Quantitative microscopy based on photon statistics (PhotoQuant)

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Quantitative information is key to understand the complex inner workings of living organisms on the molecular scale. Methods that are able to precisely determine copy numbers of different molecular species making up a structure or involved in a reaction are thus required. Ideally, such methods should provide absolute numbers, be minimally invasive enabling in situ measurements in living specimen as well as non-destructive and fast to allow for repeated quantification of dynamic complexes. Different approaches for molecular counting based on fluorescence microscopy exist, however, none of the existing methods is able to fulfill all of the aforementioned requirements at once. We have shown that individual fluorescent emitters in a diffraction-limited structure can be counted with high precision by recording and analyzing photon emission statistics from that structure (counting by photon statistics - CoPS). So far, the CoPS method enables non-destructive measurements with high time resolution and high counting range in vitro.To enable in situ counting of biomolecules, we propose to extend the CoPS methodology to account for the increased complexity of dynamic biological specimen. First, we plan to extend the currently implemented one-color point measuring scheme to allow two-color simultaneous imaging and quantification based on a pulsed interleaved excitation scheme. These works will include modifying an existing microscope setup, the microscope control and data readout electronics as well as adapting the data analysis routine. We then want to address special requirements of photon statistics measurements on non-ideal samples, i.e. elevated background fluorescence and potential interaction between individual emitters by means of sample optimization, measurements on defined structures and simulations.The established approaches will then be used to investigate two biological model systems, namely the T-cell receptor cluster upon presence of the HIV effector protein Nef and the complex formation of interferon alpha receptors and their associated kinases. Fluorescent labeling of the target proteins will be achieved using enzyme tags such as HaloTag while post-translationally modified proteins will be labeled via monoclonal antibodies. To relate the number of counted fluorophores to the underlying number of target molecules, a thorough characterization of the achieved degree of labeling will be performed.Finally, we want explore technical possibilities to extend point-based CoPS towards imaging CoPS using camera detectors. This will involve a proof-of-principle study based on an emCCD camera to characterize defined samples in imaging mode as well as establishing a theoretical model for spatially-resolved photon statistic data evaluation. Furthermore, we want to evaluate the potential of alternative detection schemes such as APD arrays for imaging CoPS with higher time resolution.