EP2572182A2 - Arrangement for measuring the optical properties of particles of a dispersion - Google Patents

Arrangement for measuring the optical properties of particles of a dispersion

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Publication number
EP2572182A2
EP2572182A2 EP11782374A EP11782374A EP2572182A2 EP 2572182 A2 EP2572182 A2 EP 2572182A2 EP 11782374 A EP11782374 A EP 11782374A EP 11782374 A EP11782374 A EP 11782374A EP 2572182 A2 EP2572182 A2 EP 2572182A2
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EP
European Patent Office
Prior art keywords
arrangement according
laser
measuring
light
cuvette
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Withdrawn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
EP11782374A
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German (de)
French (fr)
Inventor
Wolfgang GÖHDE
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.)
Partec GmbH
Original Assignee
Partec GmbH
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Filing date
Publication date
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Publication of EP2572182A2 publication Critical patent/EP2572182A2/en
Withdrawn legal-status Critical Current

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6486Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434Optical arrangements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03Cuvette constructions
    • G01N21/0303Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47Scattering, i.e. diffuse reflection
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • G01N33/487Physical analysis of biological material of liquid biological material
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N2015/1006Investigating individual particles for cytology
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00Features of devices classified in G01N21/00
    • G01N2201/06Illumination; Optics
    • G01N2201/061Sources
    • G01N2201/06113Coherent sources; lasers

Definitions

  • the task is in addition to the rapid detection of the individual particles is to measure them as accurately as possible optically.
  • lasers are used as light sources for the forward and side scattered light measurements as well as for the fluorescence excitation.
  • the laser light is allowed to act on the particles with a very small opening angle (low divergence) or as a parallel light beam.
  • the metrological problem consists in the fact that morphologically complex particles, which can often be structured like very flat, flat objects, are achieved by the laser beam at right angles to the particle surface or parallel to the particle surface. This leads, for example, to "correct" measured values of the particle fluorescence in the case of planar illumination and to "erroneous" measured values when the laser light hits the edge of the particle.
  • the invention relates to an arrangement which avoids this measurement error.
  • the flow cytometric analysis of microscopic particles has great economic importance if it is possible to make the measurements highly precise.
  • One of these Applications is the measurement of the DNA content of sperm. If this measurement is accurate enough, the 2 varieties of sperm containing X and Y chromosomes can be sorted with a device downstream of the flow cytometer and used for animal breeding.
  • the object of this invention is to find a way that in the previously known Laser-based flow cytometers reduce or completely eliminate the erroneous influence of shape factors on the measurement accuracy. This leads to a significantly increased measurement accuracy.
  • the object of the invention is also to combine the high measurement accuracy achieved with the two scattered light parameters. These can not be measured with conventional light sources.
  • a device for laser light excitation is proposed, which avoids the influence of form factors. Compared to conventional light sources with the advantage of high numerical aperture excitation, laser excitation allows a much higher density of light energy, resulting in more accurate measurements (better signal-to-noise ratio).
  • FIG. 2 is a plan view of a first embodiment of a flow cytometer
  • Fig. 5 is a plan view of a third embodiment of a flow cytometer.
  • Fig. 6 is a plan view of a fourth embodiment of a flow cytometer.
  • Fig. 1 shows in the manner of a schematic vertical section the structure of the Bruvettenteils a flow cytometer and the arrangement of the excitation beam path with the aid of a laser 1 and the measuring beam path with light-collecting optics in shape a converging lens 5, a laser light blocking filter 6 and the photodetector 7.
  • the dispersion of the measuring cuvette 3 is fed through a narrow tube 21.
  • Particle-free medium 14 is fed to the particle flow, which leads to a centering of the particles when passing the measuring range at 23.
  • the morphologically different particles have no preferential orientation. From FIGS. 2 and 4 to 6, the measuring cuvette 3 is in each case in the
  • the laser light excitation takes place from two directions, wherein the irradiation direction of the laser light beam 1 is offset from the laser light beam 2 by 90 °.
  • the laser light in the form of the two laser light beams 1 and 2 excites the fluorescence from two sides of the cuvette 3.
  • a second light collecting optic or converging lens 9 is mounted, which is offset from the first condenser lens 8 by 90 °. This type of detection of the light emanating from each particle further reduces the influence of form factors. Both beam paths for light detection are with high numerical aperture optics in
  • Fig. 3 shows an epithelial cell, which may have up to 60 ⁇ diameter in humans. Will this be on the edge? lights, the laser light reaches the nucleus 16 only weakened. If this cell is irradiated flat and angular at the same time, the jetting errors cancel each other out.
