A2E Distribution in RPE Granules in Human Eyes.

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1-F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

Integrated multimodal photoacoustic microscopy with OCT- guided dynamic focusing.

Combining different contrast mechanisms to achieve simultaneous multimodal imaging is always desirable but is challenging due to the various optical and hardware requirements for different imaging systems. We developed a multimodal microscopic optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissues. This imaging system integrated photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and confocal fluorescence microscopy in one platform. By taking advantage of the depth resolving capability of OCT, we developed a novel OCT-guided surface contour scanning methodology for dynamic focusing adjustment. We have conducted phantom, in vivo, and ex vivo tests to demonstrate the capability of the multimodal imaging system for providing comprehensive microscopic information of biological tissues. Integrating all the aforementioned imaging modalities with OCT-guided dynamic focusing for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the future.