DE102005051643A1 - Detection of microorganisms/chemically dangerous materials in e.g. a biological tissues, comprises irradiating a focus volume of the tissues by a laser pulse, and examining a radiation emitted by the tissues - Google Patents

Detection of microorganisms/chemically dangerous materials in e.g. a biological tissues, comprises irradiating a focus volume of the tissues by a laser pulse, and examining a radiation emitted by the tissues Download PDF

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DE102005051643A1
DE102005051643A1 DE102005051643A DE102005051643A DE102005051643A1 DE 102005051643 A1 DE102005051643 A1 DE 102005051643A1 DE 102005051643 A DE102005051643 A DE 102005051643A DE 102005051643 A DE102005051643 A DE 102005051643A DE 102005051643 A1 DE102005051643 A1 DE 102005051643A1
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Abstract

The laser pulse has the volume of less than 3 mu m3>, and the wavelength of infrared is 700-1200 nm. The emission is registered, which is evaluated in regards to the intensity, a spectral composition and/or emission fading away kinetics and the laser arrangement are shifted. An independent claim is also included for a detector by using the above method.

Description

Die Erfindung betrifft nach Anspruch 1 ein Verfahren zur Detektion von pathogenen Mikroorganismen und/oder chemischen Gefahrenstoffen in Flüssigkeiten oder Lebensmitteln sowie nach Anspruch 3 eine Nachweisvorrichtung für pathogene Mikroorganismen und/oder chemische Gefahrenstoffe in biologischen Geweben, Flüssigkeiten und/oder Lebensmitteln.The Invention relates according to claim 1, a method for the detection of pathogenic microorganisms and / or chemical hazardous substances in liquids or food and according to claim 3, a detection device for pathogenic microorganisms and / or chemical hazardous substances in biological tissues, fluids and / or food.

Die Erfindung betrifft ein optisches Verfahren und eine Vorrichtung zur bildgebenden Detektion von pathogenen Mikroorganismen – wie Bakterien, Sporen, Pilzen und Viren – und/oder chemischen Gefahrenstoffen in Geweben, Flüssigkeiten und/oder Nahrungsmitteln.The The invention relates to an optical method and a device for the imaging detection of pathogenic microorganisms - such as bacteria, Spores, fungi and viruses - and / or chemical hazardous substances in tissues, liquids and / or foodstuffs.

Der Nachweis von biologischen und chemischen Gefahrenstoffen mit typischen Abmessungen im Sub-Mikrometerbereich und Mikrometerbereich in biologischen Geweben wie der Haut und Schleimhäuten sowie in Gewässern und Lebensmitteln erfolgt jedoch bislang mit aufwändigen biochemischen, immun-histochemischen und gentechnischen Untersuchungsmethoden. So werden beispielsweise Abstriche gewonnen oder invasiv Gewebeproben entnommen, aufbereitet und mittels Nährlösungen und Agar-Medien die zu detektierenden Mikroorganismen üblicherweise zunächst amplifiziert, dann an spezielle Marker-Moleküle gebunden und diese dann z. B. mit optischen Methoden außerhalb der betroffenen Person bzw. außerhalb des betroffenen Lebensmittels detektiert.Of the Detection of biological and chemical hazardous substances with typical Dimensions in the sub-micrometer range and micrometer range in biological Tissues such as the skin and mucous membranes as well as in waters and Food, however, is done so far with elaborate biochemical, immune-histochemical and genetic engineering methods. For example Swabs obtained or invasively taken tissue samples, prepared and using nutrient solutions and agar media the microorganisms to be detected are usually first amplified, then to special marker molecules bound and then z. B. with optical methods outside the person concerned or outside of the food in question.

Der vorliegenden Erfindung liegt das Problem zu Grunde, ein Verfahren vorzuschlagen, mit dem die beschriebenen pathogenen Mikroorganismen und/oder chemischen Gefahrenstoffe mit einer einfachen Untersuchungsmethode in einer kurzen Zeitspanne mit vergleichsweise guter Zuverlässigkeit erkannt werden können. Weiterhin soll mit der vorliegenden Erfindung auch eine Nachweisvorrichtung vorgeschlagen werden, die sich zum Einsatz im medizinischen Bereich zur Diagnose am menschlichen Körper eignet.Of the The present invention is based on the problem of a method to propose, with the described pathogenic microorganisms and / or chemical hazardous substances using a simple method of investigation detected in a short period of time with relatively good reliability can be. Furthermore, the present invention also proposes a detection device Becoming involved in the medical field for diagnosis on the human body suitable.

