Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
  • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
  • G01J3/28—Investigating the spectrum
  • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
  • G01J3/4406—Fluorescence spectrometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N2021/6417—Spectrofluorimetric devices
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/12—Circuits of general importance; Signal processing
  • G01N2201/129—Using chemometrical methods
  • G01N2201/1293—Using chemometrical methods resolving multicomponent spectra

Definitions

• the present invention relates to multispectral fluorescence signatures for identification of chemical and biological materials.
• the excitation source can be a tunable laser or a continuous light source such as a high-pressure xenon lamp filtered by a monochromator or suitable narrow band filters.
• the usual method of measurement requires that the excitation wavelength be stepped through a set of wavelengths.
• the present invention provides a method for measuring the spectral signature by simultaneous recording of the fluorescence spectra at all excitation wavelengths.
• the present invention is of a method of and apparatus for generating a multivariate spectra over a predetermined excitation wavelength region, comprising: directing light spanning the region at a sample; simultaneously measuring intensities of resulting fluorescence spectra as a function of a plurality of fluorescence wavelengths produced at one or more excitation wavelengths in the region; and producing a multivariate spectra from the intensities, fluorescence wavelengths, and excitation wavelengths.
• the multivariate spectra are employed to identify one or more components of the sample, preferably to identify and measure a plurality of components of the sample by assuming that the multivariate spectra is a linear combination of expected multivariate spectra of the plurality of components.
• the steps may be repeatedly performed to determine reaction rates between the plurality of components in a chemical or biological reaction, or in medical diagnostic procedures or drug development procedures.
• Directing preferably employs an excitation source such as a tunable laser or a continuous light source, most preferably a high-pressure xenon lamp filtered by a monochromator or narrow band filter.
• Measuring comprises simultaneously measuring intensities of resulting fluorescence spectra as a function of a plurality of fluorescence wavelengths produced at a plurality of excitation wavelengths in the region.
• the sample may be in a cuvette or microwell plate (preferably with a clear bottom of fused silica, quartz, or other material transparent in the desired spectral range).
• the microwell plate preferably contains from 1 to 9600 wells, most preferably 12, 24, 48, 96, 384, or 1536. Processing is preferably accomplished for each well of the plate in less than approximately 1 minute, more preferably less than approximately 5 seconds, and most preferably less than approximately 1 second. Most preferably processing is accomplished for each well of the plate simultaneously.
• Measuring employs a detector such as a photomultiplier tube, CCD camera, CCD chip, fiber optic array impinging upon a photomultiplier or CCD device, or microscope.
• the sample preferably has a volume between approximately 1 picoliter to 10 milliliters.
• Fig. 1 is a multispectral fluorescent signature showing fluorescent intensity as a function of excitation and fluorescent wavelength
• Fig. 2 is a block diagram for two-dimensional imaging of excitation and fluorescent wavelengths to obtain a total fluorescent signature
• Fig. 3 is a block diagram for two-dimensional imaging of a fluorescent signature using fiber bundles for the excitation and collection;
• Fig. 4 is a block diagram of two-dimensional imaging of a fluorescent signature using a microscope lens system for the excitation and collection;
• Fig. 5 is a block diagram of a system according to the invention to measure fluorescent signatures simultaneously as a function of excitation and fluorescence wavelengths;
• Fig. 6(a) is a spectral signature of fetal bovine serum (FBS) recorded using the system of Fig. 5 showing a three-dimensional representation of the signature and a contour plot obtained by projection on the top surface; the fluorescence wavelength is measured along the x-axis and the excitation wavelength is measured along the y-axis; the fluorescent intensity at an excitation and fluorescent wavelength is measured in the z-axis; and
• FBS fetal bovine serum
• Fig. 6(b) is a contour plot of the FBS signature.
• multispectral signature along with multivariate analysis has become a viable technique for the identification of chemical and biological materials in mixtures and with strong background signals.
• a three-dimensional spectral signature is generated over a predetermined excitation wavelength by measuring the intensity of the resulting fluorescent spectra as a function of fluorescent wavelength that is produced at a number of excitation wavelength.
• An example of a spectral signature is shown in Figure 1.
• the excitation source can be a tunable laser or a continuous light source such as a high-pressure xenon lamp filtered by a monochromator or suitable narrow band filters.
• the usual method of measurement requires that the excitation wavelength be stepped through a set of wavelengths.
• the present invention is of a method and apparatus for measuring the special signature by simultaneous recording of the fluorescence spectra at all excitation wavelengths.
• the preferred method and apparatus 10 of the present invention for performing the measurement using monochromators is shown in Figure 2.
• the light from a continuous light source is focused onto the entrance slit of a monochromator that is set at a wavelength near the center of the desired excitation wavelength region 14.
• a narrow band of light at the wavelength setting passes through the slit. If the slit is removed, a broad continuous spectra is emitted from the monochromator 12.
• a slit perpendicular to the input slit is used to define the width of the light and the resulting continuous excitation is shown in Figure 2.
• the resulting excitation spectra are imaged by a lens system 16 on the sample 18 wherein each vertical position, Z 3 represents a corresponding excitation wavelength, ⁇ e .
• ⁇ e fluorescence as a function of wavelength, ⁇ f , is generated.
• the fluorescence, along with the scattered excitation light, is imaged by a lens system 16' onto the entrance slit of an imagining spectrometer 12'. The distance along the entrance slit is correlated with an excitation wavelength.
• the imaging spectrometer disperses the fluorescence in a direction perpendicular to the entrance slit and the excitation wavelength that is imaged on a two-dimensional detector 20.
