We present a technique that records transient changes in the concentration of free calcium in live neurons by TCSPC FLIM. The sample is incubated with a calcium-sensitive dye. To measure the temporal change in the calcium-ion concentration the sample is periodically stimulated by an electrical signal and scanned at high image rate with a high-frequency pulsed laser beam. Single photons of the fluorescence light are detected, and a photon distribution over the coordinates of the scan, the arrival times of the photons after the excitation pulses, and the time after the stimulation pulses is built up. The result can be interpreted as a sequence of FLIM images for different times after the stimulation pulses. The signal-to-noise ratio only depends on the available photon rate and the total acquisition time, not on the speed of the sequence. The maximum resolution at which lifetime changes can be recorded is given by the frame rate of the scanner which is currently 38 ms. Faster changes can be recorded by line scanning. Transient lifetime effects can then be resolved at a resolution of about one millisecond.
Wolfgang Becker and Samuel Frere, "Imaging of calcium transients in cultured neurons by TCSPC FLIM (Conference Presentation)," Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 97120O (Presented at SPIE BiOS: February 15, 2016; Published: 28 April 2016); https://doi.org/10.1117/12.2214083.4848767238001.
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