Fluorescence Correlation Spectroscopy and Photobleaching Recovery: Measurement of Transport and Chemical Kinetics

The dynamics of macromolecules, the subject of this symposium, are most directly studied by simply looking through a microscope and observing the molecular motion. With a microscope, we can resolve the size and shape of large particles, as well as monitor dynamic motion. For smaller particles, particularly single macromolecules, we cannot resolve the size or shape; but it is still possible to observe the motion, if we can make the particles appear as bright points of light sprinkled dilutely over a dark background. Siedentopf & Zsigmondy (1903) demonstrated this fact with a device which came to be called the ultramicroscope.

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Fluorescence correlation spectroscopy and photobleaching recovery of multiple binding reactions. II. FPR and FCS measurements at low and high DNA concentrations

Biopolymers ◽

10.1002/bip.360220809 ◽

1983 ◽

Vol 22 (8) ◽

pp. 1949-1966 ◽

Cited By ~ 31

Author(s):

R. D. Icenogle ◽

E. L. Elson

Keyword(s):

Fluorescence Correlation Spectroscopy ◽

Correlation Spectroscopy ◽

Fluorescence Correlation ◽

Multiple Binding ◽

Photobleaching Recovery

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Fluorescence microscopic analysis of molecular interactions in 3-dimensional samples by means of fluorescence correlation spectroscopy and photobleaching recovery

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102274 ◽

1988 ◽

Vol 46 ◽

pp. 38-39

Author(s):

Hong Qian ◽

Elliot L. Elson

Keyword(s):

Fluorescence Correlation Spectroscopy ◽

Chemical Interaction ◽

Microscopic Analysis ◽

Correlation Spectroscopy ◽

3 Dimensional ◽

Fluorescence Correlation ◽

Fluorescent Materials ◽

Photobleaching Recovery ◽

Open Region ◽

Fluorescence Microscopic Analysis

Fluorescence correlation spectroscopy (FCS) and fluoresence photobleaching recovery (FPR) are closely related methods which can be used to measure rates of transport (e.g. diffusion) and of chemical interaction of fluorescent materials. Both methods rely on measurements of fluorescence excited in an open region of the sample. As molecules are transported into or out of the region or their optical properties are changed by chemical interactions, the measured fluorescence changes correspondingly. Once the size of the illuminated region is known, the rates of transport (diffusion, drift, or flow) may be determined from the measured rates of change of fluorescence. There is, however, and important difference in principle between FCS and FPR. The former extracts rate information from measurements of spontaneous concentration fluctuations. The latter measures the relaxation of a macroscopic concentration gradient produced by photobleaching a portion of the fluorophores in the observation region with a brief high intensity light pulse.

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Applications of Fluorescence Correlation Spectroscopy

Quarterly Reviews of Biophysics ◽

10.1017/s0033583500002158 ◽

1976 ◽

Vol 9 (1) ◽

pp. 49-68 ◽

Cited By ~ 58

Author(s):

Watt W. Webb

Keyword(s):

Chemical Kinetics ◽

Fluorescence Correlation Spectroscopy ◽

Lateral Diffusion ◽

Preceding Paper ◽

Correlation Spectroscopy ◽

Basic Principles ◽

Fluorescence Correlation ◽

Model Based

The preceding paper by Douglas Magde has recounted the basic principles of Fluorescence Correlation Spectroscopy (FCS) as originally described (see Magde, Elson & Webb, 1972; Elson & Magde, 1974; Magde, Elson & Webb, 1974 Elson & Webb, 1975; referred to collectively as MEW), and has described the first application to chemical kinetics. In this paper I shall first illustrate the same principles of FCS with a simple graphical demonstration model based on the scheme for application to lateral diffusion in membranes as it was developed in our laboratory by Dr T. J. Herbert; I shall then proceed to discuss some current research in our group organized jointly with Professor E. L. Elson at Cornell.

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