Fluorescence Correlation Spectroscopy 2. Experimental Realization

Biopolymers (Impact Factor: 2.39). 01/1974; 13(1):29 - 61. DOI: 10.1002/bip.1974.360130103
Source: PubMed


This 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 (∼ 10−9M). 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.

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Available from: Douglas Magde, Dec 12, 2013
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    • "For Gaussian diffusion, K 2 is the mean-square displacement, while K 4 , K 6 , and higher are all zero. In this case, the spectrum reduces to the form of Magde, et al.[34] "
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    ABSTRACT: I review theoretical treatments of diffusion in crowded (i.~e., non-dilute) solutions of globular macromolecules. The focus is on the classical statistico-mechanical literature, much of which dates to before 1990. Classes of theoretical models include continuum treatments, correlation function descriptions, generalized Langevin equation descriptions, Smoluchowski and Mori-Zwanzig descriptions, and a brief but encouraging comparison with experimental results. The primary emphasis is on measurements made with quasi-elastic light scattering spectroscopy; I also discuss outcomes from fluorescence photobleaching recovery, fluorescence correlation spectroscopy, pulsed-gradient spin-echo nuclear magnetic resonance, and raster image correlation spectroscopy. I close with a list of theoretical papers on the general topic.
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    • "The radius of the detection volume was estimated to be 208 nm. The resultant autocorrelation function was fitted with a model which describes normal diffusion of monodisperse fluorophores (Magde et al., 1974): "
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    ABSTRACT: There is an immediate need to develop highly predictive in vitro cell-based assays that provide reliable information on cancer drug efficacy and toxicity. Development of biomaterial-based three-dimensional (3D) cell culture models as drug screening platforms has recently gained much scientific interest as 3D cultures of cancer cells have been shown to more adequately mimic the in vivo tumor conditions. Moreover, it has been recognized that the biophysical and biochemical properties of the 3D microenvironment can play key roles in regulating various cancer cell fates, including their response to chemicals. In this study, we employed alginate-based scaffolds of varying mechanical stiffness and adhesive ligand presentation to further explore the role of 3D microenvironmental cues on glioblastoma cell response to cytotoxic compounds. Our experiments suggested the ability of both matrix stiffness and cell-matrix adhesions to strongly influence cell responses to toxins. Cells were found to be more susceptible to the toxins when cultured in softer matrices that emulated the stiffness of brain tissue. Furthermore, the effect of matrix stiffness on differential cell responses to toxins was negated by the presence of the adhesive ligand RGD, but regained when integrin-based cell-matrix interactions were inhibited. This study therefore indicates that both 3D matrix stiffness and cell-matrix adhesions are important parameters in the design of more predictive in vitro platforms for drug development and toxicity screening. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Biotechnology and Bioengineering 07/2015; DOI:10.1002/bit.25709 · 4.13 Impact Factor
    • "The radius of the detection volume was estimated to be 208 nm. The resultant autocorrelation function was fitted with a model which describes normal diffusion of monodisperse fluorophores (Magde et al., 1974): "
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    ABSTRACT: Since the birth of tissue engineering, it has been redefined to include not only the development of tissues for clinical use, but also in vitro models for the study of tissue physiology and pathology. Great strides have been accomplished in the design of in vitro tissue models, yet one area in which they are underrepresented, but where they can have an immediate impact, is the development of platforms for drug screening. By providing more in vivo-like cell environments, such models could address the growing concerns about drug failures due to lack of efficacy or unexpected side effects. This review aims to address the interface between substrate compliance and cell responsiveness to toxins and drugs since compliance has been established as a major determinate of overall cell fate. Here, results from 2D substrates and 3D matrices are discussed. Additionally, examples of biomaterial-based high-throughput stiffness assays in drug screening are presented. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    Macromolecular Bioscience 02/2015; 15(5). DOI:10.1002/mabi.201400507 · 3.85 Impact Factor
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