Diese Aufgabe wird nach der vorliegenden Erfindung durch ein Verfahren gemäß Anspruch 1 gelöst, indem die zu untersuchende Flüssigkeit bzw. das zu untersuchende Lebensmittel einer Bestrahlung durch einen Laserpuls im nahen Infrarotbereich (Wellenlängen zwischen 700 nm und 1200 nm) mit einem Fokusvolumen des Laserpulses von weniger als 3 μm3 ausgesetzt wird, wobei nachfolgend die von der zu untersuchenden Flüssigkeit bzw. dem zu untersuchenden Lebensmittel emittierte Strahlung erfasst wird, deren Emission durch den Laserpuls angeregt wird, wobei die erfasste Strahlung hinsichtlich der Intensität, der spektralen Zusammensetzung und/oder der Emissionsabklingkinetik ausgewertet wird.This object is achieved according to the present invention by a method according to claim 1, by the liquid to be examined or the food to be examined irradiation by a laser pulse in the near infrared range (wavelengths between 700 nm and 1200 nm) with a focus volume of the laser pulse of less than 3 microns 3 is exposed, wherein subsequently detected by the examined liquid or the light emitted to be examined foods radiation whose emission is stimulated by the laser pulse, the detected radiation with respect to the intensity of the spectral composition and / or the Emissionsabklingkinetik is evaluated.

Durch dieses bildgebende, hochauflösende Verfahren zur Detektion von bestimmten pathogenen Mikroorganismen und chemischen Agenzien in Lebensmitteln und Flüssigkeiten, wie z. B. Trinkwasser und Getränken können unerwünschte Kontaminationen mit biologischen und/oder chemischen Gefahrenstoffen sensitiv und hochauflösend nachgewiesen werden.By this imaging, high-resolution method for the detection of certain pathogenic microorganisms and chemical Agents in food and liquids, such as As drinking water and drinks can undesirable Contamination with biological and / or chemical hazardous substances sensitive and high resolution be detected.

Außerdem können dadurch Aussagen zum Einfluss dieser Stoffe auf den Atmungsstoffwechsel von Zellen und zur möglichen Akkumulation dieser Agenzien in den Zellkernen getroffen werden.In addition, it can Statements on the influence of these substances on the respiratory metabolism of Cells and to the possible Accumulation of these agents are taken in the cell nuclei.

Weiterhin ergibt sich eine Lösung der Aufgabe auch im Einsatzgebiet der Medizin durch eine Nachweisvorrichtung nach Anspruch 3, indem die Nachweisvorrichtung eine Laseranordnung aufweist, mittels der ein Laserpuls im nahen Infrarotbereich (Wellenlängen zwischen 700 nm und 1200 nm) mit einem Fokusvolumen des Laserpulses von weniger als 3 μm3 emittierbar ist, wobei weiterhin eine Erfassungseinrichtung für die von dem zu untersuchenden biologischen Gewebe, der unterzusuchenden Flüssigkeit und/oder dem zu untersuchenden Lebensmittel emittierte Strahlung vorhanden ist, deren Emission durch den Laserpuls angeregt wurde, wobei weiterhin eine Auswertungseinheit vorhanden ist, mittels der die erfasste Strahlung hinsichtlich der Intensität, der spektralen Zusammensetzung und/oder der Emissionsabklingkinetik auswertbar ist.Furthermore, a solution of the problem also in the field of medicine by a detection device according to claim 3, by the detection device has a laser arrangement by means of a laser pulse in the near infrared range (wavelengths between 700 nm and 1200 nm) with a focal volume of the laser pulse of less than 3 microns 3 is emitted, further comprising a detection device for the emitted from the biological tissue to be examined, the liquid to be examined and / or the food to be examined radiation whose emission was excited by the laser pulse, wherein furthermore an evaluation unit is present by means of in that the detected radiation can be evaluated with regard to the intensity, the spectral composition and / or the emission decay kinetics.

