Final Report Summary - TOTALPHOTON (A Total Photon Camera for Molecular Imaging of Live Cells)
Our use of this next generation camera technology has focussed on microscopy, exploiting the unique property of being able to sum the binary frames noiselessly to create conventional images with much greater dynamic range. The field of super-resolution imaging is burgeoning, with a Nobel Prize in 2014 and finally the tools being made available to biologists to ‘see’ on the molecular level in living cells. A key to driving this forward is that current detectors are limited in terms of frame rate, photon counting, quantum efficiency etc. Out TotalPhoton detector(s) address these limitations head-on and we have already applied our technology to a single-molecule localisation microscopy called dSTORM (direct Stochastic Optical Reconstruction Microscopy) which captures short ‘blinks’ of fluorescent (photon) activity from single molecules to identify their location at resolutions far beneath the diffraction limit that prevents conventional microscopy from achieving ultra-high image resolution. The TotalPhoton camera’s exceptionally high frame rate allows selective summation of photons from within the duration of the millisecond molecule blink (rather than the background), which combined with a novel analysis approach, has significantly improved the localisation accuracy. Computer modelling has allowed us to better understand the possible enhancements offered by the TotalPhoton platforms to dSTORM applications and promises further advances as the project continues.
Moving forward, our ultrafast SPAD detectors provide additional information on the exact time of photon arrivals from cellular fluorescent activity. This allows for a highly sensitive imaging method called fluorescence lifetime imaging (FLIM), where the cellular image is constructed from photon arrival times, not number of photons. Inherently a slow technique due to limits of existing detectors, TotalPhoton will significantly increase imaging speed and quality, and allow, for the first time, the combination of superresolution dSTORM and FLIM imaging. As a result molecule position, species and interactions can now be studied in real time with ultra-high resolution, opening up new avenues for the biochemists and biophysicists in our project.
TotalPhoton cameras have already achieved the highest resolution and sensitivity of any SPAD camera at the target Gigaphoton/s rates. With upcoming advances in optical efficiency, by using 1000’s of miniature lenses to better couple light to the detectors and cooling, to better control the camera background signal, our next generation TotalPhoton cameras will provide a step change for cellular imaging applications and lay a new benchmark for what can be achieved in optical microscopy.