Imaging Hydoxyapatite in sub-RPE Deposits by Fluorescence Lifetime Imaging Microscopy (FLIM)

Abstract

Abstract Purpose : Purpose: Recently, we found that small spherules (~2 um) of hydroxyapatite (HAP) were present in sub-RPE deposits and have obtained evidence that they may nucleate the growth of the deposits (PMID: 25605911). The HAP spherules can be imaged with fluorescent stains such as LiCor Bone Tag or OsteoSense in vitro, but their use in vivo might be difficult especially as their safety in humans is unproven. Tetracycline antibiotics are well known to bind to HAP with a substantial concomitant increase in fluorescence, and their safety in humans and animal models is well established. Here we examined whether tetracyclines might be suitable to image the HAP spherules by fluorescence lifetime imaging microscopy (FLIM) to overcome challenges with detection due to the autofluorescence of the RPE. Methods : Methods: We measured the tetracycline lifetimes with or without binding to HAP in solution on an ISS K2 multifrequency phase fluorometer with laser excitation. We stained human fixed cadaver retinas as flat mounts with tetracyclines and imaged them by frequency domain FLIM in an ISS ALBA with 473 nm excitation and 520 +/- 20 nm emission. Data were collected and displayed as phasor plots, or fit to multiexponential decay laws using ISS Vistavision software. Results : Results: Using phase fluorometry we found that the average fluorescence lifetime of free chlortetracycline was 0.8 nsec and when bound to HAP increased to 1.7 nsec, suggesting that this approach might be used to image the spherules in human tissues. Initial results labeling sub-RPE deposits in a cadaveric flat mount RPE/choroid preparation from a 94 year old donor demonstrated that drusen could be resolved from background RPE fluorescence readily based on their differing fluorescence lifetimes by FLIM. The figure shows a color coded fluorescence intensity image of the flat mount overlain with pixels highlighted in purple having average lifetimes of 0.6 nsec (left panel) and 1.7 nsec (right). Conclusions : Based on these results it seems feasible to image tetracycline-stained HAP spherules in the retina, and based on the pioneering in vivo measurements of the Schweitzer (PMID 22511622) and Zinkernagel (IOVS 55:2106–2113(2014)) groups it may be feasible to use this in vivo following staining with an orally administered tetracycline. This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.