WO2000043775A1 - Methode de caracterisation d'entites biologiques - Google Patents

Methode de caracterisation d'entites biologiques Download PDF

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Publication number
WO2000043775A1
WO2000043775A1 PCT/US2000/001550 US0001550W WO0043775A1 WO 2000043775 A1 WO2000043775 A1 WO 2000043775A1 US 0001550 W US0001550 W US 0001550W WO 0043775 A1 WO0043775 A1 WO 0043775A1
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Prior art keywords
cells
cell
infrared
entities
dna
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PCT/US2000/001550
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English (en)
Inventor
Max Diem
Jill Bargonetti
Tamara Gopen
Susie Boydston-White
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Cytospec
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Publication date
Priority to US09/868,463 priority Critical patent/US6887711B1/en
Application filed by Cytospec filed Critical Cytospec
Publication of WO2000043775A1 publication Critical patent/WO2000043775A1/fr

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    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light

Definitions

  • the field of the invention is the field of infrared spectra of biological entities such as single exfoliated cells from normal and abnormal patients. Differences in such spectra can be used to detect cancer in samples of cells and tissues, and can be used as a screening test.
  • US Patent 5,596,992 uses infrared spectroscopy to distinguish normal from cancerous leukocytes and other cells by multivariate statistical methods. These studies use highly homogeneous samples and, therefore, have a much higher success in predicting disease from infrared data. However, they have failed to realize a source of spectral heterogeneity that confounds the interpretation of the data, and is due to the stages of cells " reproductive cycle.
  • the infrared vibrational spectra characterizing the presence of DNA in the cells is used to determine the statistical proportion of the cells in a non quiescent state, the that proportion is used to determine if the cells represent a cell population having cancerous or other anomalous characteristics.
  • Figure 1 depicts schematically the stages of the division cycles somatic cells undergo.
  • cells In cancerous cell cultures, cells often cycle directly from one division cycle into the next; i.e.. they are dividing constantly. The width of each slice shown is approximately proportional to the percentage of time the cells spend in a given stage.
  • Figure 2A shows a typical infrared absorption spectrum of a protein film. The peaks are referred to by standard nomenclature of vibrations giving rise to the absorptions.
  • Figure 2B and Fig. 2C show the infrared absorption spectra of DNA and RNA respectively.
  • the horizontal axis (abscissa) describes the wavelength of the infrared radiation, expressed in units of inverse length.
  • the ordinate denotes the amount of light absorbed by a vibration (absorbance) and is presented in arbitrary units. Single cells typically exhibit between 0.05 and 0J absorbance units.
  • Figure 3 shows the infrared traces of DNA and RNA, each superimposed on a protein background spectrum. Protein and nucleic acid intensities are adjusted such that their intensities correspond approximately to the intensity ratios observed in cells and tissues. Note that the resulting spectral composites can be easily distinguished; thus, the spectral patterns in Figure 3 can be used to judge whether DNA or RNA constitutes most of the observable nucleic acid in a cell or tissue.
  • Figure 4 shows the different spectral traces observed for cultured cells separated into different phases of their division cycle by elutriation.
  • Trace A shows the averaged spectrum obtained for an exponentially growing cell culture, that is, a mixture of all cell division phases.
  • Trace B shows a spectrum for cells in the G 1 phase, trace C in the early S phase, trace D in the late S phase and trace E in the G2 phase. Note the differences in the amount of DNA observable between Gl and G2 phase on one hand, and the S phase on the other.
  • Figure 5 shows a comparison between normal and abnormal squamous cells, clustered by cell cycle phase.
  • the traces of normal and abnormal cells are virtually indistinguishable, indicating that some cells in a sample of abnormal cells maintain normal spectral properties.
  • the spectral traces in Figure 5B are believed to be due to the Gl phase.
  • the traces due to abnormal cells agree well with those of the Gl trace observed in Figure 4, trace B, for cancerous ML-1 cells.
  • trace B for cancerous ML-1 cells.
  • Figure 6 shows a microscopic image taken of Eu 3 " stained cells through a fluorescence microscope. The arrow indicates an approximate scale of the display. The Figure demonstrates that excellent separation of cells is easily achieved, and that cells can be localized by their fluorescent emission. This localization of cells is used to speed up data manipulation for the infrared imaging, and may serve as indications whether or not single cells are in the field of view of a given infrared detector element.
  • Figure 7 shows a sketch of the most preferred embodiment of the invention.
