Abstract
Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In the standard case, fluorescence fluctuations are measured in an open detection volume defined by the confocal optics. However, if FCS measurements are carried out in cellular processes that confine the detection volume, the standard FCS model leads to erroneous results. In this paper, we derive a modified FCS model that takes into account the confinement of the detection volume. Using this model, we have carried out the first FCS measurements in dendrites of cultured neurons. We further derive, for the case of confined diffusion, the limits within which the standard two- and three-dimensional diffusion models give reliable results.
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- Berland K. M., So P. T., Gratton E. Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment. Biophys J. 1995 Feb;68(2):694–701. doi: 10.1016/S0006-3495(95)80230-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bischofberger J., Schild D. Different spatial patterns of [Ca2+] increase caused by N- and L-type Ca2+ channel activation in frog olfactory bulb neurones. J Physiol. 1995 Sep 1;487(Pt 2):305–317. doi: 10.1113/jphysiol.1995.sp020881. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brock R., Hink M. A., Jovin T. M. Fluorescence correlation microscopy of cells in the presence of autofluorescence. Biophys J. 1998 Nov;75(5):2547–2557. doi: 10.1016/S0006-3495(98)77699-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Politz J. C., Browne E. S., Wolf D. E., Pederson T. Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6043–6048. doi: 10.1073/pnas.95.11.6043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwille P., Haupts U., Maiti S., Webb W. W. Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys J. 1999 Oct;77(4):2251–2265. doi: 10.1016/S0006-3495(99)77065-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Trombley P. Q., Westbrook G. L. Excitatory synaptic transmission in cultures of rat olfactory bulb. J Neurophysiol. 1990 Aug;64(2):598–606. doi: 10.1152/jn.1990.64.2.598. [DOI] [PubMed] [Google Scholar]
- Widengren J., Rigler R. Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces. Cell Mol Biol (Noisy-le-grand) 1998 Jul;44(5):857–879. [PubMed] [Google Scholar]