Abstract
Purpose :
Previously, we showed that drusen contain microscopic calcium phosphate mineral deposits such as hydroxyapatite (HAP), and proposed that they could initiate the growth of such sub-RPE deposits (PMID: 25605911). Further, we found that larger mineral deposits called nodules are strongly associated with progression to advanced AMD within one year (odds ratio 6.4), suggesting that early detection of mineral deposits might serve as a useful early screen for AMD(PMID: 30404862). While such mineral deposits are readily detectable in vitro with stains such as OsteoSense and BoneTag, these stains have unknown human toxicity, pharmacokinetics, and metabolism and aren't administered orally. By comparison, the tetracycline family of legacy antibiotics have been used safely in humans for decades, are mostly administered orally, and were known as fluorescent mineral stains. We found that chlortetracycline and doxycycline selectively stained retinal HAP with increased quantum yield and lifetime, such that chlortetracycline-stained drusen were readily resolved from the short fluorescence lifetime of the remainder of the retina (PMID 32319262). However, some of their other optical properties are suboptimal, so in this study we tested other tetracyclines and related compounds as fluorescence stains for retinal minerals for suitability in in vivo studies.
Methods :
We measured the fluorescence spectra and lifetimes of legacy tetracyclines free in solution and bound to HAP and other minerals, and as stains for unfixed flat-mounted aging human donor retinas by fluorescence lifetime microscopy (FLIM). Of particular interest were stains spectrally compatible with existing fluorescence (lifetime) ophthalmoscopes used for fluorescein angiography and FLIO.
Results :
Multiple tetracyclines exhibit useful changes fluorescence upon HAP binding. For example, free anhydrochlortetracycline excites at 473 nm, has peak emission at 587 nm, and exhibits a principal lifetime component (two-component fit) of 0.184 ± 0.006 nsec, fractional intensity 84%. However, when bound to HAP the principal component increases more than 7-fold to 1.42 ± 0.05 nsec, fraction 70%.
Conclusions :
These data indicate retinal HAP stained with anhydrochlortetracycline or others will exhibit fluorescence resolved spectrally and in lifetime from the known background of the retina.
This is a 2021 ARVO Annual Meeting abstract.