  • Figure 8 shows a sketch of a preferred embodiment of the invention.
  • Figure 9 shows a sketch of a preferred embodiment of the invention.
  • Normal cells are found predominantly in a state, referred to as GO, in which no reproduction occurs.
  • the division process is initiated by certain biochemical signals, upon which a number of predetermined processes occur that causes a cell to duplicate itself.
  • This duplication process may take 20 to 30 hours, and can be divided into phases Gap 1 (Gl), Synthesis (S), Gap 2 (G20 and Mitosis (M).
  • Gl Gap 1
  • S Synthesis
  • Gap 2 Gap 2
  • M Mitosis
  • Cancerous cells can be cultured indefinitely if the cells are kept at proper conditions for cell growth. In such cell cultures, the cells re-enter the next division cycle as soon as the previous one is completed, and the number of cells doubles after a period of time corresponding in length to the cell division cycle.
  • Such a cell culture is said to be exponentially growing, and the cells found in a given phase of the cycle, i.e. Gl, S. G2 and M. is determined by the relative length of these stages, which is about 10 hours for the S phase. 8 hours for Gl, 6 hours for G2, and minutes for M.
  • cultured cells can be separated into fractions of good phase homogeneity by a density/size centrifugation called elutriation. Subsequently, fluorescence activated cell sorting (FACS) technology can be used to identify these fractions as Gl, S or G2 phases by the DNA content.
  • FACS fluorescence activated cell sorting
  • infrared spectroscopy the attenuation of the intensity of a beam of infrared light upon passing through a sample is measured. This attenuation is caused by the interaction of the light with the vibrational transitions of the sample molecules. These absorptions of infrared light, when plotted against the wavelength of the light, produce a unique fingerprint pattern of the molecules encountered by the beam of light.
  • the fingerprint pattern is very useful in identifying entities of biological interest, which include but are not limited to cells, proteins, viruses, fluids, etc. Such fingerprint patterns for a number of cellular components are shown later in this specification.
  • infrared spectral patterns i.e., we have demonstrated before that a nucleus of a quiescent cell is optically so dense that it may not transmit any of the incident infrared radiation, and hence no infrared spectral features due to the cell nucleus are measurable. Consequently, infrared spectroscopy and the appearance of spectral feature due to the nucleus may be used to monitor nuclear processes which result in significant changes in the packing of the constituent molecules.
  • infrared microspectroscopy also referred to as infrared microscopy
  • the infrared beam is passed through the specimen and focused by an infrared microscope that allows infrared spectral data to be collected from microscopic particles, such as single cells, or pixels of tissue the size of a cell.
  • Protein infrared absorption spectra are known to vary with the secondary structure of the protein (a-helical, pleated sheet, random, etc.), the protein's state of hydration, the solvent's ionic strength, etc. However, the averaged infrared spectra of all metabolic and structural proteins found in cells turn out the be remarkably the same for most cells. The only proteins that exhibit distinctly different spectra are found in connective tissue (e.g., collagen).
  • the averaged protein spectra found inside cells are dominated by the amide I band at ca. 1650 cm “1 , the amide II vibration at 1530 cm “1 , the amide III peak at 1245 cm “1 , and a number of side chain vibrations in the 1310, 1390 and 1450 cm “1 range.
  • the ionized PO 2 " and ribose groups exhibit a triad of peaks that occur in DNA at 1071, 1084 and 1095 cm “1 with nearly equal intensities.
  • DNA peaks are observed at 965 and 1245 cm “1 (the phosphodiester vibration).
  • the peak at 1085 cm “1 is stronger than the two other peaks in this triad, and forms a distinct "nose”.
  • the intensity ratio of this triad to the phosphodiester peak at 1245 cm “1 is about 1 : 0.7, whereas it is about unity in DNA. Since nucleic acid vibrational spectra in cells in tissues are always observed in the presence of protein, we show in Figure 3 expanded regions of the DNA and RNA spectra superimposed on protein spectra. In cells, DNA is found in the nucleus and in mitochondria.
  • RNA on the other hand, can occur in the cytoplasm as ribosomal or transfer RNA (r-RNA and t-RNA), and in the nucleus and cytoplasm as messenger RNA (m-RNA).
  • r-RNA and t-RNA ribosomal or transfer RNA
  • m-RNA messenger RNA
  • the cytoplasm is relatively rich in various RNA species, whereas the nucleus contains nearly all the DNA.
  • RNA and protein in such a nucleus is quite high, and leads to an optical density of the nucleus in excess of 1 absorbance unit.
  • DNA is not distributed uniformly throughout the nucleus; rather, it is wrapped tightly around proteins known as histones.
  • the local concentration of DNA and protein is even higher, and it is likely that the chromosomes of an inactive nucleus will appear as "black" (i.e., opaque or nearly opaque) strings.
  • carbohydrates in the form of polymeric sugars or glycoproteins
  • phospholipids in the form of polymeric sugars or glycoproteins