• the resulting spectral signature 21 is shown in the inset in Figure 2.
• the signature is recorded over a range of excitation wavelengths 14 and fluorescence wavelengths 22 without scanning the excitation monochromator or the imaging spectrometer thus producing a signature in a time much less than that required when the excitation is scanned.
• the excitation wavelength(s) are preferably between approximately 190nm to 900nm, and the emission wavelength(s) are also preferably between approximately 190nm to 900nm.
• both excitation wavelength and emission wavelength can be tuned in increments from approximately 0.1nm to 20nm.
• the invention is useful, for example, to determine the amount and/or concentration of a small fluorescent molecule (molecular weight between 100 daltons and 10,000 daltons) in a complex solution such as, e.g., human serum.
• a small fluorescent molecule molecular weight between 100 daltons and 10,000 daltons
• the invention can also be used to determine the degree of protein crystallization based on the inherent changes in fluorescence that the protein undergoes during crystallization. It can be used to determine the amount of a small molecule directly bound to serum proteins or free in solution. It can further be used to determine the binding interaction between a small molecule and a protein, solid surface, or nucleic acid.
• FIG. 5 A schematic diagram of an exemplary instrument 50 according to the invention is shown in Figure 5.
• the excitation light source for the system is a high pressure, 75 watt, Xe arc lamp 51 that gives light that is continuous in wavelength over the wavelength of interest (250 nm to 600 nm).
• the lamp is an Oriel Model 6257 in an Oriel series Q lamp housing, Model 60064 with a F/1 fused silica condensing lens.
• the continuous light from this source is focused onto the entrance slit of a 1/8 meter imaging spectrometer 52 (CVI Model CMSP110).
• the spectrometer is rotated 90 degrees to give a dispersed spectrum in the vertical direction as shown in Figure 2.
• the center wavelength of the dispersed spectrum can be set with the spectrometer.
• the dispersed output of spectrometer is then focussed into a sample cuvette 54 within a light box 53 by a fused silica lens system 59 as shown in Figure 5.
• Fused silica allows measurements in the near UV part of the spectrum (220 nm).
• the sample is in liquid suspension in a 1 cm x 1 cm fused silica or UV transmitting plastic cells, however, the system is not limited to measurements in a cell. Measurements can be made of samples on a solid substrate or with samples in a microliter plate.
• the fluorescence from the sample near the waste or best focus of the excitation spectrum at 90 degrees is focussed onto the entrance slit of the detection spectrometer 55 using a set of silica lenses 60.
• the detection spectrometer is identical to the excitation spectrometer except that the exit slit is at 90 degrees to the entrance slit.
• the dispersion of this spectrometer is at 90 degrees to the direction of dispersion of the excitation spectrometer.
• the fluorescence from the sample at each excitation wavelength is dispersed and imaged at the exit plane of the detection spectrometer. This produces the two dimensional spectral signature as previously described. This image is recorded on a two-dimensional CCD array 56.
• the detector was a Hamamatsu cooled, back thinned CCD with 512 by 256 pixels (Model S7032-0908).
• FIG. 6(a) and (b) An example of a spectral signature taken by this instrument is shown in Figures 6(a) and (b).
• the spectra was taken from a solution of 20 ⁇ l FBS (fetal bovine serum) in 1 ml of PBS (phosphate buffered solution). 3 ml of the solution was placed in a standard 1 cm x 1 cm x 3 cm fused silica cell. The cell was placed at the focus of the excitation light and the focus of the detection optics as shown in the figures.
• FBS fetal bovine serum
• PBS phosphate buffered solution
• the excitation spectrometer used a 300 line/mm grating that was set to a center wavelength of 300 nm and the detection spectrometer used a 600 line/mm grating that was set for a center wavelength of 350 nm.
• An integration time of 50 sec was used to obtain the signature.
• Figure 6(a) shows the three-dimensional signature recorded on the CCD array as a function of pixel location.
• the excitation axis has 256 pixels while the fluorescent axis has 512 pixels.
• the z-axis is a measure of the fluorescent intensity.
• the top plane has the projection of the three-dimensional image to produce a color contour plot of the signature.
• a contour plot of the signature as a function of excitation wavelength and fluorescence wavelength is shown.
• the signatures of several biological materials or chemical compounds can be taken and used as a basis set to determine the composition of an unknown solution. This includes an unknown with one or more components of the basis set.
• a variation 30 of the above process and apparatus uses fiber optic bundles 24,24' as shown in Figure 3.
• the excitation light is collected by a linear array of fibers 24 either directly or imaging with a lens system.
• the fibers collect light at different wavelengths.
• the excitation is then transmitted through the fiber to the sample.
• the emerging light is directed onto the sample with or without a lens system.
• the wavelength relationship of the transmitting fibers is preserved.
• the resulting fluorescence is collected by a fiber array 24'. Each collection fiber has a unique relationship with the excitation light.
• the light from the collection fiber is then collected by an imaging spectrometer where the fluorescence is dispersed perpendicular to the slit.
• the fibers are aligned along the slit and are correlated with the excitation light. In this manner a signature that is two dimensional in excitation and fluorescence is measured. If one is to make measurements on very small samples, micro or nanoliters of material, then a continuous excitation spectra on the scale of the samples is preferably generated.
• the excitation and fluorescence can be made co- linear with the use of a beam splitter to introduce the excitation light into the microscope system.
• the fluorescence from the sample as a function of excitation wavelength is imaged onto the slit of the detection spectrograph and is then dispersed onto the two-dimensional CCD array and recorded.

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• Physics & Mathematics (AREA)
• Spectroscopy & Molecular Physics (AREA)
• General Physics & Mathematics (AREA)
• Health & Medical Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

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• 2005
  • 2005-03-16 EP EP05729010A patent/EP1861684A1/de not_active Withdrawn
  • 2005-03-16 WO PCT/US2005/008868 patent/WO2006101484A1/en active Application Filing

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