Picosecond wide-field time-correlated single photon counting fluorescence microscopy with a delay line anode detector

Liisa M. Hirvonen, Wolfgang Becker, James Milnes, Thomas Conneely, Stefan Smietana, Alix Le Marois, Ottmar Jagutzki, Klaus Suhling

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13 Citations (Scopus)

Abstract

We perform wide-field time-correlated single photon counting-based fluorescence lifetime imaging (FLIM) with a crossed delay line anode image intensifier, where the pulse propagation time yields the photon position. This microchannel plate-based detector was read out with conventional fast timing electronics and mounted on a fluorescence microscope with total internal reflection (TIR) illumination. The picosecond time resolution of this detection system combines low illumination intensity of microwatts with wide-field data collection. This is ideal for fluorescence lifetime imaging of cell membranes using TIR. We show that fluorescence lifetime images of living HeLa cells stained with membrane dye di-4-ANEPPDHQ exhibit a reduced lifetime near the coverslip in TIR compared to epifluorescence FLIM.

Original languageEnglish
Article number071101
JournalApplied Physics Letters
Volume109
Issue number7
DOIs
Publication statusPublished - 15 Aug 2016
Externally publishedYes

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