  • the substance to be measured is located in the sperm head 17, in the middle part 18 is preferably RNA. If such an object is irradiated edgewise, the laser light does not reach all the DNA components uniformly, some of the excitation light is "scattered away", and within the sperm head 17 it absorbs light, with the result that not all DNA components contribute equally Fluorescence be excited.
  • FIG. 4 shows the arrangement with laser double excitation in a spatial representation.
  • a measuring optics with a converging lens 5 a laser light blocking filter 6 and a photodetector 7 is shown.
  • the condenser lens 5 has a high numerical aperture, so that the influence of form factors is avoided on the measurement side. Deviating from this illustrated embodiment can be provided, as in the embodiment of FIG. 2, two laser light beams 1 and 2 each assign their own measuring optics.
  • FIG. 5 shows a further embodiment of the measuring apparatus according to the invention: Since the available laser light blocking filters 6, 10, 11 do not completely block the exciting laser light at some wavelengths, an arrangement is provided in FIG. 5 which prevents the respective Laser light beam 1, 2 can go directly into the measuring optics.
  • the optical axis of the converging lens 5 with respect to the beam directions of the two excitation laser light beams 1, 2 arranged in the bisector, ie at an angle of 45 ° or 135 °.
  • a special shape of the Measuring cuvette 19 with a designated as an inclined surface 20 fifth surface in cross section is provided. This fifth surface allows the attachment of a measuring optics with high numerical aperture, without laser excitation light enters the measuring beam path.
  • the parallel alignment of the front surface of the converging lens 5 to the inclined surface 20 ensures that the fluorescent light from the measuring cuvette 19 into the converging lens 5 with low loss.
  • FIG. 6 shows a measuring cuvette which is designed essentially like that of FIG. 5 and is therefore likewise designated by 19. Due to their two aligned at an angle of 90 ° to each other, adjacent to the inclined surface 20 surfaces in addition to the converging lens 5, which is associated with the inclined surface 20, the arrangement of two converging lenses 8, 9 are provided at an angle of 90 ° to each other, similar this is shown in Fig. 2. With this arrangement, particularly precise measurement results can be achieved:
  • Notch filters 21 and 22 are provided between the measuring cuvette 19 and the converging lenses 8 and 9. They block the unwanted light components and are only permeable to stray light if possible. Since only scattered light components are to be detected, ie light components that are incident at an angle of more than 0 °, the directly incident laser light is blocked with so-called laser stops 24.
  • the alignment of the cell within the measuring cuvette 19 can be calculated so that, for example, the measurement results of cells that are unfavorably aligned are not sufficient for further investigation be taken into account, or so that the measurement results of the fluorescent light certain correction factors - depending on the orientation of the measured cell - can be assigned.
  • the illustrated embodiments may be provided to direct the light from the measuring cuvette in the direction of the photodetector by an optical element which is designed as a cylinder with a cylindrical reflection surface.
  • the cylinder may be designed as a hollow cylinder whose inner surface forms the cylindrical reflection surface, or is designed as a solid, translucent cylinder whose outer surface forms the cylindrical reflection surface.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Optical Measuring Cells (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

For an arrangement for measuring optical properties of particles of a flowable dispersion using a measuring cuvette, through the central inner chamber of which the dispersion flows and a laser light beam is shone, the invention proposes that the inner chamber of the cuvette be illuminated by two laser light beams, which are oriented at an offset of 90° from each other.

Description

"Anordnung zum Messen der optischen Eigenschaften von Partikeln einer Dispersion"  "Arrangement for measuring the optical properties of particles of a dispersion"
Beschreibung: Description:
Hintergrund background
Die genaue Messung von optischen Eigenschaften einer großen Zahl von Partikeln einer fließfähigen Dispersion - die gasförmig oder flüssig sein kann - hat in der Zytologie und bei technischen Problemstellungen eine große Bedeutung. Mit Hilfe von Durch- flusszytophotometern („Flow Cytometer"), gelingt heute die photometrische und fluoreszenzoptische Analyse von mehreren tausend Partikeln pro Sekunde. Zusätzlich wurden Vorrichtungen vorgestellt, mit denen es möglich ist, gewünschte Partikel oder Zellen aufgrund der vorausgegangenen Messungen online zu sortieren. Eine derartige Anordnung von Durchflusszytometern mit nachgeschalteter Zellsortierung dient z. B. der Trennung von X- und Y-Chromosomen enthaltenden Spermien zur weiteren Verwendung in der Tierzucht. The accurate measurement of optical properties of a large number of particles of a flowable dispersion - which may be gaseous or liquid - is of great importance in cytology and technical problems. With the aid of flow cytometry ("Flow Cytometer"), photometric and fluorescence-optical analysis of several thousand particles per second is now possible, as well as devices that allow to sort desired particles or cells online based on the previous measurements. Such an arrangement of flow cytometers with downstream cell sorting serves, for example, to separate sperm containing X and Y chromosomes for further use in animal breeding.
