Recording Ca ++ Transients in Neurons by TCSPC FLIM

Multi-dimensional time-correlated single photon counting (TCSPC) techniques are able to record Ca++ transients in live neurons via the fluorescence lifetime changes of a Ca++-sensitive dye. The technique is based on periodical stimulation of the sample, raster scanning, or line scanning with a high-frequency pulsed laser, and multi-dimensional TCSPC. The recording process builds up a photon distribution over the spatial coordinates of the scan, the times of the photons in the laser pulse period, and the times of the photons in the stimulation period. The transient-time resolution is about 40 ms for raster scanning and about 1 ms for line scanning. We demonstrate the technique for electrical stimulation of cultured neurons incubated with Oregon Green Bapta.