Application of an ultra fast single photon camera for spatial imaging of fast cellular events

Recent advances in microscopy have enabled the activities of living cells to be studied with unprecedented detail. These advances include the development of fluorescent markers that can be used to label particular structures, chemical messengers and ion concentrations in cells, together with the development of techniques such as confocal microscopy that remove unwanted information such as out-of-focus blur from the microscope image. Many cellular processes result from the coordinated activity of a diverse array of molecules, such as ion channels in the cell membranes, the components of the cell's skeleton and molecules involved in secretion and membrane turnover. Many of these interactions occur very fast, in multiple locations in the cell and on time scales of milliseconds or less. Current microscopy techniques are generally not fast or sensitive enough to record them. Recent developments in camera and detector technology are producing a generation of ultra-fast and ultra-sensitive cameras that should provide much-needed solutions for imaging of very fast cellular events. This project will adapt such a camera for imaging of fast sub-cellular events in a model cellular system. The potential application of the technology to a wider range of microscopy applications will be assessed.

Fundamentally important cellular processes such as ion channel opening and exocytotic vesicle secretion typically occur over timescales of milliseconds or less. Advances in fluorescence imaging have greatly improved understanding of the interactions involved in such activities. However, further understanding is becoming increasingly limited by the speed and sensitivity of available detectors. For example, acquiring fast spatio-temporal information with confocal and 2-photon microscopy is limited by scan speed and overall efficiency of the microscope-detector system. Pixel detectors that can combine single photon counting capability with very fast readout can potentially eliminate the trade-off between spatial resolution and acquisition speed. The aim is to equip a fluorescence microscope with an ultra fast single photon counting camera based on the deposition of n-doped, intrinsic and p-doped (n-i-p) hydrogenated amorphous silicon sensor (a-Si:H) film on top of an application-specific integrated circuit (ASIC) within an electron bombarded phototube device. The performance of the camera for monitoring fluorescence from flagella of the model organism Chlamydomonas will provide a suitable test system. This will build on the experience of the MBA in monitoring cellular calcium with fluorescent indicators. Moreover, flagella are sufficiently thin not to require optical sectioning to achieve high-resolution images. Moreover they display fast and localised changes in the second messenger calcium. The proposed work will also provide a platform for assessing the suitability for further development with optical sectioning, applications such as TIRF and fast deconvolution together with further developments in fast readout technology, for imaging of multiple fast cellular events such as membrane ion channel activity and vesicle secretion on millisecond time-scales. These developments will be pursued in further proposals.