EP1303750A1 - Procede et dispositif d'analyse de coincidence par fluorescence a deux photons polychrome - Google Patents

Procede et dispositif d'analyse de coincidence par fluorescence a deux photons polychrome

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Publication number
EP1303750A1
EP1303750A1 EP01969428A EP01969428A EP1303750A1 EP 1303750 A1 EP1303750 A1 EP 1303750A1 EP 01969428 A EP01969428 A EP 01969428A EP 01969428 A EP01969428 A EP 01969428A EP 1303750 A1 EP1303750 A1 EP 1303750A1
Authority
EP
European Patent Office
Prior art keywords
fluorescence
sample
markers
excitation
analysis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP01969428A
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German (de)
English (en)
Inventor
Katrin Heinze
Petra Schwille
Andre Koltermann
Ulrich Kettling
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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Publication of EP1303750A1 publication Critical patent/EP1303750A1/fr
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • 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
    • G01J2003/2866—Markers; Calibrating of scan
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/13—Tracers or tags

Definitions

  • the invention relates to a method for fluorescence correlation analysis, in particular methods for coincidence or cross correlation analysis on analytes marked with at least two different fluorescent markers in a sample, and measuring devices for carrying out the methods mentioned.
  • Fluorescence correlation spectroscopy is generally known as a highly sensitive optical method for the detection of dynamic properties of individual molecules or molecular compounds or of the lowest concentrations of fluorescent substances.
  • a laser beam is coupled into the sample with a microscope and focused on a measuring volume of approx. 10 "15 1 (1 fl).
  • the measuring volume is so small that, on average, less than a fluorescent one
  • the fluorescence of the sample molecules of interest is detected by a correlation analysis of the detector signals.
  • the microscope is designed for three-dimensional positioning of the measurement volume, as is possible with a confocal microscope, for example.
  • a laser beam for fluorescence excitation is focused on a diffraction-limited point in the sample.
  • a point diaphragm in the image plane, in which the excitation point is shown, serves as a field diaphragm, with which fluorescence and scattered light, which emanates from locations outside the focus, are masked out dynamic molecular properties (diffusion coefficient) in the pro- be determined.
  • relatively high excitation intensities in the range of approx. 100 kW / cm 2
  • the detector signals are subjected to a cross-correlation or coincidence analysis.
  • concentration of the sample and the size of the measuring volume are selected so that at most one molecule is present in the measuring volume at one point in time.
  • temporal correlations or coincidences in the detector signals it can be determined whether there was an analyte with one or the other or both marker dyes in the measurement volume at the time of measurement.
  • molecular association or dissociation processes such as e.g. B. the formation or breaking of chemical bonds are measured in real time.
  • the two-color technique according to WO 99/34195 also has disadvantages which limit the applicability and the accuracy of the method.
  • Various lasers 21 ', 22' are usually required to excite the fluorescence emissions of the marking dyes, the foci of which must be formed at a measuring point in a stable manner over time and with an accuracy of fractions of a femtoliter. A considerable amount of experimentation is required to adjust and stabilize the excitation laser. Furthermore, in order to achieve a sufficient spatial resolution in the radiation direction (z direction), the imaging system must have a pinhole on the imaging side, onto which the measurement volume is imaged. Another limitation concerns the available dye systems. The marking dyes must have high light stability at all excitation wavelengths. In addition, the marking dyes used must have high quantum yields.
  • the object of the invention is to provide an improved method for fluorescence measurement based on a cross-correlation and / or coincidence analysis, with which the disadvantages, in particular of the conventional two-color technique, are avoided become.
  • the method according to the invention is to be implemented with a simplified measurement setup, without having to accept restrictions in terms of accuracy and stability.
  • the object of the invention is also to provide improved correlation and / or coincidence measuring devices for fluorescence measurement with a simplified structure.
  • the basic idea of the invention is to illuminate the sample for correlation fluorescence measurement on analytes with at least two fluorescence markers on one or more substances to be analyzed with such a high excitation intensity (photon flux density) that the fluorescence excitation of the fluorescence markers by 2-photon absorption he follows.
  • the sample is preferably illuminated with a single laser line.
  • the laser beam is focused into the sample at the desired location in the measurement volume.
  • the fluorescence markers are excited simultaneously at a common excitation wavelength, but have spectrally separated fluorescence emissions that are detected with different detectors.
  • the signals from the detectors are subjected to a correlation analysis (coincidence or cross-correlation analysis).
  • the 2-photon excitation of fluorescence markers has the advantage that fluorescence markers can be used which have similar maxima in the excitation spectra of the 2-photon excitation, but which are characterized by different Stokes shifts in the emission.
  • the fluorescence measurement is aimed at a single-molecule-based analysis, in which the measurement or observation volume is so small that fluorescence fluctuations from individual molecules can be detected and evaluated.
