A fourdimensional microscope for single molecule studies in living cells using astigmatic axial resolution enhancement

An important part of life is to react to the environment and adapt to it. All living cells rely on chemical cascades that form signal pathways, from stimulus to transcription events at the DNA. To know more about these pathways we need tools to visualise the signals. Here is outlined developmental and experimental work on a bespoke microscope system primarily for fast single molecule tracing of proteins in living eukaryote cells. Both hardware and software has been developed. Frame rates and exposure times have been pushed to match the speed of freely diffusing particles in living cells. An EMCCD camera with an electronic gain of 300x and a 5 ms exposure time at 200 frames per second enables this. An astigmatic axial resolution enhancement approach has been implemented to allow tracking with high precision of single fluorescent proteins in real-time in three dimensions in a comparatively large volume. The high resolution in three spatial dimensions and one temporal aim to meet these demands. Software to extract the additional axial information has been developed that can determine axial displacement with 38 nm precision. The microscope system has proven it can find and track small clusters of YPet fluorescent protein in E. coli at short exposure times.