Precision and accuracy of absorption-corrected molecular fluorescence measurements by the cell shift method

Christmann, D. R.; Crouch, S. R.; Timnick, Andrew.

doi:10.1021/ac00236a022

Cited by 19 publications

(10 citation statements)

References 19 publications

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“…There are two ways to deal with the inner filter effect. One is to calibrate fluorometrically measured fluorescence with the sample absorbance in order to establish the linearity between the calibrated fluorescence and the fluorophore concentration (Holland et al, 1973(Holland et al, ,1977Christmann et al, 1981).…”

Section: Discussionmentioning

confidence: 99%

Kinetic assay of fluorescein mono-.beta.-D-galactoside hydrolysis by .beta.-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates

Huang¹

1991

Biochemistry

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A novel enzymatic assay method was developed for fluorogenic substrates that have significant intrinsic absorbance and fluorescence under the assay conditions. Fluorescein mono-beta-D-galactoside (FMG) was chosen as the substrate for the fluorescence enzymatic assay because of the high fluorescence of its hydrolytic product (fluorescein) and suitability of being hydrolyzed by beta-galactosidase. The fluorescence-concentration relationships for fluorescein and for FMG in both the right-angle detection mode of a fluorometer and the front-face detection mode of a fluorescence plate reader were exactly established and used to determine the kinetics of the enzyme assay. The results show that only front-face detection in the fluorescence plate reader can overcome the fluorescence concentration quenching that inevitably results from high absorbance by the intrinsically absorbing substrate in the conventional fluorometer, which utilizes right-angle detection. Only with front-face detection was the fluorescent assay of FMG hydrolysis under conditions of high optical density possible. The enzymatic measurements on the fluorescence plate reader were particularly efficient for determination of the enzyme kinetics because of the high rate of data collection. In this assay system, Michaelis-Menten constant Km and enzymatic catalysis rate k2 of FMG were determined as 117.6 microM and 22.7 mumol-(min.mg)-1, respectively. The results and methods described in this paper can be generalized for any assay using a fluorogenic substrate whether or not it has a high background absorbance.

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Section: Discussionmentioning

confidence: 99%

Kinetic assay of fluorescein mono-.beta.-D-galactoside hydrolysis by .beta.-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates

Huang¹

1991

Biochemistry

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“…In the 1970's and 1980's it was recognized that using a vibrating mirror [16,17], or shifting [18] or rotating [9,19] the cell could account for inner filter effects. Improvement of these methods, such as automating the cuvette translation [20][21][22], continued with Tucker et al publishing a related tutorial article [23] in 1992. Accounting for inner filter effects with alternative methods, such as Raman scattering [24], continue.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence measurements for evaluating the application of multivariate analysis techniques to optically thick environments.

Reichardt¹,

Timlin²,

Jones³

et al. 2010

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Laser-induced fluorescence measurements of cuvette-contained laser dye mixtures are made for evaluation of multivariate analysis techniques to optically thick environments. Nine mixtures of Coumarin 500 and Rhodamine 610 are analyzed, as well as the pure dyes. For each sample, the cuvette is positioned on a two-axis translation stage to allow the interrogation at different spatial locations, allowing the examination of both primary (absorption of the laser light) and secondary (absorption of the fluorescence) inner filter effects. In addition to these expected inner filter effects, we find evidence that a portion of the absorbed fluorescence is re-emitted. A total of 688 spectra are acquired for the evaluation of multivariate analysis approaches to account for nonlinear effects.

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“…Since then, the PCR process has become one of the major tools in genomic studies. By integrating a fluorimeter with the thermal cycler, a Real-Time PCR machine [4][5][6][7] was first introduced. This machine allows detection of DNA amplification through the detection of the fluorescence labeling dye in the PCR master mix during the early phase of reaction.…”

Section: Introductionmentioning

confidence: 99%

A novel fluorescence quantification method for polymerase chain reaction system

Chien

Lee

Cheng

et al. 2006

Optics Communications

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Precision and accuracy of absorption-corrected molecular fluorescence measurements by the cell shift method

Cited by 19 publications

References 19 publications

Kinetic assay of fluorescein mono-.beta.-D-galactoside hydrolysis by .beta.-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates

Kinetic assay of fluorescein mono-.beta.-D-galactoside hydrolysis by .beta.-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates

Fluorescence measurements for evaluating the application of multivariate analysis techniques to optically thick environments.

A novel fluorescence quantification method for polymerase chain reaction system

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