  • fluorescent markers in particular fluorescent dyes
  • the fluorescence markers for the correlation fluorescence measurement are preferably excited at an excitation wavelength at which both fluorescence markers have essentially the same fluorescence photon yield after 2-photon absorption. Since the fluorescence photon yield, defined as the count rate, which is detected per unit of time and per molecule, depends in particular on the ambient conditions (for example absorption state of the fluorescent markers, solvents and the like), a preliminary test is preferably carried out before the fluorescence measurement to determine the optimal excitation wavelength. The preliminary test is carried out once for a specific measuring system or several times before each fluorescence measurement.
  • the measurement setup is considerably simplified.
  • the simplification is that only one laser has to be used for excitation.
  • the experimental set-up is further simplified since the excitation volume of the 2-photon excitation in the direction of propagation of the laser beam (z-direction) is reduced compared to the excitation volume for 1-photon excitation.
  • the probability of 2-photon absorption depends on the square of the excitation intensity.
  • the absorption cross section is therefore reduced proportionally to z ⁇ 4 .
  • There is an inherent concentration of excitation at the focal level It is not absolutely necessary to map the measurement volume to a pinhole, since there is no flow outside the focal plane anyway. orescent light is emitted in the spectral regions of interest.
  • Another important advantage of the 2-photon excitation according to the invention is the high tolerance of biological materials (cells, cell components or cell assemblies) to infrared radiation.
  • biological materials cells, cell components or cell assemblies
  • Due to the long-wave excitation there is a further advantage for the signal-to-noise ratio, since excitation and emission light are spectrally far apart, so that disturbing stray light can be largely suppressed by optical filters without losing part of the emission light to be detected.
  • Another advantage is the reduction of false light, which mainly concerns the fluorescence of contaminants ("dirt").
  • This fluorescence is essentially critical in the short-wave visible range, ie in the case of 1-photon excitation.
  • impurities are excited with significantly less efficiency, so that the signal-to-noise ratio - compared to 1-photon excitation - is significantly higher.
  • the invention also relates to a measuring device for fluorescence measurement on analytes with at least two different fluorescence markers, in which the illumination device is formed by a single laser line which is designed to excite 2-photon absorptions of the fluorescence markers.
  • Another important feature of the device according to the invention consists in the provision of two detector devices which are set up to detect the fluorescence emission in different spectral ranges and on which the whole of the sample (in particular the excitation lumen and also fluorescent light emanating from the area surrounding the excitation volume). The detection takes place without an aperture, a pinhole aperture is not provided. A non-confocal mapping of the excitation volume onto the detectors is provided.
  • the 2-photon excitation with a single laser not only reduces the expenditure on equipment. There are also advantages for the optical adjustment. The problem of size and overlap of excitation volumes is excluded. Additional detection shutters are not required. If fluorescent dyes are used as markers, there is a further advantage in the fact that, after the 2-photon excitation, practically no triplet states are assumed, so that no signal losses occur via the triplet formation.
  • the excitation volume in the measurement method according to the invention is smaller than in the conventional 1-photon excitation. This enables measurements to be carried out at higher sample concentrations of approximately 100 nM, which has advantages for the further evaluation of the results. Concentrations in the nM range can also be determined. Short analysis times in the range of one or a few seconds are made possible. Measurements in living cells are made possible, which enables the exact determination of kinetics and concentrations of double-labeled molecules or complexes.
  • the 2-photon excitation offers the following advantages in particular: There is a physically perfect overlap of the excitation volume elements for both fluorophores.
  • the excitation volume element can counter can be reduced using conventional methods, ie measurements of higher concentrations (100 nM and higher) are possible. Detection takes place without a pinhole (excitation volume element is small enough due to 2-photon excitation). Multi-color detection of three or more fluorophores on a single-molecule basis is possible, ie monochromatic excitation via 2-photons with z.
  • FIG. 1 shows a schematic overview of a measuring device according to the invention
  • FIG. 2 shows an illustration of molecular processes which can advantageously be detected with the correlation measurement according to the invention
  • FIGS. 4, 5 measurement results to illustrate the quantum yield of marking dyes as a function of the excitation wavelength and the excitation power
  • FIGS. 6, 7 representations of curves to illustrate the accuracy and selectivity of the correlation measurement according to the invention
  • 8 shows graphs of an enzymatic degradation of a substance observed according to the invention
  • FIG. 9 shows a schematic overview of a conventional measuring device for two-color correlation measurement (prior art).
