The application of the capacitive division technique to wide-field time-resolved fluorescence microscopy

Capacitive division and other charge-sharing techniques have become ubiquitous within modern technology. Almost all touchscreen devices depend on some form of charge sharing mechanism. The Capacitive-Division Imaging Readout, C-DIR, scheme developed for space/astronomy applications, is a proven concept which has benefited from widespread publication and several iterations of prototyping. In this study, we borrowed this idea and assessed its application in the field of life sciences, specifically, fluorescence lifetime imaging microscopy (FLIM). Firstly, the composite C-DIR camera system was developed using a prototype anode developed by Lapington et al in combination with advanced photomultiplier tube technology developed by Photek Limited, and ultra-fast NINO ASIC and high performance time-to-digital converter, HPTDC, readout electronics developed by CERN. Several issues like signal noise, timing jitter and image distortion required special attention to successfully tune the C-DIR system for obtaining FLIM measurements. The C-DIR was characterized in the context of current detector technologies used for time-resolved applications. The maximum achievable global event rate was determined to be a USB 2.0 hard limit of about 1MHz. The spatial resolution and timing performance were identified as 0.5 line-pairs/mm and 200ps FWHM, respectively, which was comparable to other wide-field fluorescence lifetime cameras. These results provided the basis for using the system in a real situation. Before this was possible, however, it was necessary to engineer a bespoke software platform for data acquisition which could cope with the data rates and reduce raw data emerging from the C-DIR system, producing a format compatible with widely used fitting software. The final stage of the project involved using the C-DIR for real science by reproducing real world experiments which allow for a fitness test of the system in the field. The first experiment involved a calcium calibration where the C-DIR system was calibrated using FLIM on a series of calcium buffers of known concentrations. This C-DIR was able accurately recover the lifetime values from the calcium buffers. The second shorter experiment involved using the calibrated system for the quantification of calcium within living tissue samples using fluorescence lifetime imaging. Results were consistent with those published in the literature which solidified the position of the C-DIR as a viable option for time-resolved fluorescence microscopy.