Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis.
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 approximately 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.
Wilms
Christian D
CD
Leipzig University, Carl-Ludwig-Institute for Physiology, Liebigstr. 27, 04103 Leipzig, Germany. christian.wilms@medizin.uni-leipzig.de
Schmidt
Hartmut
H
Eilers
Jens
J
D002118:Calcium; D020013:Calcium Signaling / Q000502:physiology; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D017785:Photons
D002118:Calcium