期刊
NATURE PROTOCOLS
卷 13, 期 3, 页码 581-597出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2017.154
关键词
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资金
- Wellcome Trust Principal Fellowship [101896]
- European Research Council Advanced Grant [323113 NETSIGNAL]
- Russian Science Foundation [15-14-30000]
- grant FP7 ITN [606950 EXTRABRAIN]
- European Research Council Proof-of-Concept Grant [767372 NEUROCLOUD]
- Russian Science Foundation [15-14-30000] Funding Source: Russian Science Foundation
Nanomolar-range fluctuations of intracellular [Ca2+] are critical for brain cell function but remain difficult to measure. We have advanced a microscopy technique to monitor intracellular [Ca2+] in individual cells in acute brain slices (also applicable in vivo) using fluorescence lifetime imaging (FLIM) of the Ca2+-sensitive fluorescent indicator Oregon Green BAPTAAPTAAPTAAPTA1 (OGB-1). The OGB-1 fluorescence lifetime is sensitive to [Ca2+] within the 10-500 nM range but not to other factors such as viscosity, temperature, or pH. This protocol describes the requirements, setup, and calibration of the FLIM system required for OGB-1 imaging. We provide a step-by-step procedure for whole-cell OGB-1 loading and two-photon FLIM. We also describe how to analyze the obtained FLIM data using total photon count and gated-intensity record, a ratiometric photon-counting approach that provides a highly improved signal-to-noise ratio and greater sensitivity of absolute [Ca2+] readout. We demonstrate our technique in nerve cells in situ, and it is adaptable to other cell types and fluorescent indicators. This protocol requires a basic understanding of FLIM and experience in single-cell electrophysiology and cell imaging. Setting up the FLIM system takes similar to 2 d, and OGB-1 loading, imaging, and data analysis take 2 d.
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