Photon counting imaging and centroiding with an electron-bombarded CCD using single molecule localisation software

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.

Photon Counting Imaging with an Electron-Bombarded Pixel Image Sensor

“…The photon event brightness in EB pixel image sensors is strongly dependent on the acceleration voltage, and as the photon events typically cover an area of a few pixels, resolution lost in the detection process can be recovered by photon event centroiding—both one-iteration centre-of-mass [ 20 ] and iterative fitting [ 58 ] algorithms have been shown to produce excellent results. The local count rate is given by the frame rate of the camera; EBCMOS cameras with 500 frames per second have been described [ 46 ], and 1000 Hz planned.…”

“…With a low gain, these devices can be used in analogue mode, and at high gain, they are sensitive enough to detect single photons: they enable wide-field imaging at extremely low light levels, allowing the detection of up to hundreds or thousands of photon events per frame. Photon event centroiding can be employed to recover resolution lost in the detection process, as described in more detail in [ 58 ]. Unlike photon counting cameras employing electron-multiplying MCPs, the photon events have a narrow, acceleration-voltage-dependent pulse height distribution.…”

“…The centroiding is nowadays done in software but the algorithms employed in photon counting imaging are still usually simple, one-iteration algorithms [ 3 ]. Recently, we have applied iterative algorithms developed for super-resolution fluorescence microscopy for centroiding of photon events, and found that these algorithms yield excellent results for both MCP-intensified camera systems [ 57 ] and EBCCDs ( Figure 3 a–c) [ 58 ], providing efficient photon event recognition, low fixed pattern noise and excellent localisation results. Moreover, multi-emitter fitting algorithms–developed for super-resolution microscopy to separate fluorescent emitters whose point-spread functions overlap partially–allow separation of overlapping photon events with EBCCDs, see Figure 3 d,e, an important aspect to facilitate an increased count rate and shorter acquisition times.…”

Wide-field TCSPC: methods and applications

“…The resolution of the image is then not limited by the camera pixel size, but by the MCP pore size and the distribution of the photoelectron trajectories from the photocathode to the first MCP. We have recently demonstrated that software specifically developed for centroiding singlemolecule data for super-resolution fluorescence microscopy produces excellent results when applied to centroiding photon events from MCP-based [105] and EBCCD-based camera systems [106].…”

“…The combination of a phosphor screen and an ultrafast frame-rate CMOS camera allows up to 100s of photons to be detected simultaneously after one excitation pulse, but the timing accuracy of this method is limited by the camera frame rate to microsecond time scale. Fluorescence super-resolution software can be used to centroid photon events to sub-pixel accuracy [105,106]. MCPs are fundamentally limited by their countrate in the MHz region, and their lifespan is limited by the overall amount of charge that can be extracted from the channels.…”