Siehe zur Zellsortierung z. B. die Veröffentlichung Bessette, P. H. and Daugherty, P. S. (2004), Flow Cytometric Screening of cDNA Expression Libraries for Fluorescent Proteins. Biotechnol- ogy Progress, 20:963967. doi:10.1021 /bp034308g. Ein von der Anmelderin vertriebenes Gerät zur Zellsortierung ist aus der Praxis unter der Bezeichnung„Particle and Cell Sorter PPCS" bekannt, wobei die Arbeitsweise dieses Gerätes in dem zugehörigen„Manual" beschrieben ist. See for cell sorting z. Bessette, PH and Daugherty, PS (2004), Flow Cytometric Screening of cDNA Expression Libraries for Fluorescent Protein. Biotechnological Progress, 20: 963967. doi: 10.1021 / bp034308g. A device for cell sorting sold by the Applicant is known in practice under the name "Particle and Cell Sorter PPCS", the operation of this device being described in the associated "Manual".
Die genaue Messung der optischen Eigenschaften der Partikel ist dann erschwert, wenn diese bezüglich Form und Größe sehr unterschiedlich sind. The exact measurement of the optical properties of the particles is difficult if they are very different in shape and size.
Wichtige Einsatzgebiete von Durchflussphotozytometern finden sich zahlreich in der Biotechnologie, Überwachung von Produktionsprozessen, Zellanalyse, Zytopathologie und Immunologie. Important fields of application of flow cytometers are numerous in biotechnology, monitoring of production processes, cell analysis, cytopathology and immunology.
Hierbei besteht die Aufgabenstellung neben der schnellen Erfassung der einzelnen Partikel darin, diese möglichst genau optisch zu vermessen. In der Regel werden für die Vorwärts- und Seit- wärtsstreulichtmessungen sowie für die Fluoreszenzanregung Laser als Lichtquellen benutzt. Das Laserlicht lässt man mit sehr geringem Öffnungswinkel (geringe Divergenz) bzw. als parallelen Lichtstrahl auf die Partikel einwirken. Das messtechnische Problem besteht dabei darin, dass morphologisch komplexe Partikel, die oft wie sehr flache, flächige Objekte strukturiert sein können, von dem Laserstrahl rechtwinklig zur Partikelfläche oder parallel zur Partikelfläche erreicht werden. Dies führt zum Beispiel zu„korrekten" Messwerten der Partikelfluoreszenz bei flächiger Anleuchtung und zu„fehlerhaften" Messwerten, wenn das Laserlicht die Kante des Partikels trifft. Here, the task is in addition to the rapid detection of the individual particles is to measure them as accurately as possible optically. As a rule, lasers are used as light sources for the forward and side scattered light measurements as well as for the fluorescence excitation. The laser light is allowed to act on the particles with a very small opening angle (low divergence) or as a parallel light beam. The metrological problem consists in the fact that morphologically complex particles, which can often be structured like very flat, flat objects, are achieved by the laser beam at right angles to the particle surface or parallel to the particle surface. This leads, for example, to "correct" measured values of the particle fluorescence in the case of planar illumination and to "erroneous" measured values when the laser light hits the edge of the particle.
Die Erfindung betrifft eine Anordnung, die diesen Messfehler vermeidet. The invention relates to an arrangement which avoids this measurement error.
Stand der Technik State of the art
Die durchflußzytometrische Analyse von mikroskopisch kleinen Partikeln hat eine große wirtschaftliche Bedeutung, wenn es gelingt, die Messungen höchst präzise zu gestalten. Eine dieser Anwendungen ist die Messung des DNS-Gehaltes von Spermien. Gelingt diese Messung genau genug, lassen sich die X- und Y-Chromosomen enthaltenden 2 Sorten von Spermien mit einer dem Durchflusszytophotometer nachgeschalteten Vorrichtung sortieren und für die Tierzüchtung verwenden. The flow cytometric analysis of microscopic particles has great economic importance if it is possible to make the measurements highly precise. One of these Applications is the measurement of the DNA content of sperm. If this measurement is accurate enough, the 2 varieties of sperm containing X and Y chromosomes can be sorted with a device downstream of the flow cytometer and used for animal breeding.
