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Proceedings Paper

pH and chloride recordings in living cells using two-photon fluorescence lifetime imaging microscopy
Author(s): Mattes Lahn; Carsten Hille; Felix Koberling; Peter Kapusta; Carsten Dosche
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Paper Abstract

Today fluorescence lifetime imaging microscopy (FLIM) has become an extremely powerful technique in life sciences. The independency of the fluorescence decay time on fluorescence dye concentration and emission intensity circumvents many artefacts arising from intensity based measurements. To minimize cell damage and improve scan depth, a combination with two-photon (2P) excitation is quite promising. Here, we describe the implementation of a 2P-FLIM setup for biological applications. For that we used a commercial fluorescence lifetime microscope system. 2P-excitation at 780nm was achieved by a non-tuneable, but inexpensive and easily manageable mode-locked fs-fiber laser. Time-resolved fluorescence image acquisition was performed by objective-scanning with the reversed time-correlated single photon counting (TCSPC) technique. We analyzed the suitability of the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE for recordings in an insect tissue. Both parameters are quite important, since they affect a plethora of physiological processes in living tissues. We performed a straight forward in situ calibration method to link the fluorescence decay time with the respective ion concentration and carried out spatially resolved measurements under resting conditions. BCECF still offered only a limited dynamic range regarding fluorescence decay time changes under physiologically pH values. However, MQAE proofed to be well suited to record chloride concentrations in the physiologically relevant range. Subsequently, several chloride transport pathways underlying the intracellular chloride homeostasis were investigated pharmacologically. In conclusion, 2P-FLIM is well suited for ion detection in living tissues due to precise and reproducible decay time measurements in combination with reduced cell and dye damages.

Paper Details

Date Published: 26 February 2010
PDF: 7 pages
Proc. SPIE 7569, Multiphoton Microscopy in the Biomedical Sciences X, 75690U (26 February 2010); doi: 10.1117/12.840881
Show Author Affiliations
Mattes Lahn, Univ. of Potsdam (Germany)
Carsten Hille, Univ. of Potsdam (Germany)
Felix Koberling, PicoQuant GmbH (Germany)
Peter Kapusta, PicoQuant GmbH (Germany)
Carsten Dosche, Univ. of Potsdam (Germany)

Published in SPIE Proceedings Vol. 7569:
Multiphoton Microscopy in the Biomedical Sciences X
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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