EP1421367A1 - Analyse de correlation a plusieurs couleurs et a un canal - Google Patents

Analyse de correlation a plusieurs couleurs et a un canal

Info

Publication number
EP1421367A1
EP1421367A1 EP02797637A EP02797637A EP1421367A1 EP 1421367 A1 EP1421367 A1 EP 1421367A1 EP 02797637 A EP02797637 A EP 02797637A EP 02797637 A EP02797637 A EP 02797637A EP 1421367 A1 EP1421367 A1 EP 1421367A1
Authority
EP
European Patent Office
Prior art keywords
optical
sample
measurement volume
excitation
luminescent molecules
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.)
Withdrawn
Application number
EP02797637A
Other languages
German (de)
English (en)
Inventor
Rudolf Rigler
Per Thyberg
Adrian Honegger
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.)
Gnothis Holding SA
Original Assignee
Gnothis Holding SA
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
Priority claimed from DE10210737A external-priority patent/DE10210737A1/de
Application filed by Gnothis Holding SA filed Critical Gnothis Holding SA
Publication of EP1421367A1 publication Critical patent/EP1421367A1/fr
Withdrawn legal-status Critical Current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
    • G01N21/645—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
    • 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
    • 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"
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • 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
    • G01N2021/6419—Excitation at two or more wavelengths
    • 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
    • G01N2021/6421—Measuring at two or more wavelengths
    • 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

