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Liisa Hirvonen edited Introduction.tex
over 8 years ago
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A characteristic feature of the photon counting imaging technique is the possibility of calculating the true position of a photon event that covers several pixels with subpixel accuracy.\cite{Suhling2002,Suhling1999,Boksenberg1985} Originally developed for implementation in hardware and based on a simple center-of-mass calculation,\cite{Boksenberg1985} the centroiding is nowadays done in software but the algorithms employed in photon counting imaging are still usually simple, one-iteration algorithms.\cite{Postma2011}.
The discovery of photoswitchable and photoactivatable fluorophores in the past decade has allowed the same centroiding principle to be employed in circumventing the diffraction limit in fluorescence microscopy. Single-molecule localization fluorescence microscopy techniques are based on the activation of a small subpopulation of the fluorophores used to stain the sample. They are imaged and subsequently deactivated before the process is repeated with a different subset of fluorophores.\cite{Betzig2006,Rust2006} The centroid positions of the molecules are calculated, and the final image is formed by summing many frames. Single-molecule localization fluorescence microscopy is now a well-established technique, and much effort has been put into the development and optimization of many different types of centroiding algorithms, including iterative fitting
algorithms. algorithms.\cite{Small2014}
As recently reported, single-molecule localisation algorithms produce good results when applied to centroiding single photon events imaged with an MCP-intensified CMOS camera.\cite{Hirvonen2015_OL} Here, we extend this work and apply super-resolution software for centroiding photon events detected with an EBCCD camera. Moreover, multi-emitter fitting analysis was also implemented for separating overlapping photon events, an important aspect not reported before, which allows an increased count rate and shorter acquisition times.