Journal
CELL CALCIUM
Volume 40, Issue 1, Pages 73-79Publisher
CHURCHILL LIVINGSTONE
DOI: 10.1016/j.ceca.2006.03.006
Keywords
calcium imaging; two-photon microscopy; brain slices; fluorescence lifetime
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Two-photon microscopy (TPM) revolutionized Ca2+ imaging by allowing recordings in the depth of intact tissue and live organisms. A serious limitation in TPM, however, is the lack of an accurate and straightforward approach for the quantification of Ca2+ signals, an ability that became an invaluable tool in fluorescence microscopy. Here, we present time-correlated fluorescence lifetime imaging (tcFLIM) as a ratiometric method for the quantification of Ca2+ signals in TPM. The fluorescence lifetime of the Ca2+ indicator dye Oregon Green BAPTA-1 (OGB-1) can be recorded using the similar to 80 MHz excitation pulses utilized in TPM. It shows a Ca2+ dependence that can be explained by the Ca2+-affinity, spectral properties and purity of the dye. Pixel-wise lifetime recordings, controlled by a laser-scanning microscope, allowed quantitative Ca2+ imaging in full-frame and linescan mode. Although we focused on the high-affinity Ca2+ indicator OGB-1. our tcFLIM-based quantification is applicable to other Ca2+ dyes and to fluorescence indicators in general. (c) 2006 Elsevier Ltd. All rights reserved.
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