  • water and a few other compounds may occur in cells and tissues at levels that make them detectable via infrared spectroscopy.
  • trace A shows infrared absorption spectra collected from an exponentially growing cell culture of myeloid leukemia (ML-1) cells, and cells fractionated into the cell cycle phases by elutriation. Identification of the cells in a given fraction was performed by fluorescence activated cell sorting (FACS) analysis.
  • FACS fluorescence activated cell sorting
  • the S phase spectra in Figure 4 (trace C) resembles that of exponentially growing cells in that it exhibits strong vibrations due to DNA.
  • the spectra of cells in the Gl and G2 phases are similar to each other, and very different from the S phase spectrum in the low frequency region.
  • the shape of the peaks around 1070-1 100 cm “1 (the "nose at 1085 cm “1 ) suggests that in Gl and G2 phases, mostly RNA is observed.
  • the results for cells in the Gl and G2 phases confirmed the hypothesis of opaque DNA: Since cells in the Gl phase are diploid (2 copies of DNA), whereas cells in the G2 phase are quadruploid (4 copies of DNA), one would observe stronger DNA signals if the nuclear DNA was detectable.
  • Figure 5 A shows a comparison between healthy and diseased single cell spectra that are virtually superimposable.
  • the low nucleic acid / protein ratio of these spectra suggests that the cells are not dividing actively (GO or Gl stages).
  • reference spectra for the pure GO phase have not been observed in the ML- 1 cells reported above, since they cycle continuously and never reach the inactive phase GO. In normal samples most cells should be found in GO. and even in cancerous cells, the majority of all cells should be in GO.
  • Figure 5 B shows the differences between normal and abnormal cells in a phase whose spectra resemble those observed for the middle of the S phase.
  • the spectra differ enormously in the intensity of the peak at 1230 cm “1 to the peak at 1300 cm “1 .
  • the former of these contains protein and DNA, whereas the latter is nearly a pure protein peak.
  • any increase of the 1230/1300 cm '1 peak intensity ratio is an indication of the visibility of the DNA. This is further confirmed by the appearance of a sharp DNA peak at 970 cm "1 in the cancerous samples. Comparing individual spectra from normal and abnormal samples, spectral pairs similar to the ones depicted in Figures 5A and 5B can be found.
  • Each spectral pair is thought to arise from a given phase in the cell cycle, and the spectra always differ in the amount of spectral contribution of DNA. This is an indication that normal and cancerous single cells in a number of different stages of their reproductive cycle, can be distinguished by their detectable DNA content.
  • the intensity ratio of the peaks at 1230/1300 cm "1 is not a uniformly applicable indicator of disease.
  • any cells actively involved in replication or transcription has a higher visible DNA content than inactive cells.
  • cell cycle dependent data of cells from various organs need to be collected to create a baseline of how much DNA contribution constitute an abnormality.
  • Epithelial cell samples can be derived by scraping the surface of the epithelium with suitable devices such as brushes, spatulas, etc., used presently to collect specimens.
  • Samples from internal organs can be obtained by thin needle aspiration, needle biopsies or surgical biopsies.
  • Cellular components of body fluids can be isolated directly from these body fluids. Standard methods, such as digestion with collagenase to break up tissues into individual cells, is utilized to obtain single cells suitable for spectroscopic analysis.
  • Other biological specimens such as proteins and fragments of DNA, RNA, or other molecules may be obtained by methods very well known in the art.
  • exfoliated cells may contain cells at different stages of maturation. whereas lymphocytes and leukocytes may be found at different stages of maturation and differentiation.
  • Cells derived from tissue sections may contain endothelial cells (for example, from blood vessels). This heterogeneity is expected and will not present a significant problem if the spectral data are collected on a cell-by-cell basis, rather than an averaged spectral collection.
  • the cells or other entities obtained as described above are treated to remove impurities due to blood, lymph, mucus or other confounding constituents. Simple separation procedures are useful to enhance the percentage of desirable entities for the investigation.
  • the spectroscopic sample for the most preferred embodiment consists of a partial layer of cells with good separation between the cells.
  • 10 4 cells each occupying about 2 x 10 "4 mm 2 and distributed uniformly on an area of 50 mm 2 , result in a sample partial layer with an occupation of about 5 % of the surface of the substrate.
  • Such a sample is suitable for analysis by the method of the invention, since the infrared absorption spectrum of each cell alone may be recorded.
  • the samples reported by Zakim and Lord often had sample populations 1000- 10000 fold higher. The high population creates non-linear absorption effects, retains the solvent in a manner that cannot be controlled, and may be responsible for many of the artifacts reported by them.