  • the invention is described below with reference to 2-photon excitations in test systems with two fluorescent markers. Corresponding implementations of the invention result in multi-color applications. Three or more suitable fluorophores can be excited for emission with a monochrome 2-photon excitation. This allows the measurement of complex molecular and cellular processes in which more than two analytes are involved.
  • the optical structure of a 2-photon fluorescence correlation spectrometer according to the invention is illustrated schematically in FIG. 1.
  • the spectrometer 100 comprises a sample chamber 10, an illumination device 20, a detector device 30, a correlator 40 and an imaging system 50.
  • the imaging system 50 is preferably formed by an inverted microscope construction (eg with an Olympus IX70 microscope).
  • the sample chamber 10 is an application-selected container, in which the sample 11 is arranged at rest or flowing.
  • the sample 11 is a solution or suspension of the substances or particles to be examined. It can be provided that the sample chamber 10 is arranged to be movable in one or more spatial directions. The agility of the
  • the sample chamber can rely on a scan movement relative to the imaging system 50 for taking three-dimensional images, e.g. B. three-dimensional concentration distributions in the sample. It is also possible to use the sample chamber 10 to impress a periodic modulation movement as described in WO 99/34195.
  • the wall of the sample chamber 10 facing the imaging system 50 has such a small thickness that the focus 12 of the excitation light can be formed by the objective 51 at a short distance of approximately 400 to 500 ⁇ m.
  • the corresponding wall preferably has the thickness of a cover slip, as is used in microscopy. The thickness is, for example, approx. 150 to 190 ⁇ m.
  • the illuminating device 20 is a single laser which is designed for the 2-photon excitation of the fluorescence markers used in each case.
  • a tunable pulse laser is preferably used, such as.
  • the parallel laser light is directed via the dichroic mirror 52 (eg of the type 710 DCSPXR, AHF Analysentechnik, Tübingen, Germany) into the lens 51 (eg 60 x 1.2 lens UplanApo Olympus) and in the sample chamber 10 focused.
  • the dimensions of the excitation volume r 0 and z 0 in the focal plane are known from calibration measurements.
  • the diameter of the focus in the focal plane is z. B.
  • r 0 0.48 ⁇ m.
  • a fluorescence emission is excited in this excitation volume, which is emitted via the objective 51, the dichroic mirror 52, an emission filter 53 (e.g. of the type 600 DF 200, AHF analysis technology) to suppress the excitation light and an optics 54 a second dichroic mirror 55 (e.g. type 595 DCLP, AHF analysis technology) is directed.
  • the short-wave part of the fluorescent light is reflected at the second dichroic mirror 55 and is passed through a bandpass filter 56 onto the long-wave Leader detector 31 directed to the detector device.
  • the fluorescent light transmitted through the dichroic mirror is also filtered (edge filter 57) and directed onto the shorter-wave detector 32.
  • the coupling into the detectors takes place with optical fibers 58 or 59.
  • the detectors are, for example, avalanche photodiodes (type: SPCM-200, EG & G Optoelectronics, Canada).
  • the optical coupling fibers have a diameter of 100 ⁇ m and are individually adjustable in all three spatial directions.
  • the detectors 31, 32 are connected to a correlator 40.
  • a correlator card type: ALV-5000, manufacturer LAV Langen, Germany
  • the coupling fibers can also be dispensed with and the fluorescent light can be imaged directly on the detectors.
  • the optical structure can be equipped with a fiber-coupled spectrometer (manufacturer Ocean Optics, USA).
  • the sample 11 in the sample chamber 10 contains at least two analytes marked with different fluorescent markers and / or at least one analyte marked with at least two fluorescent markers.
  • the subject of the fluorescence measurement according to the invention is, for example, a coincidence analysis of the fluorescence emissions of the different fluorescence markers detected with the detectors 31, 32. This is illustrated schematically in FIG. 2.
  • the sample contains, for example, the analytes AI and A2, which are each marked with fluorescent markers M1 and M2.
  • the analytes are, for example, pairs of antibodies and antigens, the binding behavior of which is to be examined. As long as the analytes AI and A2 are not bound to each other, they pass through the excitation volume separately at different times.
  • the detectors 31, 32 deliver fluorescence signals separated in time, which are symbolized schematically in FIG. 2 (left, center) by arrows P1, P2.
  • the fluorescence signals are measured uncorrelated at any time. relations or coincidence signal G cannot be derived.
  • a correlation or coincidence signal can be derived accordingly (FIG. 2, bottom right).
  • the decomposition of the analyte A3 into subcomponents can also be detected, as is of interest, for example, when observing the enzymatic degradation of a substrate labeled twice with fluorescent markers.
  • the measuring method according to the invention preferably captures all chemical reactions or physical processes in which a chemical bond is established between separate analytes or an existing bond is cut open or a physical association or dissociation is carried out accordingly. All analytes (substances) that can be marked with fluorescent markers on the different sides of the compound to be produced or separated are accessible to the measuring method.
  • the signal detection with the detectors and the correlation analysis are carried out in a manner known per se from the FCS techniques.
  • a fluorescence measurement is carried out with the detectors in predetermined time windows.
  • the width of the time window is chosen depending on the application. It is preferably set to the mean residence time of the analytes in the measurement volume.
  • the length of stay is particularly dependent on the molecular or particle size and mobility and can be measured or theoretically estimated.
