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Title: Fluorescence Correlation Spectroscopy, Raster Image Correlation Spectroscopy, and Number and Brightness on a Commercial Confocal Laser Scanning Microscope with Analog Detectors (Nikon C1)
Contributor(s): Moens, Pierre  (author)orcid ; Gratton, Enrico (author); Salvemini, Iyrri (author)
Publication Date: 2011
Open Access: Yes
DOI: 10.1002/jemt.20919Open Access Link
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Abstract: Fluorescence correlation spectroscopy (FCS) was developed in 1972 by Magde, Elson and Webb. Photon counting detectors and avalanche photodiodes have become standards in FCS to the point that there is a widespread belief that these detectors are essential to perform FCS experiments, despite the fact that FCS was developed using analog detectors. Spatial and temporal intensity fluctuation correlations using analog detection on a commercial Olympus Fluoview 300 microscope have been reported by Brown et al. (2008). However, each analog instrument has its own idiosyncrasies that need to be understood before using the instrument for FCS. In this work, we explore the capabilities of the Nikon C1, a low-cost confocal microscope, to obtain single point FCS, Raster-scan image correlation spectroscopy (RICS), and Number and Brightness data both in solution and incorporated into the membrane of giant unilamellar vesicles. We show that it is possible to obtain dynamic information about fluorescent molecules from single point FCS, RICS, and Number and Brightness using the Nikon C1. We highlighted the fact that care should be taken in selecting the acquisition parameters to avoid possible artifacts due to the detector noise. However, due to relatively large errors in determining the distribution of digital levels for a given microscope setting, the system is probably only adequate for determining relative brightness within the same image.
Publication Type: Journal Article
Grant Details: NHMRC/568301
Source of Publication: Microscopy Research and Technique, 74(4), p. 377-388
Publisher: John Wiley & Sons Inc
Place of Publication: United States of America
ISSN: 1059-910X
Field of Research (FOR): 029901 Biological Physics
Socio-Economic Outcome Codes: 920102 Cancer and Related Disorders
Peer Reviewed: Yes
HERDC Category Description: C1 Refereed Article in a Scholarly Journal
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