Unlocking the potential of single-photon wide-field microscopy

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This project aims at transforming wide-field microscopy by adding single photon capabilities that are so far only available in confocal microscopy. We will use the latest generation of single photon sensitive imaging sensors with improved photon detection efficiency and embedded data processing circuits to enable the recording of single photons at MHz rate with picosecond accuracy. This will significantly reduce the readout noise and enable different new imaging modes in wide-field microscopy such as fluorescence lifetime imaging and photon correlation imaging which can be used for multiplexing or molecular counting. In this project, we will focus on improving different super-resolution microscopy techniques for imaging fixed and living cells. For instance, the localisation precision in single-molecule localisation microscopy (SMLM) will benefit not only from the reduced noise levels but also from the possibility to distinguish and reject instantly scattered light from the delayed light emission of the fluorescent labels. Moreover, the embedded photon histogramming capabilities will allow to implement fluorescence lifetime-based multiplexing and thereby increase the number of different labels that can be used in a sample. We will also explore the potential for probing molecular interactions in cells using single-molecule Forster Resonance Energy Transfer. Overall, we expect that this will improve the achievable resolution, add inherent quantitative information to the image data and increase the number of proteins that can be simultaneously imaged. We will explore the application of single-photon SMLM by imaging protein complexes of tetraspanins in the plasma membrane of fixed cells.Furthermore, we want to increase the spatial resolution of super-resolution optical fluctuation imaging (SOFI) and enhance its capabilities in live-cell super-resolution microscopy. Here, the high time resolution of the single photon sensitive imaging sensor will allow us to extend the timescale that can be used for correlating the intensity fluctuations in SOFI. This is very relevant because the majority of optical fluctuations occur on the microsecond timescale which is not covered by current imaging sensors such as EMCCD and sCMOS cameras. Thereby, we expect a significant improvement in the achievable resolution and at the same time a better separation of fluorophores with different properties. We also expect to overcome a major limitation in SOFI by increasing the number of fluorescent labels that can be used in SOFI experiments. Like for single-photon SMLM, we will implement time gating and fluorescence lifetime multiplexing to further reduce noise and increase the number of probes that can be simultaneously imaged. We will explore the suitability of single-photon SOFI by imaging visualising the same protein complexes of tetraspanins and their dynamics in the plasma membrane of living cells.Overall, we expect that the single-photon super-resolution microscopy developed in this project will significantly improve the achievable spatial resolution due to a significantly reduced noise level and rejection of immediate scattering. At the same time, single-photon wide-field microscopy will enable additional imaging modes such as fluorescence lifetime imaging microscopy (FLIM) which will increase the number of simultaneously imaged targets, and photon correlation imaging which will enable quantitative molecular imaging as the first quantum imaging technique in wide-field fluorescence microscopy. The successful development of single-photon super-resolution microscopy will be door opener for other imaging modes such as fluorescence correlation imaging of fast molecular processes. In summary, this will lead to a step change in wide-field microscopy with great prospect to transform the way we can image molecular scale structures and processes in the life sciences and in biomedical research.