Two Photon Excited Fluorescence Imaging of Human RPE Cells Using a Femtosecond Ti:Sapphire Laser

Abstract

Purpose: : Imaging and delineation of retinal pigment epithelium (RPE) cells in the living human eye have marked limitations with currently available technologies including OCT or fluorescence angiography. We evaluated the feasibility of imaging human RPE cells and the intracellular lipofuscin (LF) granule distribution with newly developed non–invasive two photon excited fluorescence (TPEF) technique.

Methods: : TPEF imaging of the RPE of human donor eyes was conducted using a multiphoton scanning laser microscope which employs a femtosecond Ti:sapphire laser as excitation laser source. The spectrum of autofluorescence of LF granules was analyzed using a confocal scanning laser microscope coupled to a UV argon laser.

Results: : Using TPEF it was possible to delineate individual RPE cells and morphology as well as the intracellular distribution of LF granules with high contrast and sub–micron resolution. The typical diameter of individual LF granules was found to be below 1 µm, with single RPE cells possessing larger granules. These larger LF granules demonstrated different autofluorescence emission with enhanced blue–green fluorescence and an emission peak at 36 nm shorter than regular LF granules.

Conclusions: : TPEF imaging represents a novel tool to investigate morphological and spectral characteristics of human RPE cells. Spectral variations of individual LF granules may indicate differences in the complex yet incompletely understood molecular composition. Because of reduced photo–damage, deeper sensing depth and larger wavelength tuneable range, TPEF imaging may be superior to conventional single photon excited fluorescence techniques. Particularly, it may allow imaging the RPE cell monolayer and LF accumulation on the sub–microscopic level in the living eye. As the RPE plays a central role in many retinal diseases and as excessive accumulation of LF/A2–E is a common downstream pathogenetic pathway this may help to elucidate pathophysiological mechanisms and to monitor therapeutic interventions. Based on these investigations it is now planned to combine TPEF imaging with a confocal scanning laser ophthalmoscope for application in vivo.

Keywords: microscopy: confocal/tunneling • laser • retinal pigment epithelium