Definitions

  • the invention relates to a method for determining luminescent molecules by optical excitation in confocal measurement volumes, wherein different species of luminescent molecules in a sample are excited at different times and the emission radiation originating from the different species from a measurement volume is collected by a single detector. Furthermore, a device suitable for carrying out the method is disclosed.
  • FCS fluorescence correlation spectroscopy
  • the fluorescence correlation spectroscopy can be carried out in conjunction with a so-called cross-correlation analysis, in which two species of luminescent molecules which differ with respect to at least one optical property are excited in a confocal measurement volume and the presence or absence of a correlation between the two measurement signals is analyzed.
  • a disadvantage of cross-correlation methods used to date is that the emission radiation originating from the different luminescent species had to be detected by * -separate detectors.
  • One object on which the present application was based was to provide methods and devices for determining luminescent moles, in particular the color-fluorescence correlation spectroscopy, which can be used to determine in a simple manner Allow cross correlations.
  • the invention thus relates to a method for determining luminescent molecules by optical excitation in confocal measurement volumes, comprising the steps: (a) providing a sample comprising luminescent molecules,
  • Molecules in at least one confocal measurement volume which is part of the sample, and (c) collecting and evaluating emission radiation from the at least one measurement volume, wherein in one measurement volume several species of luminescent molecules that are different with regard to an optical property are excited at different times and the emission radiation from the different species is captured and evaluated by a single detector.
  • the method according to the invention represents a single-channel multicolor correlation analysis, several, e.g. 2, 3, 4 or even more, with respect to at least one optical property, such as emission wavelength and / or luminescence decay time, different species of luminescent molecules are excited in a single measurement volume and the signals can be evaluated on a single detector.
  • at least one optical property such as emission wavelength and / or luminescence decay time
  • 2 or more, for example 3, separate light sources in particular lasers with different wavelengths, for example ⁇ 1, ⁇ 2, ⁇ 3, are preferably used each used separate excitation of different species.
  • These separate light sources can be irradiated or coupled into the confocal measurement volume at one or more times by means of one or more adjustable optical switching elements and used therein to excite the emission.
  • suitable adjustable optical switching elements are acousto-optical modulators, adjustable reflection elements, such as, for example, piezo-controlled mirrors, adjustable diffraction elements, adjustable diffraction-optical elements and Kerr cells.
  • a single detector is used per measurement volume, which alternately records the signals emitted by the different excited species and defines them by suitable measures, for example a digital coding signal.
  • a signal processor, a data storage unit and a correlator are preferably also connected to the detector.
  • the signal picked up by the detector is preferably evaluated in a correlator with a coding signal corresponding to the clock frequency of the different light sources.
  • energy-dispersive detectors can also be used, which can differentiate between the individual types of emission radiation that come from the differently excited species.
  • the clock frequency with which the various light sources are radiated onto the measurement volume is related to the measurement signal to be expected. So when determining diffusion events, where the diffusion time, e.g. is in the range of about 1 ms, a clock frequency with a significantly shorter clock interval, e.g. with a clock interval of 0.1-10 ⁇ s (corresponding to 10-0.1 MHz), e.g. of approximately 1 ⁇ s (corresponding to 1 MHz). With other types of determinations, the cycle time is set accordingly.
  • ⁇ 1, ⁇ 2, ⁇ 3 can be used to calculate autocorrelation functions (only taking a single signal into account) and cross correlations (taking into account several signals together), e.g. ⁇ 1 x ⁇ 2, ⁇ 2 x ⁇ 3, ⁇ 1 x ⁇ 3 or ⁇ 1 x ⁇ 2 x ⁇ 3 It is therefore a multiplexing method that can basically be transferred to a larger number of laser frequencies.
  • Corresponding electronic background analysis can be used to calculate a correlation or / and coincidence curve in which "correctly" occurring signals can be distinguished from signals which arise from undesired interference (crosstalk) from several marker groups.
  • dichroic filters and / or blocking filters e.g. Interference filters of various orders or notch filters for regulating the radiation, e.g. for selective transmission at certain wavelengths.
  • a particularly preferred embodiment of the method according to the invention comprises a parallel determination of luminescent molecules in several samples.
  • the light emitted by the optical excitation device can be split into multiple light beams or foci, which are focused on measurement volumes in several samples.
  • the emitted light is split up by using one or more diffractive optical elements, as described in DE 1 01 26 083.0.
  • a diffractive optical element, which is arranged in the beam path before the beam combination, is particularly preferably used for each light source.
  • the diffractive optical elements that can be used are, for example, three-dimensional optical gratings, optionally applied to an optically transparent support, which diffract light that passes through and generate a predetermined diffraction pattern in the object plane through constructive and destructive interference, ie a desired arrangement of multiple optical foci, in any arrangement can.
  • the multiple optical foci are favorably caused by interference 1. Order formed, with only slight light losses due to interferences of the 0th or higher orders.
  • a preferred embodiment of the method according to the invention relates to the detection of molecules luminescent in the confocal measurement volumes by fluorescence correlation spectroscopy.
  • the method can in principle be carried out according to the method described in EP-B-0 679 251.
  • the measurement is carried out preferably by one or a few analyte molecules in a measuring volume, wherein the concentration of the analyte to be determined is preferably 10 "6 mol / l and the measuring volume, preferably ⁇ 10" 1.
  • Substance-specific parameters are determined, which are determined by luminescence measurement on the analyte molecules.
