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

Introduction

The detection of single photons is a technique used in many fields of science and technology, including fluorescence microscopy and spectroscopy, bioluminescence studies, optical tomography, DNA sequencing, lidar, quantum information science and encryption, and optical communications both on earth and in space.(Hadfield 2009, Buller 2010, Eisaman 2011, Seitz 2011) 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 fr