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.

Fluorescence lifetime imaging microscopy (FLIM) is a tool that has become of important use to many biologists and medical researchers. It allows for the imaging of fluorescent markers within cells and as well their decay lifetime information. This lifetime information yields useful information about the chemical properties of the local region surrounding the fluorophore marker. It is therefore desirable to be able to measure this information and localise it to produce either a 2D or 3D image stack of the lifetimes and intensity information. Two major problems must be overcome to achieve good resolution, both spatially and temporally. There must be a method to provide optical depth sectioning and the imaging must be fast. Sectioning can be done with confocal or two photon microscopes however, these are point scanning devices and are relatively slow.
We will detail a new microscope system based on light sheet illumination that uses a micro channel plate (MCP) device called a Capacitive Division Imaging Readout (CDIR) which has been developed by Photek Ltd. The device uses an array of capacitors to move the charge site from the MCP to four pre-amplifiers and time- over-threshold discriminators. This camera has the ability to image photons as well as measure the arrival time, enabling high speed FLIM imaging of biological samples. The optical sectioning is achieved by using a diffraction limited sheet of light, that illuminates the sample from the side in the plane orthogonal to the objectives optical axis.
We will present the first results taken from the microscope.

Measuring the spatial position and lifetime distribution of the fluorophores enables users to discern vital information about the chemical environment within a biological system. It however, remains challenging since the photon count is usually extremely limited, requiring a high degree of efficiency within the system. The timing precision to measure the lifetimes are of the order of picoseconds. Traditional methods to achieve two dimensional images involve point scanning with a confocal or two photon microscope and collecting the photon timings with a point collector. This has the downside of being comparatively slow. High speed, photon timing counters with spatial imaging capabilities therefore offer a desirable option to decrease measurement times.
We have developed a new solution to this problem. Our system side illuminated our sample with a sheet of excitation light produced by a pulsed laser diode at 470nm. The imaging path is then performed by two cameras: a high resolution EMCCD captures an intensity image allowing for alignment of the sample. The second path contains a multichannel plate (MCP) photomultiplier.
To read the charge, time and position from the MCP remains a challenge. We have demonstrated an alternative technology; a Capacitive Division Imaging Readout (C-DIR) which has been developed by Photek Ltd. The device uses an array of capacitors to move the charge site from the MCP to four preamplifiers and time-over-threshold discriminators.
The work here will present data on the measured frame rate,point spread function, and spatial and temporal resolution of the device. The microscope shows a excellent degree of time resolution and temporal repeatability. The spatial imaging exhibits some degree of geometric distortion and post measurement correction methods have been implemented to reduce this. Images of biological samples will be presented showing the two dimensional life time imaging of the system along with measurements of FRET.

Fluorescence lifetime imaging microscopy (FLIM) has allowed scientists to discern information about the chemical properties of biological processes and has become a vital tool in the life sciences and medical research communities. Measuring the spatial lifetime distribution of the fluorophores as well as the intensity distribution enables users to discern vital information about the chemical environment. It however, remains challenging and often involves slow scanning. We present a new microscope system based on light sheet illumination that uses a micro channel plate (MCP) device called a Capacitive Division Imaging Readout (CDIR) which has been developed by Photek Ltd. The device uses an array of capacitors to move the charge site from the MCP to four pre-amplifiers and time-over-threshold discriminators. This camera has the ability to image photons as well as measure the arrival time, enabling high speed FLIM imaging of biological samples.