  • a preferred feature of the method according to the invention is that the distance between the measurement volume in the sample liquid and the focusing optics of the light source is> 1 mm, preferably 1.5 to 10 mm and particularly preferably 2 to 5 mm. It is further preferred that a gas phase region is arranged between the carrier containing the sample liquid and the optical focusing device, which can contain air, protective gas or vacuum. Methods and devices for performing FCS with a large distance between focusing optics and confocal measurement volume are described in DE 101 1 1 420.6. However, for certain applications it is of course also possible to choose a smaller distance between focusing optics and measuring volume of ⁇ 1 mm. Direct contact between the specimen and the focusing optics, e.g. by using an immersion liquid.
  • the method according to the invention is basically suitable for carrying out any determination method.
  • a preferred embodiment relates to the determination of an analyte in a sample, for example for diagnostic applications or for screening to identify active substances which interact with a target substance.
  • one or more analyte-binding substances are added to the sample, which carry a marking group that can be detected by luminescence measurement, in particular a fluorescence marking group.
  • the method according to the invention preferably comprises determining the binding of the labeling substance to the analyte to be detected.
  • This proof can be, for example, by Multi-color cross-correlation determination is carried out, using at least two different markings, in particular fluorescence markings, the correlated signal of which is determined within the measurement volume.
  • This cross-correlation determination is, for example, by Schwüle et al. (Biophys. J. 72 (1 997), 1 878-1 886) and Rigler et al. (J. Biotechnol. 63 (1 998), 97-1 09).
  • the method according to the invention is particularly suitable for the detection of biomolecules, e.g. Nucleic acids, proteins or other analyte molecules occurring in living organisms, especially in mammals such as humans.
  • biomolecules e.g. Nucleic acids, proteins or other analyte molecules occurring in living organisms, especially in mammals such as humans.
  • analytes that have been generated in vitro from biological samples can also be detected, e.g. cDNA molecules which have been produced by reverse transcription from mRNA or proteins which have been produced by in vitro translation from mRNA or from DNA.
  • the method is also suitable for the detection of analytes which are present as elements of a library and have predetermined characteristics, e.g. Binding to the detection reagent. Examples of such libraries are phage libraries or ribosomal libraries.
  • the determination comprises nucleic acid hybridization, one or more luminescence-labeled probes binding to a target nucleic acid as analytes.
  • Hybridization methods of this type can be used, for example, to analyze gene expression, for example to determine a gene expression profile, to analyze mutations, for example single nucleotide polymorphisms (SNP).
  • SNP single nucleotide polymorphisms
  • the method according to the invention is also suitable for determining enzymatic reactions and / or for determining nucleic acid amplifications, in particular in a thermocycling process.
  • Preferred methods for determining nucleic acid polymorphisms are in DE 100 56 226.4 and DE 100 65 631 .5 described. A two-color or multicolor cross-correlation determination is particularly preferably carried out.
  • the determination comprises the detection of a protein-protein or protein-ligand interaction, the protein ligands being e.g. low molecular weight active substances, peptides, nucleic acids etc. can be used.
  • a two- or multi-color correlation measurement is preferably also carried out for such determinations.
  • so-called “molecular beacon” probes or primers can be used which, when they are in free form, give a measurement signal which differs in terms of luminescence intensity or / and decay time than in the bound state.
  • a further preferred embodiment of the invention comprises a method for the selection of particles in a substance library, wherein a particle with a predetermined property is selected from a population comprising a large number of different particles.
  • a population of different particles is preferably provided, particles which have a predetermined property are marked, the particles are guided in a microchannel through a detection element, comprising multiple confocal volume elements, in order to differentiate between marked and unmarked particles and separated marked particles.
  • the steps of guiding and separating are preferably repeated at least once, the concentration of the particles in a subsequent cycle being preferably reduced by at least a factor of 10 4 compared to a previous cycle.
  • the particles can be selected, for example, from cells, parts of cell surfaces, cell organelles, viruses, nucleic acids, proteins and low-molecular substances.
  • the method is also suitable for Selection of particles from a combinatorial library, which can contain genetic packaging such as phages, cells, spores or ribosomes.
  • the particle population preferably contains more than 10 6 and particularly preferably more than 10 10 different particles.
  • the particles are preferably labeled with a luminescent labeling group.
  • Yet another embodiment comprises performing a sequence analysis of polymers, in particular biopolymers, wherein luminescent fragments of an analyte present in the sample are determined.
  • This embodiment is particularly suitable for performing nucleic acid sequencing.
  • a carrier particle with a nucleic acid molecule immobilized thereon is preferably provided, with essentially all nucleotide building blocks of at least one base type bearing a fluorescent label in at least one strand of the nucleic acid molecule.
  • the carrier particle is introduced into a sequencing device comprising a microchannel, there, e.g. held with an IR capture laser and, e.g.
  • nucleic acid building blocks are then passed through a microchannel, preferably by means of a hydrodynamic flow, and there the base sequence of the nucleic acid molecule is determined in confocal volume elements on the basis of the sequence of the cleaved nucleotide building blocks.
  • the light beams originating from the light sources are split into several optical foci.
  • the light beam is preferably split into 2-32, in particular 4-16 optical foci.
  • confocal volume elements are imaged in the sample from these optical foci.
  • the confocal volume elements advantageously have one Size from 10 "18 to 10 " 9 I, preferably from 10 " 18 to 10 " 12 1 and particularly preferably from 10 -16 to 10 "14 I.
  • a separate detector per volume unit or a spatially resolving detection matrix e.g. an avalanche photodiode matrix or an electronic detector matrix, e.g. a CCD camera.