  • the spectrum of mostly single entities should be recorded. Overlapping a cancerous with a non cancerous cell, for example, would lead to a spectrum which is not definitive for either type of cell.
  • Cells are most preferably visualized by staining with fluorescent, monatomic dyes (for example, isotonic Eu 3+ ion solution). Such staining does not perturb the infrared spectra within the limits of detect ability, but permits the cells to be detected using fluorescence excitation. Identification of cells is necessary to avoid data collection from cell debris. An image of a typical cell sample under fluorescence excitation in the most preferred embodiment of the invention is shown in Figure 6 which demonstrates the excellent separation of cells in this sample preparation procedure.
  • the area of the substrate (ca. 50 mm 2 ) that contains the cells is imaged using fluorescent and infrared wavelengths as follows.
  • the substrate is inserted in the focal position 70 of a combination infrared microspectrometer (also known as an infrared microscope) and fluorescence microscope shown schematically in figure 7.
  • Such instrumentation is commercially available.
  • Fig. 7 shows is the optical arrangement for the infrared/fluorescent microscope combination which is the most preferred embodiment of the invention.
  • Infrared light from the interferometer is passed through a Cassegrain condenser 73 A into the sample, collected via an identical Cassegrain objective 73B, and focused with an infrared transmitting lens such as a ZnSe lens 77 onto the confocal array detector 71. Reflecting optics could be used as well in place of the ZnSe lens.
  • In order to reduce computation time for the analysis of up to 10,000 individual cells only pixels that contain cellular spectral information is processed. This information is obtained by imaging the sample via fluorescence microscopy prior to infrared data acquisition. To this end, the cells are illuminated by introducing an ultraviolet beam of light 74 from a UV lamp 75 into the optical train using a movable mirror 78.
  • the UV light induces intense fluorescence from the cells in the focal region 70 that have been treated with Eu 3+ ions. Since this stain consists of monatomic ions, it does not exhibit an infrared absorption spectrum; consequently, there is no spectral difference in the infrared spectrum due to stained and unstained cells.
  • the fluorescent light collected in the reflection mode as shown in fig. 7 is filtered to remove the excitation wavelength by a movable dichroic mirror 75, and is processed via a CCD detector 76. This image taken by the fluorescence light will indicate, by the fluorescent intensities, the position of cells on the sample substrate (cf. Figure 6).
  • the UV fluorescent image may equally well be taken in transmission mode, and/or the spectra of the cells may be taken (at less resolution) in reflection mode after reflection from an IR reflecting substrate holding the cells
  • the analysis of the cells on the substrate proceeds as follows.
  • the sample substrate is first illuminated with UV light that is absorbed by the Eu 3+ stain with which the cells have been treated.
  • the fluorescence induced by the UV excitation is observed in reflection mode of the microscope, and collected through selective bandpass filters to reject the excitation wavelength.
  • the fluorescence of the stained cells is detected by the CCD camera, and the positions of all cells on the substrate is computed by the positions of the pixels of the CCD camera detecting the fluorescent radiation.
  • the illumination of the sample is switched to infrared radiation.
  • the movable mirrors 75 and 78 are removed from the optical path of the microscope and the infrared light is passed through a step-scanning interferometer (not shown) to modulate the wavelength patterns of the light prior to being directed into the microscope.
  • the movable mirror 78 and the dichroic mirror 75 could be formed on IR transparent substrates to reflect the appropriate light and transmit the infrared light.
  • Each of the detector elements of the confocal infrared array detector 71 is exposed to the infrared radiation passing through the sample cells which are imaged 1 : 1 onto the detector resulting in about 1 cell or less per detector element.
  • Interferograms are collected for all 65536 detector elements, and converted to infrared absorption spectra via a mathematical process known as Fourier transform (FT). Only the interferograms from detector pixels onto which cells are imaged are Fourier transformed to save data analysis time. These pixels are identified by the fluorescence picture obtained in the previous step. Selecting only picture elements that are known to contain spectral information reduces the number of individual spectra to be calculated from the interferograms from 65000 to between 10,000 and 20,000.
  • FT Fourier transform
  • the resulting spectra are uniformly expanded, smoothed and corrected for water vapor abso ⁇ tion.
  • the spectra (or their derivative spectra) are clustered using vector correlation methods.
  • the clustering reveals the degree of "relatedness" of spectral patterns, and will reduce the number of independent observations to a few dozen spectral patterns for each sample.