Mittels dieser Vorrichtung können bestimmte pathogene Mikroorganismen und/oder chemische Agenzien auch in tierischen und humanen Geweben mittels der Laser-Anordnung bildgebend und hochauflösend detektiert werden.through this device can certain pathogenic microorganisms and / or chemical agents also in animal and human tissues by means of the laser arrangement Imaging and high resolution be detected.

Die Wirkungsweise des Verfahrens und der Nachweisvorrichtung lässt sich durch die nichtlinearen Prozesse der Zweiphotonen-Fluoreszenzanregung, der Dreiphotonen- Fluoreszenzanregung, der Second Harmonic Generation (SHG) und der Triple Harmonic Generation (THG) erklären.The Operation of the method and the detection device can be by the non-linear processes of the two-photon fluorescence excitation, the three-photon fluorescence excitation, the Second Harmonic Generation (SHG) and the Triple Harmonic Generation Explain (GHG).

Die zu detektierenden Substanzen werden selektiv zur Strahlungsemission angeregt. Die emittierte Strahlung der Substanzen unterscheidet sich von der emittierten Strahlung der Mikroumgebung hinsichtlich der Intensität und/oder der spektralen Zusammensetzung und/oder der Emissionsabklingkinetik. Diese Größen können messtechnisch erfasst und ausgewertet werden.The substances to be detected become selective for radiation emission stimulated. The emitted radiation of the substances differs from the emitted radiation of the microenvironment the intensity and / or the spectral composition and / or the emission decay kinetics. These sizes can be measured recorded and evaluated.

Die nichtlinearen Prozesse werden dabei durch fokussierte Femtosekunden-Laserstrahlung im nahen infraroten (NIR) Spektralbereich von 700 nm bis 1200 nm in einem geringen Fokusvolumen von weniger als 3 μm3 ausgelöst.The nonlinear processes are triggered by focused femtosecond laser radiation in the near infrared (NIR) spectral range from 700 nm to 1200 nm in a low focus volume of less than 3 μm 3 .

Bei der Ausgestaltung des Verfahrens nach Anspruch 2 werden die zu untersuchende Flüssigkeit bzw. das zu untersuchende Lebensmittel und die Laseranordnung derart gegeneinander verschoben, dass aus den untersuchten Teilvolumina ein untersuchtes Gesamtvolumen zusammen gesetzt wird.at the embodiment of the method according to claim 2 to be examined Liquid or the food to be examined and the laser arrangement such shifted against each other, that from the examined subvolumes an examined total volume is put together.

Durch das Beam-Scanning, bei dem der Laserstrahl über das zu untersuchende Target verschoben wird, oder durch das Objekt-Scanning, bei dem bei fest positioniertem Laserstrahl das Target verschoben wird, wird das zu untersuchende Target abgerastert und die emittierte Strahlung in Abhängigkeit von der Position des Anregungsvolumens detektiert.By the beam scanning, in which the laser beam over the target to be examined is moved, or by object scanning, when fixed Positioned laser beam is moved to the target, the scanned target to be examined and the emitted radiation in dependence of the position of the excitation volume detected.

Das Verfahren und die Vorrichtung nach der vorliegenden Erfindung sind wegen der einfachen Untersuchungsmethode und dem schnellen Vorliegen von Ergebnissen vorzugsweise zum Nachweis des Eindringens von pathogenen Mikroorganismen in Geweben und Lebensmitteln im Bereich Bioterrorismus und Lebensmittelsicherheit einsetzbar.The Method and apparatus according to the invention are because of the simple examination method and the quick presence of results preferably for detecting the penetration of pathogenic Microorganisms in tissues and foods in the area of bioterrorism and food safety.