  • these confocal volume elements are each provided in separate containers of a carrier, preferably a microstructure.
  • the volume of these containers is preferably in the range from 10 "6 I and particularly preferably ⁇ 10 " 8 I to 10 ⁇ 12 I.
  • the carrier can comprise a microwave structure with several wells for receiving sample liquid, for example, a diameter between 10 and 1000 microns exhibit. Suitable microstructures are described, for example, in DE 100 23 421 .6 and DE 100 65 632.3. These microstructures can be used, for example, to determine nucleic acid hybridization in solution.
  • the carrier preferably comprises at least one temperature control element, for example a Peltier element, which enables temperature regulation of the carrier and / or individual sample containers therein.
  • the carrier used for the method is expediently designed in such a way that it enables optical detection of the sample.
  • a carrier which is optically transparent at least in the region of the sample containers is therefore preferably used.
  • the carrier can either be completely optically transparent or an optically transparent base and contain an optically opaque cover layer with cutouts in the sample containers.
  • Suitable materials for supports are, for example, composite supports made of metal (for example silicon for the cover layer) and glass (for the base). Such supports can be produced, for example, by applying a metal layer to the glass with predetermined cutouts for the sample containers.
  • plastic carriers made of polystyrene or polymers based on acrylate or methacrylate can be used. It is further preferred that the carrier has a cover for the sample containers in order to provide a closed system which is essentially isolated from the surroundings during the measurement.
  • a carrier which contains a lens element which is arranged in the beam path between the measurement volume and the light source or detector of the optical device.
  • the lens element can be attached to the bottom of a microwave structure.
  • a lens element can be made, for example, by heating and molding a photoresist using a master mold, e.g. made of metal such as silicon, and then applied to the carrier.
  • a master mold e.g. made of metal such as silicon
  • supports made of fully plastic structure e.g. when using supports made of fully plastic structure - the lens elements integrated in the support, e.g. are produced during injection molding.
  • the numerical aperture of the optical measuring arrangement can be enlarged by using a lens element, preferably a convex lens element. This numerical aperture is preferably in the range from 0.5 to 1.2.
  • the support is also preferably coated with a transparent anti-reflective coating to produce a higher refractive index.
  • a transparent anti-reflective coating for example, transparent oxides or nitrides can be used as antireflection coatings.
  • Anti-reflective coatings are preferably also used on the optics.
  • electric fields can be generated in the carrier, in particular in the area of the sample containers, in order to achieve a concentration of the analytes to be determined in the measurement volume. Examples of electrodes that are suitable for generating such electrical fields are described, for example, in DE 101 03 304.4.
  • the molecule to be determined can be bound to a carrier particle, in particular in the case of a determination in microwave format or in the case of single molecule sequencing.
  • the carrier particle has a size that enables movement in microchannels and retention in a desired position within a sequencing device.
  • the particle size is preferably in the range from 0.5-1 ⁇ m and particularly preferably from 1-3 ⁇ m.
  • suitable materials for carrier particles are plastics, such as polystyrene, glass, quartz, metals or semimetals, such as silicon, metal oxides, such as silicon dioxide, or composite materials which contain several of the aforementioned components.
  • Optically transparent carrier particles for example made of plastics or particles with a plastic core and a silicon dioxide shell, are particularly preferably used.
  • Nucleic acid molecules are preferably immobilized on the carrier particle via their 5 'end, e.g. through covalent or non-covalent interaction.
  • the binding of polynucleotides to the support is particularly preferably carried out by high-affinity interactions between the partners of a specific binding pair, e.g. Biotin / streptavidin or avidin etc.
  • nucleic acid molecules can also be bound to the support by adsorption or covalently.
  • Carrier particles to which only a single nucleic acid molecule is bound are preferably used. Such carrier particles can be produced in that the intended for the determination
  • Nucleic acid molecules in a molar ratio of preferably 1: 5 to 1:20, for example 1:10, are brought into contact with the carrier particles under conditions in which the nucleic acid molecules are immobilized on the carrier.
  • the nucleic acid molecules bound to a support e.g. DNA molecules or RNA molecules can be in single-stranded form or double-stranded form.
  • the nucleic acid molecules are preferably in single-stranded form.
  • a fluorescent labeling group e.g. at least 90%, preferably at least 95% of all nucleotide building blocks of at least one base type, a fluorescent labeling group.
  • all nucleotide building blocks of at least 2 base types, for example 2, 3 or 4 base types can carry a fluorescent label, each base type advantageously containing a different fluorescent label group.
  • Such labeled nucleic acids can be obtained by enzymatic primer extension on a nucleic acid template using a suitable polymerase, e.g. a thermostable DNA polymerase. A detailed description of this method can be found in DE 100 31 840.1 and DE 100 65 626.9 and the literature citations given there.
  • Yet another object of the present invention is a device for determining luminescent molecules, in particular for carrying out a method as described above
  • an optical excitation and focusing device comprising a plurality of separate light sources, and an adjustable optical switching element for coupling the separate light sources at different times to a confocal measurement volume, which is part of the sample, and (c) an optical detection device, each comprising a single detector per measurement volume for the detection of luminescence.
  • the carrier is preferably a microstructure with a plurality, preferably at least 10, particularly preferably at least 10 2 containers for holding a sample liquid, the sample liquid in the separate containers being able to come from one or more sources.
  • the sample liquid can be introduced into the containers of the carrier, for example by means of a piezoelectric liquid dispensing device.
