Multispectral autofluorescence lifetime imaging of RPE cells using two-photon excitation

Abstract

ABSTRACT In this paper, we present our investigation on multispectral autofluorescence lifetime imaging of RPE cells using two-photon excitation. Morphological characters of RPE cells are obtained with high spatial resolution. Different autofluorescence lifetime parameters have been compared at different emission bands. Spatial distribution of dominant endogenous fluorophores in RPE cells, such as FAD, A2E and AGE etc have been obtained by the analysis of W m and a 1 /a 2 ratio in the whole emission spectrum. Keywords: Retinal pigment epithelium, RPE, fluorescence lifetime imaging microscopy, two-photon excitation, autofluorescence, retina 1. INTRODUCTION The retinal pigment epithelium (RPE) is a single layer of ce lls between the neurosensory retina and the choroid which itself is separated from the RPE by Bruch´s membrane [1]. Histology study in vitro shows that the fundus contains a wide variety of fluorophores among which lipofuscin is dominant. It is a byproduct of incomplete digestion of the distal ends of photoreceptors and accu mulates in the RPE cells when aging [2]. In modern ophthalmology, it is believed that the causes of many retinal diseases, such as age-related macular degeneration, diabetic retinopathy and retinitis are related to the progressive accumulation of autofluorescent membrane-bound lipofuscin [3]. Although it is not yet clear why RPE cells are damaged with aging, a previous study proposes that lipofuscin granules are most likely harmful when present in sufficient amount in the RPE cells. Since lipofuscin granules in the RPE cells demonstrate strong autofluorescence when excited by blue and green light [1], a number of pioneering works have focused on the measurement of retinal autofluorescence charact eristics and retinal autofluorescence imaging. In this work, we present our investigation on the morphology and endogenous fluorophores characteristics of RPE cells at sub cellular level by using two-photon excitation and fluorescence lifetime imaging microscopy (FLIM). One of the advantages of two-photon excitation is the simultaneous excitation of many fluorophores present in the RPE cell. And FLIM measurement can provide functional information about the fluorophores that is complimentary to fluorescence intensity and spectrum measurement. Our preliminary results reveal the components of the fluorophores and their distribution inside the RPE cells.