Die unerwünschten biologischen und chemischen Stoffe können beispielsweise Anthrax-Sporen, gefährliche Bakterien und Viren sowie Schimmel- und Hefepilze in biologischen Geweben sowie in Lebensmitteln sein. Üblicherweise beträgt die Längendimension einzelner Bakterien 0,25 μm bis 10 μm (z. B. Bacillus anthracis 3 – 10 μm, Clostridium botulinum 3 – 8 μm, Escheria coli 1.5 μm, Staphylococcus aureus 0.9 μm, Salmonella species 1 – 3 μm, Mycoplasma pneumoniae 0.25 μm, Sporen 0.5 – 2 μm). Virendimensionen liegen im Bereich 0,02 μm bis 1 μm (z. B. Ebola virus 1 μm, Poxviridae 0.14 – 0.45 μm, Rhinovirus 0.023 μm) und Pilzdimensionen im Längenbereich bis 10 μm (z. B. Aspergillus 3 – 8 μm, Candida albicans 3 – 8 μm). Die genannten Mikroorganismen können zudem dreidimensionale Cluster bilden. Um eine schnelle in situ Frühdiagnose durchführen zu können, sind hochauflösende in situ Nachweisverfahren gefordert.The undesirable For example, anthrax spores, dangerous ones, can be biological and chemical substances Bacteria and viruses as well as mold and yeast fungi in biological Tissues as well as in food. Usually the length dimension is of individual bacteria 0.25 μm up to 10 μm (eg Bacillus anthracis 3 - 10 μm, Clostridium botulinum 3 - 8 μm, Escheria coli 1.5 μm, Staphylococcus aureus 0.9 μm, Salmonella species 1 - 3 μm, mycoplasma pneumoniae 0.25 μm, Spores 0.5 - 2 μm). virus dimensions are in the range of 0.02 μm to 1 μm (z. B. Ebola virus 1 μm, Poxviridae 0.14 - 0.45 μm, rhinovirus 0.023 μm) and fungal dimensions in the length range up to 10 μm (eg Aspergillus 3-8 μm, Candida albicans 3 - 8 μm). The mentioned Microorganisms can also form three-dimensional clusters. To get a quick in situ early diagnosis carry out to be able to are high resolution required in situ detection method.

Durch die mit der vorliegenden Erfindung vorgeschlagene Nachweisvorrichtung wird es beispielsweise möglich, bildgebend und dreidimensional hochauflösend in situ Sporen und gefährliche Bakterien auf bzw. in kontaminierten Hautarealen möglicherweise betroffener Personen zu untersuchen und mit den ermittelten Daten eine Schnelldiagnose zu treffen.By the detection device proposed by the present invention is it possible, for example, Imaging and three-dimensional high resolution in situ spores and dangerous Bacteria on or in contaminated skin may be examine the data subjects and with the data collected to make a quick diagnosis.

Mikroorganismen und viele chemische Agenzien besitzen eine Autofluoreszenz im sichtbaren Spektralbereich. Typischerweise zeichnen sich Mikroorganismen durch eine Fluoreszenz im blaugrünen Spektralbereich aus, die üblicherweise mit ultravioletter (UV) Strahlung induziert werden kann. Einige pathogene Mikroorganismen synthestisieren zudem gelb und rot fluoreszierende Zink-Porphyrine und metallfreie Porphyrine, die optimal mit Strahlung um 400 nm angeregt werden können (König, Schneckenburger: Laser-Induced Autofluorescence for Medical Diagnosis. J. Fluorescence 4(1)(1994)17-40). Jedoch ist die Eindringtiefe von UV und kurzwelliger sichtbarer Strahlung in Geweben und Lebensmitteln aufgrund hoher Absorptions- und Streukoeffizienten gering und ermöglichen keine sensitive hochauflösende Detektion von pathogenen Mikroorganismen und chemischen Stoffen in der Objekttiefe.microorganisms and many chemical agents have visible autofluorescence Spectral range. Typically, microorganisms are known a fluorescence in blue-green Spectral range, which is usually can be induced with ultraviolet (UV) radiation. Some Pathogenic microorganisms also synthesize yellow and red fluorescent Zinc porphyrins and metal-free porphyrins that are optimal for radiation 400 nm can be excited (King, Schneckenburger: Laser-Induced Autofluorescence for Medical Diagnosis. J. Fluorescence 4 (1) (1994) 17-40). However, the penetration depth of UV and shortwave visible radiation in tissues and foods due to high absorption and scattering coefficients low and allow no sensitive high-resolution Detection of pathogenic microorganisms and chemical substances in the object depth.