  • the containers of the carrier are designed in such a way that they enable the detection reagent to be bound with the analyte in solution.
  • the containers are preferably depressions in the carrier surface, whereby these depressions can in principle have any shape, for example circular, square, diamond-shaped, etc.
  • the carrier can also comprise 10 3 or more separate containers.
  • the carrier can also contain a microchannel structure with one or more microchannels, which is particularly suitable for a single-molecule sequencing method, as described in DE 100 31 840.1 and DE 100 65 626.9, or for a particle selection method, as described in DE 100 31 028.1, are suitable.
  • the optical excitation and focusing device comprises a plurality of strongly focused light sources, preferably laser beams, which are focused on the measurement volume in the sample liquid by means of corresponding optical devices.
  • the individual laser beams are focused on the measurement volume with adjustable optical switching elements at different times in a predetermined time cycle.
  • the optical device preferably contains dichroic filters and / or blocking filters and - for splitting the laser beams into multiple foci - one or more diffractive optical elements.
  • the diffractive optical elements can be arranged before or / and after the combination of the different laser beams.
  • a diffractive optical element is preferably used for each laser before the beam combination.
  • the detection device can contain, for example, a fiber-coupled avalanche photodiode detector or an electronic detector. However, excitation and / or detection can also be used. Matrices consisting of a dot matrix of laser dots generated by diffractive optics or a quantum well laser and a detector matrix generated by an avalanche photodiode matrix or an electronic detector matrix, e.g. a CCD camera can be used.
  • the carrier can be provided in a prefabricated form, with luminescence-marked next-to-re-g a n e zi e s being filled into several separate containers of the carrier, and prior to this sweeping hybridization probes or primers.
  • the carrier containing the detection reagents is then advantageously dried.
  • a prefabricated carrier which contains a multiplicity of separate, for example 100 containers, in which different detection reagents, for example reagents for detecting nucleic acid hybridization such as primers and / or probes, are present.
  • This carrier can then be filled with a sample originating from an organism to be examined, for example a human patient, so that different analytes from a single sample are determined in the respective containers.
  • Such carriers can be used, for example, to create a gene expression profile, for example for the diagnosis of diseases, or for Determination of nucleic acid polymorphisms, for example for the detection of a certain genetic predisposition, can be used.
  • FIG. 1 shows the schematic representation of an embodiment of the method according to the invention.
  • Three light sources e.g. Lasers (1, 2,3) with wavelengths ⁇ 1, ⁇ l and ⁇ 3 are activated by means of suitable optical switching elements, e.g. acousto-optical modulators, connected to enable the individual laser beams to be coupled into the beam path over a predetermined time cycle.
  • the laser beam which alternates between the wavelengths ⁇ 1, ⁇ l and ⁇ 3 - in the specified time cycle - strikes a dichroic filter (6), which directs it via a focusing lens (8) to a confocal measuring volume in the sample (10).
  • Emission radiation emanating from luminescent molecules in the sample (10) is passed through the dichroic filter (6) and a blocking filter (1 2) and then via a pinhole (14) into a detector (1 6) with a signal processor, data storage and correlator.
  • FIG. 2 shows a schematic representation of the time-dependent signal flow l (t) at the detector.
  • the signal originating from the emission radiation ⁇ 1, ⁇ 2, ⁇ 3 from luminescent molecules in the sample is recorded by the detector and provided with a coding signal for the respective time cycle of the wavelengths ⁇ 1, ⁇ l and ⁇ 3.
  • the corresponding auto and cross correlation functions can be calculated from the time profiles of ⁇ 1, ⁇ 2 and ⁇ 3.
  • FIG. 3A shows the reflection (R) or transmission (T) of the dichroic filter (6) according to FIG. 1, which is dependent on the wavelength ⁇ .
  • the transmission is minimal at the excitation wavelengths ⁇ 1, ⁇ l and ⁇ 3.
  • FIG. 3B shows the wavelength-dependent transmission (T) of a blocking filter (1 2) according to FIG. 1.
  • the transmission is minimal in the range of the excitation wavelengths ⁇ 1, ⁇ l and ⁇ 3, but maximum in the range of the emission wavelengths ⁇ 1, ⁇ 2 and ⁇ 3 (not shown).
  • FIG. 4 shows the schematic representation of a particularly preferred embodiment of the method according to the invention.
  • 3 lasers 21, 22, 23
  • the laser beam passes through a diffractive optical element (26) and is there divided into a plurality, e.g. 9 optical foci (shown in cross section), with a changing wavelength ⁇ 1, ⁇ 2 and ⁇ 3.
  • the split light beam is directed onto a sample matrix (1 2) via a dichroic mirror (28) and a focusing lens (1 0), which may consist of several sub-elements.
  • This sample matrix consists e.g. corresponding to the matrix of the multiple optical foci from several separate samples, each containing a measurement volume.
  • Emission radiation originating from the measurement volumes of the sample matrix (32) is directed through the dichroic filter (28) and a blocking filter (34) onto a detector matrix (36).
  • the matrix of emission beams with a wavelength of ⁇ 1, ⁇ 2, ⁇ 3 - depending on the timing and the molecules excited in the sample - is shown in cross section.
  • the signals originating from the individual measurement volumes are collected and evaluated in separate detectors in the detector matrix (36).
  • Figure 5 shows another particularly preferred embodiment.
  • a diffractive optical element 34, 35, 36
  • Each laser beam can thus be split into multiple optical foci before the beams are combined by means of optical switching elements (37a, 37b). Otherwise the determination according to FIG. 4 is carried out.
  • the embodiments according to FIGS. 4 and 5 are suitable for a single-channel multiplex multicolor correlation determination.
  • a fluorescence cross-correlation determination of 2 marker groups in a sample with 2 laser beams was carried out on the Confocor 2 (Zeiss) instrument.
  • the data were evaluated in such a way that a temporally alternating change in the wavelength of the excitation light was simulated.
  • the alternating frequency was 1 MHz, i.e. the fluorescence from a first marker group was set to 0 for a time segment of 1 ⁇ s and the fluorescence from the second marker group was set to 0 for the next time segment of 1 ⁇ s.
  • the frequency for a complete cycle was 0.5 MHz (corresponding to a period of 2 ⁇ s).