Da der Unterschied des DNS-Gehaltes dieser zwei Spermienar- ten z. B. beim Rind nur 1 ,6% beträgt (Gesamtmenge der DNS pro Zelle: 3,3 pg) kommt es auf eine sehr genaue Messung der Zellen zu deren Unterscheidung an. Since the difference in the DNA content of these two sperm species z. B. in cattle is only 1, 6% (total amount of DNA per cell: 3.3 pg), it depends on a very accurate measurement of the cells to distinguish them.
Um diese zu gewährleisten, hat es Vorschläge gegeben, die Zel len vor Passieren des die Fluoreszenz anregenden Laserstrahles so zu orientieren, dass diese vorzugsweise von dem Laserstrahl auf ihrer Fläche getroffen werden. Diese Orientierung erreicht man mehr oder weniger nur durch eine flächige bzw. elliptische Austrittsöffnung des Röhrchens, das die Zellsuspension zur Messung hinführt. In order to ensure this, there have been proposals to orient the cells before passing the fluorescence-exciting laser beam in such a way that they are preferably struck by the laser beam on their surface. This orientation is achieved more or less only by a flat or elliptical outlet opening of the tube, which leads the cell suspension to the measurement.
Ein anderer Weg zur Vermeidung des„Orientierungsfehlers" wurde dadurch begangen, dass man die Zellen zur Fluoreszenzanregung mit einer besonders großen numerischen Apertur (großer Raumwinkel) beleuchtet. Das gelingt bisher nur mit konventionellen Lichtquellen, z. B. mit Entladungslampen. Another way of avoiding the "orientation error" was by illuminating the cells for fluorescence excitation with a particularly large numerical aperture (large solid angle), which has so far only been possible with conventional light sources, for example with discharge lamps.
Damit wird jede Zelle allseitig gleichmäßig beleuchtet, was den Formfaktor weitgehend ausschaltet. This illuminates each cell evenly on all sides, which largely shuts off the form factor.
Dieser Weg der Beleuchtung mit einer großen numerischen Apertur vermindert zwar den Formfaktor-Einfluss, erlaubt aber nicht die Erfassung anderer oft wichtiger Parameter der Zellen wie Vorwärtsstreulicht (Partikelgröße) oder Seitwärtsstreulicht (Homogenitätsfaktor). Although this approach of illumination with a large numerical aperture reduces the shape factor influence, it does not permit the detection of other often important parameters of the cells, such as forward scattered light (particle size) or side scattered light (homogeneity factor).
Die dieser Erfindung zugrunde liegende Aufgabenstellung besteht darin, einen Weg zu finden, den bei den bisher bekannten Laser-basierten Durchflußzytophotometern fehlerhaften Einfluss von Formfaktoren auf die Messgenauigkeit zu vermindern oder ganz auszuschließen. Dies führt zu einer deutlich gesteigerten Messgenauigkeit. Aufgabe der Erfindung ist es zudem, die dadurch erreichte hohe Messgenauigkeit mit den beiden Streulichtparametern zu kombinieren. Mit konventionellen Lichtquellen lassen sich diese nicht messen. Erfindungsgemäß wird eine Vorrichtung zur Laserlichtanregung vorgeschlagen, die den Einfluss von Formfaktoren vermeidet. Gegenüber konventionellen Lichtquellen mit dem Vorteil der Anregung mit hoher numerischer A- pertur erlaubt die Laseranregung eine sehr viel höhere Lichtenergiedichte, was zu genaueren Messungen führt (besseres Signal-Rausch-Verhältnis). The object of this invention is to find a way that in the previously known Laser-based flow cytometers reduce or completely eliminate the erroneous influence of shape factors on the measurement accuracy. This leads to a significantly increased measurement accuracy. The object of the invention is also to combine the high measurement accuracy achieved with the two scattered light parameters. These can not be measured with conventional light sources. According to the invention, a device for laser light excitation is proposed, which avoids the influence of form factors. Compared to conventional light sources with the advantage of high numerical aperture excitation, laser excitation allows a much higher density of light energy, resulting in more accurate measurements (better signal-to-noise ratio).