  • certain spectral traces that are clearly associated with cells of low information content are discarded. For example, spectra from red blood cells or fully mature. inactive cells may be discarded.
  • the resultant spectral patterns referred henceforth as the "reduced spectral set" is analyzed as follows.
  • the reduced spectral set is compared to the cell cycle dependent reference sets that have been collected beforehand for the organ sample under investigation. This analysis reveals whether or not the distribution of the cell cycle stages falls within normal limits. Subsequently, all spectra in the reduced data set are searched for the occurrence of spectral patterns associated with a fast growing and rapidly dividing cell. The occurrence of such cells in a sample is indicative of abnormality.
  • the analysis described above requires knowledge of the cellular spectra, and cellular distribution, observed for normal samples.
  • the analysis depends to a lesser degree on bases (reference) sets than the one described by Zakim and Lord, which used spectral averages over large number of cells. It is clear that such an averaging process reduces the sensitivity of the method enormously, since only a small number of cells exhibit spectral traces modified by disease.
  • the method presented here uses both a statistical analysis of the spectral patterns of the individual cells (i.e.. changes in the distribution of cells at various stages of their development), as well as variations in the spectral patterns due to disease, for a vastly enhances sensitivity of the infrared spectral method.
  • a preferred embodiment of the invention is sketched in Fig. 8.
  • a specimen generator 80 produces a plurality of specimen 82 in the same fashion as a FACS machine or an ink jet printer.
  • the specimen 82 may be droplets containing the entities to be measured, or may be cells or other biological entities.
  • the specimen are produced and fly through an atmosphere with a defined velocity.
  • the atmosphere may be ordinary laboratory air, or may be air with a controlled water vapor pressure or partial pressure of another vapor such as alcohol, or may be an inert gas such as nitrogen or argon, or vacuum.
  • the specimen are generally electrically charged so that the specimen generator may accelerate them to a relatively high speed. However, such electrical charging is optional.
  • the specimen are optionally marked with a fluorescent marker as detailed above for the marking of cells, and UV light source 81 and fluorescence detector 83 may be used to mark the position of a specimen 82, or indeed may be used to decide whether specimen 82 contains entities to be investigated.
  • the position and speed of each specimen is now known.
  • a vibrational spectrum recording apparatus is now used to record the spectrum of each specimen, and the resulting plurality of spectra is treated as detailed above to characterize the statistics of all the specimen.
  • the preferred vibrational spectrum recording device is two infrared light sources 84 and 85 which direct infrared light on to a specimen 82C, and two detectors 86 and 87 which measure the light transmitted through specimen 82C.
  • Light sources 84 and 85 are preferably pulsed laser sources which produce infrared light at frequencies characterizing the infrared abso ⁇ tion spectrum of DNA and RNA respectively. A plurality of such lasers may be used to produce an entire vibrational spectrum for each specimen as detailed in the most preferred embodiment.
  • a computer 88 is shown controlling specimen generator 80 and infrared sources 84 and 85, and monitoring fluorescence detector 83. Lines indicating control and monitor functions for light source 81 and detectors 86 and 87 are not shown to avoid complexity in fig. 8.
  • a preferred embodiment of the invention is shown in figure 9, where the separate infrared light sources 84 and 85 are replaced by a broad band infrared light source 94.
  • the infrared light from the source 94 which is preferably a pulsed broad band laser, is focused on a specimen 82C which has been marked as noted above for figure 8.
  • the broadband infrared light is then analyzed spectrally as indicated in fig. 9 by a focusing grating 96 which images light transmitted through the cell onto an array detector 98 according to the infrared wavelength. Rays of two different infrared wavelengths are traced in fig. 9 in order to guide the eye.
  • the grating 96 could be replaced by an imaging system used to image the light from the specimen 82C on to the slit of a normal spectrometer and infrared recording array detector, or indeed by any other spectral recording device as known in the spectroscopic art. Lenses conventionally shown in fig. 8 and 9 are figurative only, and imaging and light handling devices as known in the art are anticipated by the inventors.
  • vibrational spectrum recording devices of the invention are not limited to the devices described above, but may be any vibrational spectrum recording devices known in the spectroscopic art to record IR abso ⁇ tion spectra or Raman spectra. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, withing the scope of the appended claims, the invention may be practiced otherwise then as specifically described.

