Abstract
Photon event centroiding in photon counting imaging and single-molecule localisation in super-resolution fluorescence microscopy share many traits. Although photon event centroiding has traditionally been performed with simple single-iteration algorithms, we recently reported that iterative fitting algorithms originally developed for single-molecule localisation fluorescence microscopy work very well when applied to centroiding photon events imaged with an MCP-intensified CMOS camera. Here, we have applied these algorithms for centroiding of photon events from an electron-bombarded CCD (EBCCD). We find that centroiding algorithms based on iterative fitting of the photon events yield excellent results and allow fitting of overlapping photon events, a feature not reported before and an important aspect to facilitate an increased count rate and shorter acquisition times.
Highlights
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 – a process termed centroiding [9–11]
We recently reported that single-molecule localisation algorithms produce excellent results when applied to centroiding single photon events imaged with an microchannel plate (MCP)-intensified complementary metal-oxide-semiconductor (CMOS) camera [18]
The ion events are incorrectly localised as photon events, but due to the relatively rare occurrence of ion events compared to photon events this does not have a noticeable effect on the results
Summary
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 – a process termed centroiding [9–11]. Single-molecule localisation fluorescence microscopy techniques are based on the activation of a small subpopulation of the fluorescent proteins or fluorophores used to stain the sample. They are imaged and subsequently deactivated before the process is repeated with a different subset of fluorophores [14– 16]. Single-molecule localisation fluorescence microscopy is a well-established technique, and much effort has been put into the development and optimisation of many different types of centroiding algorithms, including iterative fitting algorithms [17]. We recently reported that single-molecule localisation algorithms produce excellent results when applied to centroiding single photon events imaged with an MCP-intensified CMOS camera [18]. Multi-emitter fitting analysis was used for separating overlapping photon events, an important aspect not reported before, which allows an increased count rate and shorter acquisition times
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