DOI: 10.1002/bip.1974.360130103. Fluorescence correlation spectroscopy. II. An experimental realization

Fluorescence correlation spectroscopy. II. An experimental realization

10.1002/bip.1974.360130103

Crossref journal-article

Wiley

Biopolymers (311)

Abstract

AbstractThis paper describes the first experimental application of fluorescence correlation spectroscopy, a new method for determining chemical kinetic constants and diffusion coefficients. These quantities are measured by observing the time behaviour of the tiny concentration fluctuations which occur spontaneously in the reaction system even when it is in equilibrium. The equilibrium of the system is not disturbed during the experiment. The diffusion coefficients and chemical rate constants which determine the average time behaviour of these spontaneous fluctuations are the same as those sought by more conventional methods including temperature‐jump or other perturbation techniques. The experiment consists essentially in measuring the variation with time of the number of molecules of specified reactants in a defined open volume of solution. The concentration of a reactant is measured by its fluorescence; the sample volume is defined by a focused laser beam which excites the fluorescence. The fluorescent emission fluctuates in proportion with the changes in the number of fluorescent molecules as they diffuse into and out of the sample volume and as they are created or eliminated by the chemical reactions. The number of these reactant molecules must be small to permit detection of the concentration fluctuations. Hence the sample volume is small (10−8 ml) and the concentration of the solutes is low (∼ 10−9 M). We have applied this technique to the study of two prototype systems: the simple example of pure diffusion of a single fluorescent species, rhodamine 6G, and the more interesting but more challenging example of the reaction of macromolecular DNA with the drug ethidium bromide to form a fluorescent complex. The increase of the fluorescence of the ethidium bromide upon formation of the complex permits the observation of the decay of concentration fluctuations via the chemical reaction and consequently the determination of chemical rate constants.

Bibliography

Magde, D., Elson, E. L., & Webb, W. W. (1974). Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers, 13(1), 29â61. Portico.

Authors 3

Douglas Magde (first)
Elliot L. Elson (additional)
Watt W. Webb (additional)

References 43 Referenced 1,137

10.1103/PhysRevLett.29.705
10.1002/bip.1974.360130102
{'key': 'e_1_2_1_4_2', 'first-page': '377', 'volume-title': 'Statistical Physics', 'author': 'Landau L. D.', 'year': '1958'} / Statistical Physics by Landau L. D. (1958)
10.1021/ma60023a019
10.1016/S0079-6638(08)70188-4
{'key': 'e_1_2_1_6_3', 'first-page': '49', 'volume-title': 'Polarisation, Matiere et Rayonnement', 'author': 'Benedek G. B.', 'year': '1969'} / Polarisation, Matiere et Rayonnement by Benedek G. B. (1969)
10.1063/1.1712445
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10.1063/1.1671255
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10.1016/B978-1-4832-3114-3.50008-8
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10.1038/169506a0
10.1099/00221287-36-2-225
10.1016/0022-2836(67)90276-8
10.1016/0022-2836(68)90286-6
10.1016/0022-2836(68)90207-6
10.1016/0022-2836(67)90353-1
D. M.Crothers private communication.
{'key': 'e_1_2_1_18_2', 'first-page': '153', 'volume-title': 'Laser Physics', 'author': 'Maitland A.', 'year': '1969'} / Laser Physics by Maitland A. (1969)
S.Weinreb MIT Research Lab. of Electronics Technical Report#412 Appendix B (1963).
10.1063/1.1654464
D. E.Koppel personal communication.
{'key': 'e_1_2_1_22_2', 'first-page': '277', 'volume-title': 'Thermodynamics', 'author': 'Callen H. B.', 'year': '1960'} / Thermodynamics by Callen H. B. (1960)
{'key': 'e_1_2_1_23_2', 'first-page': '297', 'volume-title': 'The Principles of Chemical Equilibrium', 'author': 'Denbigh K.', 'year': '1964'} / The Principles of Chemical Equilibrium by Denbigh K. (1964)
{'key': 'e_1_2_1_24_2', 'volume-title': 'Photophysics of Aromatic Molecules', 'author': 'Birks J. B.', 'year': '1970'} / Photophysics of Aromatic Molecules by Birks J. B. (1970)
10.1016/S0022-2836(65)80096-1
10.1098/rspa.1972.0071
{'key': 'e_1_2_1_27_2', 'volume-title': 'Techniques of Organic Chemistry', 'author': 'Eigen M.', 'year': '1963'} / Techniques of Organic Chemistry by Eigen M. (1963)
10.1021/ja01136a059
{'key': 'e_1_2_1_29_2', 'first-page': '358', 'volume': '87', 'author': 'Waring M. J.', 'year': '1964', 'journal-title': 'Biochem. Biophys. Acta'} / Biochem. Biophys. Acta by Waring M. J. (1964)
S. M.TarrandA. H.Ross private communication.
10.1016/0022-2836(69)90420-3
{'key': 'e_1_2_1_32_2', 'first-page': '24a', 'volume': '13', 'author': 'Breshoff J.', 'year': '1973', 'journal-title': 'Biophys. Soc. Abstracts'} / Biophys. Soc. Abstracts by Breshoff J. (1973)

Dates

Type	When
Created	20 years, 7 months ago (Dec. 29, 2004, 7:55 p.m.)
Deposited	1 year, 10 months ago (Oct. 19, 2023, 11:39 a.m.)
Indexed	2 weeks, 4 days ago (Aug. 5, 2025, 8:26 a.m.)
Issued	51 years, 7 months ago (Jan. 1, 1974)
Published	51 years, 7 months ago (Jan. 1, 1974)
Published Online	21 years, 6 months ago (Feb. 1, 2004)
Published Print	51 years, 7 months ago (Jan. 1, 1974)

Funders 0

None

BibTeX

@article{Magde_1974, title={Fluorescence correlation spectroscopy. II. An experimental realization}, volume={13}, ISSN={1097-0282}, url={http://dx.doi.org/10.1002/bip.1974.360130103}, DOI={10.1002/bip.1974.360130103}, number={1}, journal={Biopolymers}, publisher={Wiley}, author={Magde, Douglas and Elson, Elliot L. and Webb, Watt W.}, year={1974}, month=jan, pages={29–61} }

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