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STORM below the localisation limit

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Localisation-based imaging methods such as stochastic optical reconstruction microscopy (STORM) and photoactivatable localization microscopy have allowed fluorescence imaging with a resolution of tens of nm. These methods have two limitations. First, the resolution is limited by the number of photons emitted in one frame, so even if a fluorophore appears in more than one frame the localisation accuracy is not improved. Secondly, since the image is reconstructed from single fluorophore localisations, only a very small number of fluorophores can be in an emitting state in each frame. This results in long imaging times, and the data from overlapping fluorophores being discarded. Here we describe how these limitations can be overcome with Bayesian analysis by modelling the entire dataset as being generated by a number of blinking fluorophores. This means that a single fluorophore can contribute to the data in many frames, improving the localisation. This technique can also deal with a large number of overlapping fluorophores, decreasing the number of frames required to form an image. Using this new method we are able to demonstrate resolution as fine as 6 nm on biological samples with non-photoswitchable fluorophores.

This talk is part of the Physics of Medicine (PoM) Seminar Series series.

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