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Journal of Biomedical Optics • Open Access

Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution
Author(s): Colin G. Coates; Donal J. Denvir; Noel G. McHale; Keith D. Thornbury; Mark Hollywood

Paper Abstract

The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512×512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD.

Paper Details

Date Published: 1 November 2004
PDF: 9 pages
J. Biomed. Opt. 9(6) doi: 10.1117/1.1805559
Published in: Journal of Biomedical Optics Volume 9, Issue 6
Show Author Affiliations
Colin G. Coates, Andor Technology Ltd. (United Kingdom)
Donal J. Denvir, Andor Technology Ltd. (United Kingdom)
Noel G. McHale, Queen's Univ. Belfast (United Kingdom)
Keith D. Thornbury, Queen's Univ. Belfast (United Kingdom)
Mark Hollywood, Queen's Univ. Belfast (United Kingdom)

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