Abstract
The technique of image cross-correlation spectroscopy has been applied to measurements of diffusion of fluorescent beads in polymer solution. The technique is based on measurement and analysis of temporal fluctuations of the intensity observed in fluorescence confocal microscope images. As with other fluctuation techniques, fluorescence fluctuations arise from stochastic concentration fluctuations about the equilibrium concentration. The dynamics of the fluctuations depend on the number of fluorescent molecules in the observation volume. This work presents the theory and illustrates how the technique can be applied to measurement of diffusion of fluorescent beads in solutions of various viscosities. Further we extend the concept of cross-correlation to studies on the cell surface to detect the relative distribution of molecules (receptors) on the cell surface, in which intensity fluctuations arising from samples containing two distinct types of labelled molecules are cross-correlated using two detectors with different wavelength sensitivities.
Similar content being viewed by others
References
Magde D, Elson EL, Webb WW (1974). Fluorescence correlation spectroscopy, II: An experimental realization. Biopolymers 13: 29–61.
Elson EL, Magde D (1974). Fluorescence correlation spectroscopy, I: Conceptual basis and theory. Biopolymers 13: 1–27.
Kask P (1987). Fluorescence correlation spectroscopy and its biophysical application. Studia biophysica 118: 7–24.
Meyer T, Schindler H (1988). Particle counting by fluorescence correlation spectroscopy: Simultaneous measurement of aggregation and diffusion of molecules in solutions and in membranes. Biophys J 54: 983–993.
Nicoli DF, Briggs J, Ellings VB (1980). Fluorescence immunoassay based on long time correlations of number fluctuations. Proc Natl Acad Sci USA 77: 4904–4980.
Palmer AG, Thompson NL (1987). Theory of sample translation in fluorescence correlation spectroscopy. Biophys J 51: 339–343.
Palmer AG, Thompson NL (1987). Molecular aggregation characterized by high order autocorrelation spectroscopy. Biophys J 52: 257–270.
Petersen NO (1984). Diffusion and aggregation in biological membranes. Can J Biochem Cell Biol 62: 1158–1166.
Petersen NO (1986). Scanning fluorescence correlation spectroscopy, I: Theory and simulation of aggregation measurements. Biophys J 49: 809–815.
Petersen NO, Johnson DC, Schlesinger MJ (1986). Scanning fluorescence correlation spectroscopy, II: Application to virus glycoprotein aggregation. Biophys J 49: 817–820.
Petersen NO, Elson EL (1986). Measurements of diffusion and chemical kinetics by fluorescence photobleaching recovery and fluorescence correlation spectroscopy. Methods Enzymol 130: 454–484.
Thompson NL, Axelrod D (1983). Immunoglobulin surface binding kinetics studied by total internal reflection with fluorescence correlation spectroscopy. Biophys J 43: 103–114.
St-Pierre PR, Petersen NO (1992). Average density and size of microclusters of epidermal growth factor receptors on A431 cells. Biochemistry 31: 2459–2463.
Petersen NO, Hoddelius PL, Wiseman PW, Seger O, Magnusson K-E (1993). Quantitation of membrane receptor distributions by Image Correlation Spectroscopy: Concept and application. Biophys J 65: 1135–1146.
Koppel DE, Morgan F, Cowan AE, Carson JH (1994) Scanning concentration correlation spectroscopy using the confocal laser microscope. Biophys J 66: 502–507.
Elson EL (1985). Fluorescence correlation spectroscopy and photobleaching recovery. Annu Rev Phys Chem 36: 379–406.
Ehrenberg M, Rigler R (1976). Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules. Quart Rev Biophys 9: 69–81.
Koppel DE, Axelrod D, Schlessinger J, Elson EL, Webb WW (1976). Dynamics of fluorescence marker concentration as a probe of mobility. Biophys J 16: 1315–1329.
Magde D, Webb WW, Elson EL (1978). Fluorescence correlation spectroscopy, III: Uniform translation and laminar flow. Biopolymers 17: 361–376.
Scalettar BA, Hearst JE, Klein MP (1989). FRAP and FCS studies of self-diffusion and mutual diffusion in entangled DNA solutions. Macromolecules 22: 4550–4559.
Webb WW (1976). Applications of fluorescence correlation spectroscopy. Quart Rev Biophys 9: 49–68.
Barisas BG, Leuther MD (1979). Fluorescence photobleaching recovery measurement of protein absolute diffusion constants. Biophys Chem 10: 221–229.
Jacobson K, Derzko Z, Wu E-S, Hou Y, Poste G (1967). Measurement of the lateral mobility of cell surface components in single, living cells by fluorescence recovery after photobleaching. J Supramol Struct 5: 565–576.
Koppel DE (1979). Fluorescence redistribution after photobleaching: a new multipoint analysis of membrane translational dynamics. Biophys J 28: 281–291.
Lee GM, Ishihara A, Jacobson KA (1991). Direct observation of brownian motion of lipids in a membrane. Proc Natl Acad Sci USA 88: 6274–6278.
Magnusson K-E, Wojcieszyn J, Dahlgren C, Stendahl O, Sundqvist T, Jacobson K (1983). Lateral diffusion of wheat germ agglutinin labeled glycoconjugates in the membrane of differentiating HL-60 and U-937 cells assessed with fluorescence recovery after photobleaching (FRAP). Cell Biophys 5: 119–128.
Anderson CM, Georgiou GN, Morrison IG, Stevenson GVW, Cherry RJ (1992). Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera. Low density lipoprotein and influenza virus receptor mobility at 4 °C. J Cell Sci 101: 415–425.
Ghosh RN, Webb WW (1994). Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules. Biophys J 66: 1301–1318.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Srivastava, M., Petersen, N.O. Image cross-correlation spectroscopy: A new experimental biophysical approach to measurement of slow diffusion of fluorescent molecules. Methods Cell Sci 18, 47–54 (1996). https://doi.org/10.1007/BF00123523
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00123523