Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Patent
1997-04-28
1999-08-03
Evans, F. L.
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
2504581, G01N 2164
Patent
active
059332336
DESCRIPTION:
BRIEF SUMMARY
The present invention pertains to a method for the determination of material-specific parameters of one or a few molecules by means of correlation spectroscopy, and a device for performing the method.
WO 94/16313 describes the method of fluorescence correlation spectroscopy (FCS) as an examination method. Due to a confocal arrangement, extremely small volume elements can be examined in a sample of quite different material compositions. Thus, by measuring the spectroscopical parameters of single or a few molecules, information can be achieved which permits conclusions on the material composition of such small volume elements to be drawn. However, this method requires relatively low concentrations of the molecules to be examined. If one desires to transfer the fluorescence correlation spectroscopy to systems in which relatively high concentrations of the molecule to be measured exist, the method reaches its limits since fluctuation analysis is no longer possible then. For example, if concentrations of more than 1 .mu.m of the molecules to be examined are present in a measuring volume of 10.sup.-14 l, the fluorescence correlation spectroscopy employed so far is unsuitable.
From Fischer (J. Opt. Soc. Amb. B., Vol. 3, No. 10, pp. 1239-1244, October 1986), it is known that the concept of "surface-enhanced fluorescence spectroscopy" can be used to detect small changes in the optical properties of the microenvironment of a single small scattering object. Submicron apertures in thin silver or gold films coated on a microscope slide are used to analyze the properties of the solution to be examined.
Srivastava and Badyopadhyay (Rev. Sci. Instrum. 61 (2), 1990) describe a device for measuring the photoluminescence using optical fibers for guiding the excitation and emission radiations. This spectrometer is employed for determining photoluminescence spectra of several III-V components.
Miniaturized examination systems are described, e.g., in DD 271 953 A1. There is disclosed a means for the automated photometric analysis of minute sample quantities which can perform transmission (absorbance) and fluorescence measurements. Wave guide sheets which are in optical contact with at least one sample at their surface at least in sections thereof are implemented in a support. For each wave guide sheet, means implemented in the support are provided for coupling electromagnetic radiation into the wave guide sheets. For integration in peripheric technology, the support is fastened on a transport means which provides contact with means for the preparation and after-processing of the sample.
However, none of the methods described enables an extension of the measuring limits of correlation spectroscopy towards higher concentrations. In particular, no miniaturized device for performing correlation spectroscopy has been described previously. Such a device would be desirable in modern biotechnology, especially evolutive biotechnology.
Thus, it has been the object of the invention to develop a powerful examination system which enables the determination of material-specific parameters of molecules in relatively highly concentrated solutions by means of correlation spectroscopy, in particular fluorescence correlation spectroscopy. This examination system should be miniaturizable.
Surprisingly, the object of the invention is achieved by a method for the determination of material-specific parameters of one or a few molecules in a sample in which the molecule(s) to be determined is (are) present in relatively high concentrations wherein said molecule or molecules is (are) excited by electromagnetic radiation (excitation radiation) to emit electromagnetic radiation (emission radiation) wherein said excitation and/or emission radiation passes a means which is permeable to the corresponding wavelength of this electromagnetic radiation which means is disposed between an excitation or emission radiation source and an excitation or emission radiation detector, said means for transmitting electromagnetic waves having at least one region the largest
REFERENCES:
patent: 5446534 (1995-08-01), Goldman
Evans F. L.
Evotec BioSystems GmbH
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