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Content archived on 2024-06-18

A Total Photon Camera for Molecular Imaging of Live Cells

Final Report Summary - TOTALPHOTON (A Total Photon Camera for Molecular Imaging of Live Cells)

TotalPhoton aims to construct a high-resolution camera capable of imaging the time-of-arrival, polarisation and wavelength of each of the maximal 10 Gigaphoton emitted from a labelled, biological cell every second. Capturing this complete optical fingerprint will significantly enhance our ability to observe the organisation, movement and interactions of cellular components at the fundamental molecular scales, enhancing our understanding of disease, function and the very mechanisms responsible for everyday life. In the first period of the project, we have tackled the central challenge of designing a camera operating at a sufficient speed and sensitivity to process every photon individually, requiring rates 10-100 thousand times faster than existing technology. Our camera is founded on silicon CMOS technology (the same as used in mobile phone cameras and microprocessors) but employs a unique, ultra-high speed, ultra-sensitive light sensor known as a SPAD (single photon avalanche diode). It operates at 100 thousand frames per second and generates binary images composed of black or white pixels which indicate not only the presence or absence of single photons (individual packets of quantised light) but the actual time they hit the detector. This incredible ability, to detect the arrival time of single packets of light, at such incredible speeds, provides an entirely new avenue to cutting edge imaging applications.
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.