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} Single photon detection is also possible with electron-bombarded (EB) sensors, where the single photoelectron liberated from the photocathode is accelerated directly into the CCD or CMOS sensor.\cite{Spring1998} The resulting photon events are smaller and dimmer than MCP-intensified photon events, but have a narrow, voltage-dependent pulse height distribution and avoid distortion of the image due to the coupling of the MCP to the camera, spectral matching of the camera sensitivity and the phosphor and image lag due to the phosphor decay time.\cite{Hirvonen2014_rsi}  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. 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 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.\cite{Betzig2006,Rust2006} 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 much effort has been put into the development and optimization of many different types of centroiding algorithms, including iterative fitting algorithms.  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} In this work, we have applied super-resolution software for centroiding single photon eventsevents  detected with an EBCCD camera. Multi-emitter fitting analysis was also tested for separating overlapping photon events, an important aspect which allows an increased count rate and shorter acquisition times.