Abstract
This article describes the use of confocal fluorescence correlation microscopy as a way to measure the translocation and dynamics of molecules on a sub-cellular scale. Using this technique it is possible with a single measurement to determine not only the size of labelled molecules in terms of their translational diffusion coefficient (DT) but also their number density <N> within a sample1,2. The sample volume is defined precisely by confocal optics, and in our system is a quasi cylindrical space of approximately 1 fL, with a length to width ratio of between 6 to 8. The probe volume is therefore of subcellular dimensions. FCM has a great potential in cell biology, since most cellular events involve changes in the physical size and/or number of individual sub-cellular components e. g., the binding and release of sub-units or effectors, changes in the level of gene expression, the processes of polymerisation and depolymerisation, and changes in microviscosity or compartmentation. In such complex systems time-dependent molecular interactions can be resolved from the correlated intensity fluctuations of individual specifically-labelled, or intrinsically fluorescent, molecules in the probe volume.
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References
D. Magde, E.L. Elson, and W.W. Webb, Fluorescence correlation spectroscopy. II. An experimental realisation, Biopolymers 13:29 (1974)
R. Rigler, Ü. Mets, J. Widgren, and P. Kask, Fluorescence correlation spectroscopy with high count rate and low background: Analysis of translational diffusion, Eur. Biophys. J. 22:169 (1993)
B.R. Terry and E.K. Matthews, G-actin concentrations and F-actin dynamics measured within femtolitre volumes by fluorescence correlation microscopy, Biophys J., submitted (1995)
Z. Huang, S. Yue, W. You and R.P. Haugland, Anal. Biochem., 214:272 (1993)
S. Broersma, Viscous force constant for a closed cylinder, J. Chem. Phys., 32:1632 (1960)
S. Broersma, Viscous force and torque constants for a cylinder, J. Chem. Phys., 74:6989 (1981)
B.R. Terry, E.K. Matthews, and J. Haseloff, Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy, Biophys. Biochem. Res. Comms. submitted (1995)
D.C. Prasher, V.K. Eckenrode, W.W. Ward, F.G. Prendergast, and M.J. Cormier, Primary structure of the Aequorea victoria green-fluorescent protein, Gene 111:229 (1992)
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© 1996 Springer Science+Business Media New York
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Terry, B.R., Matthews, E.K. (1996). Dynamics of Actin Measured by Fluorescence Correlation Microscopy (FCM). In: SlavÃk, J. (eds) Fluorescence Microscopy and Fluorescent Probes. Springer, Boston, MA. https://doi.org/10.1007/978-1-4899-1866-6_46
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DOI: https://doi.org/10.1007/978-1-4899-1866-6_46
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4899-1868-0
Online ISBN: 978-1-4899-1866-6
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