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Monitoring intracellular nanomolar calcium using fluorescence lifetime imaging


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 BAPTA1 (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 2 d, and OGB-1 loading, imaging, and data analysis take 2 d.

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Figure 1: Schematic of the time-resolved imaging system.
Figure 2: Photon count versus free calcium calibration and experimental noise assessment.
Figure 3: FLIM readout variability assessment in vitro and in situ for Ca2+-independent indicators and OGB-1.
Figure 4: Illustration of a normalized total count (NTC) technique to map intracellular [Ca2+] in the plane of view.
Figure 5: Improving the Ca2+ signal dynamic range and the signal-to-noise ratio using the gated-intensity approach.
Figure 6: Suppression of artifactual autofluorescence using the tail-gated intensity approach.


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This work was supported by a Wellcome Trust Principal Fellowship (101896), a European Research Council Advanced Grant (323113 NETSIGNAL), a Russian Science Foundation grant (15-14-30000, computing cluster setup), grant FP7 ITN (606950 EXTRABRAIN), and a European Research Council Proof-of-Concept Grant (767372 NEUROCLOUD) to D.A.R.

Author information




K.Z. designed and set up the optical system arrangements and software, and analyzed the data; K.Z. and D.A.R. conceived and designed the experiments; T.P.J. and K.Z. performed the experiments; and K.Z., T.P.J., and D.A.R. wrote and illustrated the manuscript.

Corresponding authors

Correspondence to Kaiyu Zheng or Dmitri A Rusakov.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Optimized protocol to prepare calibration solution battery.

See Supplementary Methods for details.

Supplementary Figure 2 Half-gated NTC calibration for improved monitoring of [Ca2+] fluctuations.

Normalised total count values (open circles) obtained by using the integrated count over the 5-10 ns post-pulse interval in the fluorescence decay, normalised against the count peak value (Fig. 5c). Red solid line, best-fit logistic function (as in Fig. 2b); reduced χ2, 6.5278∙10−6; adjusted R2, 0.997.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, and the Supplementary Methods. (PDF 513 kb)

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Zheng, K., Jensen, T. & Rusakov, D. Monitoring intracellular nanomolar calcium using fluorescence lifetime imaging. Nat Protoc 13, 581–597 (2018).

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