title: Fluorescence correlation spectroscopy and photon-counting histogram analysis of receptor-receptor interactions.
abstract: Fluorescence correlation spectroscopy (FCS) performed using a laser scanning confocal microscope is a technique with single-molecule sensitivity that is becoming more accessible to cell biologists. In this chapter, we describe the use of FCS for the analysis of diffusion coefficients and receptor-receptor interactions in live cells in culture. In particular, we describe a protocol to collect fluorescence fluctuation data from fluorescence-tagged receptors as they diffuse into an out of a small laser-illuminated observation volume using a commercially available system such as the Zeiss ConfoCor 3 or LSM-780 microscope. Autocorrelation analysis of the fluctuations in fluorescence intensity provides information about the diffusion time and number of fluorescent molecules in the observation volume. A photon-counting histogram can be used to examine the relationship between fluorescence intensity and the number of fluorescent molecules to estimate the average molecular brightness of the sample. Since molecular brightness is directly proportional to the number of fluorescent molecules, it can be used to monitor receptor-receptor interactions and to decode the number of receptor monomers present in an oligomeric complex.
authors.affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York, USA.
authors.forename: Katharine
authors.initials: K
authors.lastname: Herrick-Davis
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Mazurkiewicz
mesh_terms: D000818:Animals; D051940:B7-2 Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018106:CD28 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D003412:Cricetulus; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D057054:Molecular Imaging; D017785:Photons; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods / Q000706:statistics & numerical data*; D013194:Staining and Labeling; D014162:Transfection
chemical_list: D051940:B7-2 Antigen; D001426:Bacterial Proteins; D018106:CD28 Antigens; D008164:Luminescent Proteins; D018343:Receptors, Adrenergic, beta-2; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins
keywords: Fluorescence correlation spectroscopy; G-protein-coupled receptors; Photon-counting histogram; Receptor–receptor interactions; Tyrosine kinase receptors