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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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Abstract

L'invention se rapporte à un appareil et à une méthode permettant de mesurer les caractéristiques spectrales vibratoires dans l'infrarouge de chacune des entités d'une pluralité d'entités telles que des cellules, et de déterminer statistiquement à partir de ces mesures la présence d'anomalies telles qu'un cancer.
PCT/US2000/001550 1999-01-22 2000-01-22 Methode de caracterisation d'entites biologiques WO2000043775A1 (fr)

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US60/116,755 1999-01-22

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008110974A1 (fr) * 2007-03-13 2008-09-18 Koninklijke Philips Electronics N. V. Analyse d'un materiau cellulaire biologique en fonction des diffrentes interactions entre une lumiere d'eclairage et des composants cellulaires
CN104990930A (zh) * 2015-07-09 2015-10-21 中国科学院上海光学精密机械研究所 光学元件缺陷激光近场调制检测装置及诱导损伤预测方法

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US3704342A (en) * 1970-01-22 1972-11-28 Dynarad Infrared scanning system
US4922104A (en) * 1987-11-30 1990-05-01 501 Hitachi, Ltd. Infrared microspectrometer
US5596992A (en) * 1993-06-30 1997-01-28 Sandia Corporation Multivariate classification of infrared spectra of cell and tissue samples
US5733739A (en) * 1995-06-07 1998-03-31 Inphocyte, Inc. System and method for diagnosis of disease by infrared analysis of human tissues and cells
US5733507A (en) * 1995-06-07 1998-03-31 Inphocyte, Inc. Biological cell sample holder for use in infrared and/or Raman spectroscopy analysis holder
US5841139A (en) * 1997-02-28 1998-11-24 Bio-Rad Laboratories, Inc. Optical instrument providing combined infrared and Ramen analysis of samples

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3704342A (en) * 1970-01-22 1972-11-28 Dynarad Infrared scanning system
US4922104A (en) * 1987-11-30 1990-05-01 501 Hitachi, Ltd. Infrared microspectrometer
US5596992A (en) * 1993-06-30 1997-01-28 Sandia Corporation Multivariate classification of infrared spectra of cell and tissue samples
US5733739A (en) * 1995-06-07 1998-03-31 Inphocyte, Inc. System and method for diagnosis of disease by infrared analysis of human tissues and cells
US5733507A (en) * 1995-06-07 1998-03-31 Inphocyte, Inc. Biological cell sample holder for use in infrared and/or Raman spectroscopy analysis holder
US5841139A (en) * 1997-02-28 1998-11-24 Bio-Rad Laboratories, Inc. Optical instrument providing combined infrared and Ramen analysis of samples

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008110974A1 (fr) * 2007-03-13 2008-09-18 Koninklijke Philips Electronics N. V. Analyse d'un materiau cellulaire biologique en fonction des diffrentes interactions entre une lumiere d'eclairage et des composants cellulaires
CN104990930A (zh) * 2015-07-09 2015-10-21 中国科学院上海光学精密机械研究所 光学元件缺陷激光近场调制检测装置及诱导损伤预测方法
CN104990930B (zh) * 2015-07-09 2017-10-20 中国科学院上海光学精密机械研究所 光学元件缺陷激光近场调制检测装置及诱导损伤预测方法

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