Ein neueres Verfahren ist die Multiphotonen-Fluoreszenzmikroskopie, bei der durch Zweiphotonen-Anregung mittels naher infraroter Femtosekunden-Laserstrahlung exogene Fluorophore in Einzelzellen nachgewiesen werden können (Patent US 5.034.613). Zudem ist es möglich, schwache Eigenfluoreszenzen in den Mitochondrien durch Präsenz des reduzierten Koenzyms NAD(P)H zu detektieren (König et al. Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress. J. Microsc. 183(1996)197-204). Zellkerne zeigen keine sichtbare Autofluoreszenz.One the newer method is multiphoton fluorescence microscopy, in the case of two-photon excitation by means of near-infrared femtosecond laser radiation Exogenous fluorophores can be detected in single cells (US Pat 5034613). It is also possible weak autofluorescences in the mitochondria due to presence of the reduced coenzyme NAD (P) H (König et al., Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress. J. Microsc. 183 (1996) 197-204). Nuclei show no visible Autofluorescence.

Ein Ausführungsbeispiel der Erfindung ist in der Zeichnung dargestellt. Die Figuren zeigen dabei:One embodiment the invention is shown in the drawing. The figures show attended:

1. Die Strahlung eines NIR Femtosekundenlasers (1) wird mittels Optik (2) in einen flexiblen Wellenleiter, z. B. eine Photonic Crystal Fiber, (3) mit Kollimator (4) fokussiert und zu einem x,y Galvoscanner (5), und durch eine Scanoptik (6) und einen Strahlteiler (7) sowie eine piezoverstellbare Fokussieroptik (8) geleitet und auf das zu untersuchende Objekt (9) geleitet. Die biologischen und/oder chemischen Gefahrenstoffe sowie die Mikroumgebung emittieren. Die Emissionsstrahlung wird durch den Strahlteiler (7) von der Laser-Streustrahlung separiert und mittels wellenlängen-selektivem Element (10) zeitaufgelöst mittels schnellem Detektor (11) sensitiv, z. B. mittels des Verfahrens der zeitkorrelierten Einzelphotonenzählung mit Zeitauflösungen im Pikosekundenbereich, detektiert. Die Bildverarbeitung erfolgt in dem Modul (12) anhand der Detektorsignale und der Position des x,y Scanners (5) und der Fokussieroptik (8). 1 , The radiation of a NIR femtosecond laser ( 1 ) by means of optics ( 2 ) in a flexible waveguide, z. B. a photonic crystal fiber, ( 3 ) with collimator ( 4 ) and converted to an x, y galvoscanner ( 5 ), and by a scanning optics ( 6 ) and a beam splitter ( 7 ) and a piezoverstellbare focusing optics ( 8th ) and to the object to be examined ( 9 ). The biological and / or chemical hazardous substances as well as the microenvironment emit. The emission radiation is transmitted through the beam splitter ( 7 ) separated from the laser scattered radiation and by means of wavelength-selective element ( 10 ) time-resolved by means of a fast detector ( 11 ) sensitive, z. Example by means of the method of time-correlated single photon counting with time resolutions in the picosecond range, detected. The image processing takes place in the module ( 12 ) based on the detector signals and the position of the x, y scanner ( 5 ) and the focusing optics ( 8th ).

2. Die Strahlung eines NIR Femtosekundenlasers (1) wird mittels Optik (2) in einen flexiblen Wellenleiter, z. B. eine Photonic Crystal Fiber, (3) mit piezo-verstellbarer Fokussieroptik (13) fokussiert. Die Strahlposition ist fixiert. Es wird das auf einer Verstelleinheit (14) befindliche Objekt (9) bewegt (Objektscanning). 2 , The radiation of a NIR femtosecond laser ( 1 ) by means of optics ( 2 ) in a flexible waveguide, z. B. a photonic crystal fiber, ( 3 ) with piezo-adjustable focusing optics ( 13 ) focused. The beam position is fixed. It will be on an Ver position unit ( 14 ) object ( 9 ) moves (object scanning).

