Liisa Hirvonen edited Introduction.tex  almost 9 years ago

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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; importance of this field was recognized with the 2014 Nobel Prize in Chemistry awarded “for the development of superresolved fluorescence microscopy.” Single-molecule localization microscopy techniques are based on the activation of a small subpopulation of the fluorophores which can then be imaged and subsequently deactivated before the process is repeated with a different subset of fluorophores. Rather than detecting a single photon, the detected event consists of many photons that are emitted from a single fluorophore with a size much smaller than the resolution of the microscope, but similar to photon counting imaging, the centroid positions of the molecules are calculated, and the final image is formed by summing many frames.  Single-molecule localization microscopy is now a well-established technique, and a variety of free software is available for image reconstruction. As the usefulness of this method crucially depends on both the localization accuracy and precision, much effort has been put into the development and optimization of many different types of centroiding algorithms, including iterative fitting algorithms. As recently reported, super-resolution software for single-molecule localisation gives good results when applied to centroiding single photon events imaged with an MCP-intensifed CMOS camera.\cite{Hirvonen_2015} In this work, we have applied super-resolution software for centroiding single photon events events detected with an EBCCD camera. Multi-emitter fitting analysis was also tested for separating overlapping photon events. events, an important aspect which allows an increased count rate and shorter acquisition times.