  • the photon numbers recorded in the time windows are
  • a concentration measurement is possible on the basis of the coincidence analysis.
  • a measure of the number of double-labeled molecules or particles in the sample is derived from the strength of the detected coincidences (amplitude of coincidence signals).
  • the cross-correlation or coincidence analysis of the detector signals carried out with the correlator 40 is preferably carried out in a known manner, as described in WO 99/34195.
  • the details of signal analysis disclosed in this patent application are fully incorporated by reference into the present description.
  • a relative movement is set between the sample and the illumination device during the fluorescence analysis by means of a beam scanner and / or a sample drive.
  • the fluctuation movements increase and the diffusion times become shorter.
  • the measuring volume element can be scanned through the sample. If this relative movement is set, the time window of the coincidence analysis may have to be adjusted.
  • Fluorescent dyes such as are known, for example, from fluorescence microscopy are preferably used as fluorescence markers M1, M2.
  • Dye pairs are selected which have similar absorption cross sections at a selected wavelength and which have spectrally separable fluorescence spectra with high photostability.
  • the marker pairs are, for example, the dyes rhodamine green / Texas red, fluorescein derivatives (eg Alexa 488 / Alexa 594) or molecular biological dyes such as green fluorescent proteins (GFP) / red fluorescent proteins (RFP) used.
  • GFP green fluorescent proteins
  • RFP red fluorescent proteins
  • autofluorescent proteins such as GFP, dsRED, autofluorescent biomolecules, e.g. As tryptophan, tyrosine, or flavins, or autofluorescent organic molecules can be used.
  • the method according to the invention can also be designed to detect Raman scattering or surface enhanced Raman scattering (SERS).
  • FIG. 3 shows the spectral properties of the marker system rhodamine green / Texas red. Both dyes show a similarly high fluorescence photon yield and sufficient light stability to tolerate the excitation intensities used according to the invention.
  • the spectra (1) and (2) show the fluorescence emissions of rhodamine green and Texas red ( ⁇ M solutions) at an excitation wavelength of 830 nm.
  • Curve (3) shows the transmission curve of the dichroic mirror 55. In the region of the shorter-wave Fluorescence (1) results in the reflection to the detector 31.
  • the curves (4) and (5) show the transmission characteristics of the filters 56 and 57, respectively, which are intended to further improve the signal-to-noise ratio, but not a mandatory feature of the invention are.
  • the excitation of the 2-photon absorptions takes place at a predetermined excitation wavelength, which is selected as follows. After determining the excitation spectra of the fluorescent markers used (see FIG. 3), the fluorescence photon yield is determined for each fluorescence marker as a function of the excitation wavelength. Since the excitation spectra of the fluorescence markers overlap, there are also overlapping curve profiles of the wavelength-dependent fluorescence photons. prey.
  • the optimal wavelength is selected according to the wavelength or the wavelength interval in which the fluorescence photon yields of the two fluorescent markers essentially match or the deviation between the fluorescence photon yields is less than a predetermined ratio, e.g. B. is less than factor 3. This is illustrated below using the example of fluorescent dyes.
  • FIGS. 4 and 5 illustrate spectral properties of the marker pair rhodamine green / Texas red.
  • an excitation spectrum in the range 740 nm to 900 nm is recorded for each dye.
  • the curve profiles in FIG. 4 show an excitation maximum at 780 nm for Texas red (crosses) and at 850 nm for rhodamine green (triangles).
  • an excitation wavelength is selected in which both dyes can be excited with almost the same efficiency and in which both dyes exhibit comparatively strong fluorescence emissions. In the example shown, the excitation wavelength is 830 nm.
  • Figure 5 shows that the excitation at 830 nm actually causes 2-photon absorption.
  • the fluorescence intensity as a function of the excitation power was measured separately for both dyes. For both dyes below the saturation limit there is the expected square dependence of the fluorescence intensity on the incident power for 2-photon processes.
  • the double logarithmic representation provides the corresponding linearized form with slope 2.
  • FIG. 6 shows the course of autocorrelation curves, which were recorded with a test solution from Rhodamin Grün in the two detection channels, and a corresponding cross-correlation curve between the two detection channels. All three curves are essentially the same. This shows that the detection volumes are identical or the detection beam paths are precisely adjusted to the excitation volume.
  • Cross-correlation measurements on double-labeled (upper curve) and single-labeled (lower curve) DNA samples are illustrated in FIG. 7.
  • One advantage of the measurement method is that cross-correlation signals G result for the non-correlated samples, which are less than 10% of the corresponding correlated signals.
  • the structure according to the invention is thus superior to the conventional 1-photon measurements.
  • FIG. 8 illustrates a preferred application of the measurement method according to the invention for determining concentrations in the sample.
  • the real-time measurement of enzyme kinetics is shown.
  • a double-labeled substrate (DNA sample) is broken down enzymatically into individually labeled products. Accordingly, the number of double-labeled molecules detected decreases over time. With increasing concentration of the added enzyme (endonuclease EcoRI), the decrease in substrate concentration is accelerated.

