title: Time-resolved autofluorescence imaging of human donor retina tissue from donors with significant extramacular drusen.
abstract: OBJECTIVE
  Time and spectrally resolved measurements of autofluorescence have the potential to monitor metabolism at the cellular level. Fluorophores that emit with the same fluorescence intensity can be discriminated from each other by decay time of fluorescence intensity after pulsed excitation. We performed time-resolved autofluorescence measurements on fundus samples from a donor with significant extramacular drusen.
  
  
  METHODS
  Tissue sections from two human donors were prepared and imaged with a laser scanning microscope. The sample was excited with a titanium-sapphire laser, which was tuned to 860 nm, and frequency doubled by a BBO crystal to 430 nm. The repetition rate was 76 MHz and the pulse width was 170 femtoseconds (fs). The time-resolved autofluorescence was recorded simultaneously in 16 spectral channels (445-605 nm) and bi-exponentially fitted.
  
  
  RESULTS
  RPE can be discriminated clearly from Bruch's membrane, drusen, and choroidal connective tissue by fluorescence lifetime. In RPE, bright fluorescence of lipofuscin could be detected with a maximum at 510 nm and extending beyond 600 nm. The lifetime was 385 ps. Different types of drusen were found. Most of them did not contain lipofuscin and exhibited a weak fluorescence, with a maximum at 470 nm. The lifetime was 1785 picoseconds (ps). Also, brightly emitting lesions, presumably representing basal laminar deposits, with fluorescence lifetimes longer than those recorded in RPE could be detected.
  
  
  CONCLUSIONS
  The demonstrated differentiation of fluorescent structures by their fluorescence decay time is important for interpretation of in vivo measurements by the new fluorescence lifetime imaging (FLIM) ophthalmoscopy on healthy subjects as well as on patients.
authors.affiliation: Department of Ophthalmology, University Hospital Jena, Jena, Germany. schweitzer@med.uni-jena.de
authors.forename: Dietrich
authors.initials: D
authors.lastname: Schweitzer
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Gaillard
authors.forename: James
authors.initials: J
authors.lastname: Dillon
authors.forename: Robert F
authors.initials: RF
authors.lastname: Mullins
authors.forename: Stephen
authors.initials: S
authors.lastname: Russell
authors.forename: Birgit
authors.initials: B
authors.lastname: Hoffmann
authors.forename: Sven
authors.initials: S
authors.lastname: Peters
authors.forename: Martin
authors.initials: M
authors.lastname: Hammer
authors.forename: Christoph
authors.initials: C
authors.lastname: Biskup
mesh_terms: D000369:Aged, 80 and over; D005260:Female; D005453:Fluorescence; D006801:Humans; D008062:Lipofuscin / Q000378:metabolism*; D018613:Microscopy, Confocal; D008875:Middle Aged; D012160:Retina / Q000378:metabolism*; D015593:Retinal Drusen / Q000378:metabolism* / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014019:Tissue Donors
chemical_list: D008062:Lipofuscin