Double‐pulse fluorescence lifetime imaging in confocal microscopy

M. MÜLLER, R. GHAUHARALI, K. VISSCHER, G. BRAKENHOFF

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides high time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non‐steady‐state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime determination. 1995 Blackwell Science Ltd

Original languageEnglish (US)
Pages (from-to)171-179
Number of pages9
JournalJournal of Microscopy
Volume177
Issue number2
DOIs
StatePublished - Feb 1995
Externally publishedYes

Keywords

  • CCD
  • Fluorescence
  • bilateral scanning
  • confocal microscopy
  • lifetime imaging
  • photobleaching
  • photodegradation
  • ratio‐imaging
  • saturation

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology

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