3. Die Strahlung eines NIR Femtosekundenlasers (1) wird durch einen x,y Galvoscanner (5), Scanoptik (6), Strahlteiler (15), piezoverstellbarer Fokussieroptik (16) und einer stäbchenförmigen Optik bestehend aus Grinlinsen (17), die minimal invasiv in das Gewebe bzw. die Flüssigkeit bzw. das Lebensmittel eingeführt wird, transmittiert und in das Objekt (9) fokussiert. Die emittierte Strahlung wird über den Strahlteiler (15) einem schnellen Detektor mit wellenlängen-selektiven Element (18) zugeführt. 3 , The radiation of a NIR femtosecond laser ( 1 ) is replaced by an x, y Galvoscanner ( 5 ), Scan optics ( 6 ), Beam splitters ( 15 ), piezoverstellbarer focusing optics ( 16 ) and a rod-shaped optic consisting of grin lenses ( 17 ), which is introduced minimally invasively into the tissue or the liquid or the food, and transmitted into the object ( 9 ) focused. The emitted radiation is transmitted via the beam splitter ( 15 ) a fast detector with wavelength-selective element ( 18 ).

Claims (3)

Verfahren zur Detektion von pathogenen Mikroorganismen und/oder chemischen Gefahrenstoffen in Flüssigkeiten und/oder Lebensmitteln, dadurch gekennzeichnet, dass die zu untersuchende Flüssigkeit bzw. das zu untersuchende Lebensmittel einer Bestrahlung durch einen Laserpuls im nahen Infrarotbereich (Wellenlängen zwischen 700 nm und 1200 nm) mit einem Fokusvolumen des Laserpulses von weniger als 3 μm3 ausgesetzt wird, wobei nachfolgend die von der zu untersuchenden Flüssigkeit bzw. dem zu untersuchenden Lebensmittel emittierte Strahlung erfasst wird, deren Emission durch den Laserpuls angeregt wird, wobei die erfasste Strahlung hinsichtlich der Intensität, der spektralen Zusammensetzung und/oder der Emissionsabklingkinetik ausgewertet wird.A method for the detection of pathogenic microorganisms and / or chemical hazardous substances in liquids and / or foods, characterized in that the liquid to be examined or the food to be examined irradiation by a laser pulse in the near infrared range (wavelengths between 700 nm and 1200 nm) is exposed to a focus volume of the laser pulse of less than 3 microns 3 , wherein subsequently emitted by the liquid to be examined or the food to be examined radiation is detected whose emission is excited by the laser pulse, wherein the detected radiation with respect to the intensity, the spectral Composition and / or the emission decay rate is evaluated. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass die zu untersuchende Flüssigkeit bzw. das zu untersuchende Lebensmittel und die Laseranordnung derart gegeneinander verschoben werden, dass aus den untersuchten Teilvolumina ein untersuchtes Gesamtvolumen zusammen gesetzt wird.Method according to claim 1, characterized in that that the liquid to be examined or the food to be examined and the laser arrangement in such a way be shifted against each other, that from the examined subvolumes an examined total volume is put together. Nachweisvorrichtung für pathogene Mikroorganismen und/oder chemische Gefahrenstoffe in biologischen Geweben, Flüssigkeiten und/oder Lebensmitteln, dadurch gekennzeichnet, dass die Nachweisvorrichtung eine Laseranordnung aufweist, mittels der ein Laserpuls im nahen Infrarotbereich (Wellenlängen zwischen 700 nm und 1200 nm) mit einem Fokusvolumen des Laserpulses von weniger als 3 μm3 emittierbar ist, wobei weiterhin eine Erfassungseinrichtung für die von dem zu untersuchenden biologischen Gewebe, der unterzusuchenden Flüssigkeit und/oder dem zu untersuchenden Lebensmittel emittierte Strahlung vorhanden ist, deren Emission durch den Laserpuls angeregt wurde, wobei weiterhin eine Auswertungseinheit vorhanden ist, mittels der die erfasste Strahlung hinsichtlich der Intensität, der spektralen Zusammensetzung und/oder der Emissionsabklingkinetik auswertbar ist.Detection device for pathogenic microorganisms and / or chemical hazardous substances in biological tissues, liquids and / or foods, characterized in that the detection device has a laser arrangement by means of a laser pulse in the near infrared range (wavelengths between 700 nm and 1200 nm) with a focus volume of the laser pulse of less than 3 microns 3 is emitted, further comprising a detection device for the emitted from the biological tissue to be examined, the liquid to be examined and / or the food to be examined radiation whose emission was excited by the laser pulse, further comprising an evaluation unit is, by means of which the detected radiation in terms of intensity, the spectral composition and / or the emission decay kinetics is evaluable.
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