Volumetric and time-sequenced live-cell imaging

This proposal aims to develop, customise and demonstrate a new technique for imaging dynamic 3-dimensional processes in living cells that may be either unstained or imaged using fluorescent markers. The technique proposed will achieve wide-field, 3-D imaging with a time-sequence capability, leading to a 3-D 'movies' of live cells that can be obtained with minimal photo-damage to those cells.Our current view of many processes occurring in cells is based on static, snapshot views of dead cells. Increasingly, cell-biologists with to study dynamic events occurring in living cells. Whatever imaging technique is used to obtain this information, the effects of the toxicity of stains and of photo-damage (which may be accentuated by the generation of free radicals during the photobleaching of flurophores) must be considered. Although techniques like confocal scanning microscopy are widely and successfully used, some experiments where photo-damage is a problem are still better performed using wide-field imaging techniques.This proposal is for the use of a passive 3-dimensional imaging technique based on the use of diffractive optical elements. This technique can provide flux-efficient (i.e. low damage) 'movies' of living cells using stains or flurophores, but also through 3-D phase-contrast imaging of unmodified cells. Through collaboration between Physicists and two live-cell imaging groups, we intend to demonstrate the techniques.