Single-molecule localization software applied to photon counting imaging

Abstract

Centroiding in photon counting imaging has traditionally been accomplished by a single-step, noniterative algorithm, often implemented in hardware. Single-molecule localization techniques in superresolution fluorescence microscopy are conceptually similar, but use more sophisticated iterative software-based fitting algorithms to localize the fluorophore. Here, we discuss common features and differences between single-molecule localization and photon counting imaging and investigate the suitability of single-molecule localization software for photon event localization. We find that single-molecule localization software packages designed for superresolution microscopy-QuickPALM, rapidSTORM, and ThunderSTORM-can work well when applied to photon counting imaging with a microchannel-plate-based intensified camera system: photon event recognition can be excellent, fixed pattern noise can be low, and the microchannel plate pores can easily be resolved.

Photon Counting Imaging with an Electron-Bombarded Pixel Image Sensor

“…The global count rate depends on the number of pixels in the sensor, and as both CMOS and CCD sensors can be manufactured in large, megapixel arrays, the detection of hundreds or thousands of photons per frame is possible. The imaging speed is usually limited by the CCD or CMOS read-out time, and with photon event centroiding, the localisation time depends on the complexity of the algorithm [ 57 ].…”

“…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.…”

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

“…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] . Here, we extend this work and apply super-resolution software for centroiding photon events detected with an EBCCD camera.…”

Resonant Electro-Optic Imaging for Microscopy at Nanosecond Resolution

“…They also suffer from gain noise and reduced image quality unless operated in lowthroughput photon counting mode. 29 Similarly, modulated pixels suffer from high read noise and dark current. Confocal scanning with time-correlated single photon counting detectors remains the standard method for FLIM microscopy.…”

“…Gated intensifiers have high photon loss due to the photocathode quantum efficiency and because they only capture the gated light fraction. They also suffer from gain noise and reduced image quality unless operated in low-throughput photon counting mode . Similarly, modulated pixels suffer from high read noise and dark current.…”