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

Abstract

La présente invention concerne un procédé pour détecter des molécules luminescentes par excitation optique dans des volumes de mesure à foyer commun. Selon ce procédé, différentes espèces de molécules luminescentes se trouvant dans un échantillon sont excitées à différents moments et le rayonnement émis par les différentes espèces provenant d'un volume de mesure est capté par un seul détecteur. La présente invention concerne également un dispositif permettant de mettre en oeuvre ledit procédé.
EP02797637A 2001-08-28 2002-08-28 Analyse de correlation a plusieurs couleurs et a un canal Withdrawn EP1421367A1 (fr)

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DE10141950 2001-08-28
DE10141950 2001-08-28
DE10210737A DE10210737A1 (de) 2001-08-28 2002-03-12 Einkanal-Mehrfarben-Korrelationsanalyse
DE10210737 2002-03-12
PCT/EP2002/009610 WO2003021240A1 (fr) 2001-08-28 2002-08-28 Analyse de correlation a plusieurs couleurs et a un canal

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WO2005008212A2 (fr) * 2003-06-20 2005-01-27 The Regents Of The University Of California Analyse par fluorescence a excitation modulee
DE10357584B4 (de) * 2003-12-08 2006-06-14 Leica Microsystems Cms Gmbh Verfahren zum Trennen unterschiedlicher Emissionswellenlängen in einem Scanmikroskop
US7242009B1 (en) * 2005-06-22 2007-07-10 Hach Ultra Analytics, Inc. Methods and systems for signal processing in particle detection systems
KR20120071453A (ko) * 2010-12-23 2012-07-03 삼성전자주식회사 미생물 검출장치
US9920364B2 (en) 2012-03-06 2018-03-20 Rudolf Rigler Cyclic single molecule sequencing process
US11320380B2 (en) * 2020-04-21 2022-05-03 Sartorius Bioanalytical Instruments, Inc. Optical module with three or more color fluorescent light sources and methods for use thereof
EP4290220A1 (fr) * 2022-06-10 2023-12-13 Leica Microsystems CMS GmbH Contrôleur pour un dispositif d'imagerie et procédé
EP4375644A1 (fr) * 2022-11-22 2024-05-29 Gnothis Holding AG Analyse d'empreintes digitales d'événements moléculaires uniques

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US7223985B2 (en) 2007-05-29
WO2003021240A1 (fr) 2003-03-13

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