Calcium-sensitive fluorescent lifetime imaging using wide field time-resolved detection of emission from optical sections excited by light sheet illumination

Calcium sensitive dyes are valuable tools in the study of intracellular Ca+ signalling. But application of these sensors using conventional wide field fluorescence intensity imaging is limited in model organisms, embryos and other moderately thick tissues by blurring due to detection of emitted light from outside the focal plane, unproductive photobleaching above and below the focal plane as well as depth-dependent variation in intensity. In order to overcome these difficulties we are combining wide field fluorescence lifetime imaging which is intensity-independent with planar illumination orthogonal to the axis of detection. We will demonstrate the accurate calibration of Ca+ sensitivity of the fluorescence lifetime of Oregon Green Bapta1 in buffer systems and its utility in detection of rapid Ca+ dynamics in vivo. In this implementation of our Single Plane Illumination Microscope (SPIM), we are testing a novel detector, the Capacitive Division Imaging Readout (CDIR) detector, consisting of a Microchannel Plate (MCP) vacuum detector with spatial resolution (102 pixels) and timing resolution of the order of 100 ps. The CDIR detector, capacitively couples the charge output of the MCP between four readout nodes. The charge collected at each node is then measured using the NINO ASIC, a charge sensitive amplifier/discriminator, and the HPTDC ASIC, a high time resolution time-to-digital convertor, allowing the charge position to be calculated using a centroiding imaging algorithm.