Abstract
Two-photon microscopy (TPM) revolutionized Ca 2+ 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 Ca 2+ 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 Ca 2+ signals in TPM. The fluorescence lifetime of the Ca 2+-indicator dye Oregon Green BAPTA-1 (OGB-1) can be recorded using the ∼80 MHz excitation pulses utilized in TPM. It shows a Ca 2+ dependence that can be explained by the Ca 2+-affinity, spectral properties and purity of the dye. Pixel-wise lifetime recordings, controlled by a laser-scanning microscope, allowed quantitative Ca 2+ imaging in full-frame and linescan mode. Although we focused on the high-affinity Ca 2+ indicator OGB-1, our tcFLIM-based quantification is applicable to other Ca 2+ dyes and to fluorescence indicators in general.
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