Liisa Hirvonen edited Introduction.tex  over 8 years ago

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Photon counting imaging is a well-established low light level imaging technique where an image is assembled from individually detected photons. In conventional photon counting imaging, photon events on the phosphor screen of a microchannel plate (MCP)-based image intensifier are imaged with a charge-coupled device (CCD) or a complementary metal-oxide-semiconductor (CMOS) camera at high frame rates, and many frames are accumulated to build up an image.\cite{Hirvonen2014_ol} Photon counting imaging is also possible with electron-bombarded (EB) sensors, where single photoelectrons liberated from the photocathode are accelerated by a high voltage directly into the CCD or CMOS sensor to produce a photon event.\cite{Spring1998} These are smaller and dimmer than MCP-intensified photon events, with a narrow, voltage-dependent pulse height distribution.\cite{Hirvonen2014_rsi} EBCCD or EBCMOS-based photon counting imaging avoids distortion of the image due to the coupling of the intensifier to the camera, and image lag due to the phosphor decay time, and there is no need for spectral matching of the camera sensitivity to the phosphor.  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}. algorithms.\cite{Postma2011}  The discovery of photoswitchable and photoactivatable fluorophores in the past decade \cite{Fernandez_2008} 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.\cite{Small2014}