging hydroxyapatite in sub-retinal pigment epithelial deposits by fluorescence lifetime imaging microscopy with tetracycline staining

Significance: Recent evidence suggests that hydroxyapatite (HAP) in sub-retinal pigment epithelial (sub-RPE) deposits in aged human eyes may act to nucleate and contribute to their growth to clinically detectable size. Sub-RPE deposits such as drusen are clinical hallmarks of age-related macular degeneration (AMD), therefore enhanced and earlier detection is a clinical need. We found that tetracycline-family antibiotics, long known to stain HAP in teeth and bones, can also label the HAP in sub-RPE deposits. However, HAP-bound tetracycline fluorescence excitation and emission spectra overlap with the well-known autofluorescence of outer retinal tissues, making them difficult to resolve. Aim: In this initial study, we sought to determine if the HAP-bound tetracyclines also exhibit enhanced fluorescence lifetimes, providing a useful difference in lifetime compared with the short lifetimes observed in vivo in the human retina by the pioneering work of Schweitzer, Zinkernagel, Hammer, and their colleagues, and thus a large enough effect size to resolve the HAP from background by fluorescence lifetime imaging. Approach: We stained authentic HAP with tetracyclines and measured the lifetime(s) by phase fluorometry, and stained aged, fixed human cadaver retinas with drusen with selected tetracyclines and imaged them by fluorescence lifetime imaging microscopy (FLIM). Results: We found that chlortetracycline and doxycycline exhibited substantial increase in fluorescence lifetime compared to the free antibiotics and the retinal background, and the drusen were easily resolvable from the retinal background in these specimens by FLIM. Conclusions: These findings suggest that FLIM imaging of tetracycline (and potentially other molecules) binding to HAP could become a diagnostic tool for the development and progression of AMD.