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.

Keywords: Photon counting imaging, single-molecule localisation, electron-bombarded CCD

OCIS codes: (040.3780) Detectors: Low light level, (030.5260) Coherence and statistical optics: Photon counting, (100.6640) Image processing: Superresolution, (110.0180) Imaging systems: Microscopy, (170.2520) Medical optics and biotechnology: Fluorescence microscopy