title: Two-photon-excited fluorescence imaging of human RPE cells with a femtosecond Ti:Sapphire laser.
abstract: OBJECTIVE
  To record the distribution and spectrum of human retinal pigment epithelial cell lipofuscin (LF) by two-photon-excited fluorescence (TPEF) and confocal laser scanning microscopy.
  
  
  METHODS
  Ex vivo TPEF imaging of the human retinal pigment epithelium (RPE) of human donor eyes was conducted with a multiphoton laser scanning microscope that employs a femtosecond Ti:sapphire laser as an excitation laser source. The spectrum of autofluorescence of LF granules was analyzed with a confocal laser scanning microscope coupled to a UV argon laser.
  
  
  RESULTS
  TPEF examination allowed for imaging of RPE cell morphology and intracellular distribution of LF granules with high-contrast and submicrometer resolution. Variations in cell size and shape as well as in autofluorescence spectra of individual LF granules were recorded. The typical diameter of LF granules was found to be below 1 mum, with some RPE cells possessing larger granules. Remarkably, enhanced blue-green autofluorescence was observed from these larger LF granules.
  
  
  CONCLUSIONS
  TPEF imaging represents a novel tool for the investigation of morphologic and spectral characteristics of human RPE cells. Spectral variations of individual LF granules may indicate differences in the complex molecular composition. Compared to conventional single-photon excited autofluorescence, TPEF with a tunable laser source allows for reduced photo damage and deeper sensing depth. It may help to elucidate further the pathophysiological role of LF accumulation as a common downstream pathogenetic pathway in retinal diseases. With the proof of principle from this ex vivo study, further work is now planned to evaluate the safety of TPEF RPE imaging in RPE cultures and animal models.
authors.affiliation: Department of Ophthalmology, University of Bonn, Germany.
authors.forename: Almut
authors.initials: A
authors.lastname: Bindewald-Wittich
authors.forename: Meng
authors.initials: M
authors.lastname: Han
authors.forename: Steffen
authors.initials: S
authors.lastname: Schmitz-Valckenberg
authors.forename: Sarah R
authors.initials: SR
authors.lastname: Snyder
authors.forename: Günter
authors.initials: G
authors.lastname: Giese
authors.forename: Josef F
authors.initials: JF
authors.lastname: Bille
authors.forename: Frank G
authors.initials: FG
authors.lastname: Holz
mesh_terms: D000328:Adult; D000369:Aged, 80 and over; D006801:Humans; D007834:Lasers; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology*