2 May 2000 Method for intracellular imaging of ion concentrations using confocal microscopy and fluorophore lifetimes
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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.
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Kjell Carlsson, Kjell Carlsson, Anders Liljeborg, Anders Liljeborg, Ronnie M. Andersson, Ronnie M. Andersson, Hjalmar Brismar, 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); doi: 10.1117/12.384198; https://doi.org/10.1117/12.384198
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