Method for intracellular imaging of ion concentrations using confocal microscopy and fluorophore lifetimes

Kjell Carlsson; Anders Liljeborg; Ronnie M. Andersson; Hjalmar Brismar

doi:10.1117/12.384198

2 May 2000 Method for intracellular imaging of ion concentrations using confocal microscopy and fluorophore lifetimes

Kjell Carlsson, Anders Liljeborg, Ronnie M. Andersson, Hjalmar Brismar

Proceedings Volume 3919, Three-Dimensional and Multidimensional Microscopy: Image Acquisition Processing VII; (2000) https://doi.org/10.1117/12.384198
Event: BiOS 2000 The International Symposium on Biomedical Optics, 2000, San Jose, CA, United States

Abstract

There exist a number of fluorescent probes whose lifetimes change in response to ion concentrations (for example H⁺ and Ca²⁺) in the surrounding medium. We describe a technique for utilizing this property in a confocal scanning laser microscope. The technique is based on intensity-modulated laser illumination of the specimen, and phase-sensitive lock-in detection of the fluorescent light. In this way we get a lifetime-dependent output signal which, after calibration, can provide information concerning ion concentrations. In the current study we have used a pH sensitive fluorophore, SNAFL-2, to study the performance of this technique. We find that the sensitivity is such that a pH difference of 0.1 units can easily be detected in an 8- bit digital image. Noise measurements show that under realistic conditions we can expect a pixel-to-pixel standard deviation of approximately one to two pH units.

Citation Download Citation

Kjell Carlsson, Anders Liljeborg, Ronnie M. Andersson, and Hjalmar Brismar "Method for intracellular imaging of ion concentrations using confocal microscopy and fluorophore lifetimes", Proc. SPIE 3919, Three-Dimensional and Multidimensional Microscopy: Image Acquisition Processing VII, (2 May 2000); https://doi.org/10.1117/12.384198

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