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Abstract

L"invention concerne un procédé de mesure par fluorescence sur des analytes d"un échantillon, marqués au moyen de différents marqueurs fluorescents présentant des émissions de fluorescence spectralement différentes. Ledit procédé consiste à illuminer l"échantillon (11) dans un volume de mesure au moyen d"un laser (20) destiné à exciter l"émission de fluorescence des deux marqueurs fluorescents au moins, l"illumination de l"échantillon dans le volume de mesure étant effectuée au moyen d"une ligne laser individuelle au plus, présentant une intensité d"excitation telle que les marqueurs fluorescents sont excités en commun par absorption à deux photons. Ledit procédé consiste également à détecter l"émission de fluorescence au moyen d"au moins deux dispositifs de détection (31, 32) servant à détecter la lumière dans différentes gammes spectrales en fonction des propriétés de fluorescence spectrales des marqueurs fluorescents, et à effectuer une analyse de coïncidence et/ou de corrélation croisée de signaux de détecteur des dispositifs de détection (31, 32). L"invention concerne également un dispositif de mesure destiné à la mise en oeuvre du procédé selon l"invention.
EP01969428A 2000-07-20 2001-07-18 Procede et dispositif d'analyse de coincidence par fluorescence a deux photons polychrome Ceased EP1303750A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10035190A DE10035190C5 (de) 2000-07-20 2000-07-20 Verfahren und Vorrichtung zur Fluoreszenzmessung
DE10035190 2000-07-20
PCT/EP2001/008328 WO2002008732A1 (fr) 2000-07-20 2001-07-18 Procede et dispositif d"analyse de coincidence par fluorescence a deux photons polychrome

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EP1303750A1 true EP1303750A1 (fr) 2003-04-23

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US (1) US7507582B2 (fr)
EP (1) EP1303750A1 (fr)
AU (1) AU2001289690A1 (fr)
DE (1) DE10035190C5 (fr)
WO (1) WO2002008732A1 (fr)

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CN100451677C (zh) * 2005-11-18 2009-01-14 北京航空航天大学 高光谱全偏振成像遥感系统
CN101889192B (zh) * 2007-10-25 2012-07-04 纽约州立大学研究基金会 光子光谱仪

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US20040022684A1 (en) 2004-02-05

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