Optimization of spinning disk confocal microscopy: synchronization with the ultra-sensitive EMCCD

F. K. Chong; Colin G. Coates; Donal J. Denvir; Noel G. McHale; Keith D. Thornbury; Mark A. Hollywood

doi:10.1117/12.529220

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13 July 2004 Optimization of spinning disk confocal microscopy: synchronization with the ultra-sensitive EMCCD

F. K. Chong; Colin G. Coates; Donal J. Denvir; Noel G. McHale; Keith D. Thornbury; Mark A. Hollywood

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Proceedings Volume 5324, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XI; (2004) https://doi.org/10.1117/12.529220
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

Abstract

The advent of Electron Multiplying Charge Coupled Device (EMCCD) technology and it's ability to overcome previous hurdles in low-light fluorescence microscopy, such as phototoxicity to live cells, photobleaching of fluorophores and exposure time restrictions, has resulted in a significant resurgence of interest in use of confocal spinning disk techniques for live cell microscopy. Here provide an understanding of, and technical solutions to, the issues of synchronization that have previously marred the coupling of fast CCD camera technology to confocal spinning disk arrangements. We examine the challenges arising from both old and new models of the Nipkow spinning disk confocal unit and suggest solutions throughout based on a sound comprehension of both (a) relative scan/exposure times; (b) relative orientation of the coupled devices; (c) optimisation of EMCCD clocking parameters.

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F. K. Chong, Colin G. Coates, Donal J. Denvir, Noel G. McHale, Keith D. Thornbury, and Mark A. Hollywood "Optimization of spinning disk confocal microscopy: synchronization with the ultra-sensitive EMCCD", Proc. SPIE 5324, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XI, (13 July 2004); https://doi.org/10.1117/12.529220

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