Lösung solution
Der vorliegende Vorschlag wird anhand der rein schematischen Zeichnungen nachfolgend näher erläutert. Dabei zeigt The present proposal is explained in more detail below with reference to the purely schematic drawings. It shows
Fig. 1 einen schematischen Vertikalschnitt durch ein Durch- flusszytometer, 1 shows a schematic vertical section through a flow cytometer,
Fig. 2 eine Draufsicht auf ein erstes Ausführungsbeispiel eines Durchflusszytometers,  2 is a plan view of a first embodiment of a flow cytometer,
Fig. 3 Ansichten aus jeweils zwei verschiedenen Richtungen auf zwei unterschiedliche Arten zu messender Zellen, 3 views from two different directions in two different types of cells to be measured,
Fig. 4 eine perspektivische Ansicht auf ein zweites Ausführungsbeispiel, 4 is a perspective view of a second embodiment,
Fig. 5 eine Draufsicht auf ein drittes Ausführungsbeispiel eines Durchflusszytometers, und  Fig. 5 is a plan view of a third embodiment of a flow cytometer, and
Fig. 6 eine Draufsicht auf ein viertes Ausführungsbeispiel eines Durchflusszytometers.  Fig. 6 is a plan view of a fourth embodiment of a flow cytometer.
Fig. 1 zeigt in Art eines schematischen Vertikalschnitts den Aufbau des Küvettenteils eines Durchflusszytometers sowie die Anordnung des Anregungsstrahlenganges mit Hilfe eines Lasers 1 und des Messstrahlenganges mit lichtsammelnder Optik in Form einer Sammellinse 5, einem Laserlichtsperrfilter 6 und dem Photodetektor 7. Durch ein enges Röhrchen 21 wird die Dispersion der Messküvette 3 zugeführt. Um den Partikelstrom wird partikelfreies Medium 14 zugeführt, das zu einer Zentrierung der Partikel beim Passieren des Messbereichs bei 23 führt. Die morphologisch unterschiedlichen Partikel haben keine Vorzugsorientierung. Aus den Fig. 2 sowie 4 bis 6 ist die Messküvette 3 jeweils in derFig. 1 shows in the manner of a schematic vertical section the structure of the Küvettenteils a flow cytometer and the arrangement of the excitation beam path with the aid of a laser 1 and the measuring beam path with light-collecting optics in shape a converging lens 5, a laser light blocking filter 6 and the photodetector 7. The dispersion of the measuring cuvette 3 is fed through a narrow tube 21. Particle-free medium 14 is fed to the particle flow, which leads to a centering of the particles when passing the measuring range at 23. The morphologically different particles have no preferential orientation. From FIGS. 2 and 4 to 6, the measuring cuvette 3 is in each case in the
Aufsicht oder perspektivisch ersichtlich. Dabei wird deutlich, dass vorschlagsgemäß die Laserlichtanregung aus zwei Richtungen erfolgt, wobei die Einstrahlrichtung des Laserlichtstrahls 1 gegenüber dem Laserlichtstrahl 2 um 90° versetzt ist. Das führt dazu, dass„flache" Partikel von Laser 1 kantig und von Laser 2 flächig beleuchtet werden - oder je nach Orientierung des flächigen Partikels im Flusskanal auch umgekehrt. Da die Partikel jede Orientierung haben können, ist die Summe des Lichtes der beiden Laserlichtstrahlen 1 und 2, die das fluoreszierende Mate- rial erreicht, stets gleich groß. Top view or perspective view. It becomes clear that according to the proposal, the laser light excitation takes place from two directions, wherein the irradiation direction of the laser light beam 1 is offset from the laser light beam 2 by 90 °. This results in flat "particles" being illuminated at edges by Laser 1 and by Laser 2 flatly - or, conversely, depending on the orientation of the two-dimensional particle in the flow channel, since the particles can have any orientation, the sum of the light of the two laser light beams is 1 and 2, which reaches the fluorescent material, always the same size.
Bei dem Ausführungsbeispiel der Fig. 2 regt das Laserlicht in Form der beiden Laserlichtstrahlen 1 und 2 die Fluoreszenz von zwei Seiten der Küvette 3 an. Zusätzlich zur Lichtsammeioptik in Form einer ersten Sammellinse 8 ist eine zweite Lichtsammelop- tik bzw. Sammellinse 9 angebracht, die gegenüber der ersten Sammellinse 8 um 90° versetzt ist. Diese Art der Erfassung des von jedem Partikel ausgehenden Lichts vermindert den Einfluss von Formfaktoren noch zusätzlich. Beide Strahlengänge zur Lichterfassung sind mit Optiken hoher numerischer Apertur inIn the embodiment of FIG. 2, the laser light in the form of the two laser light beams 1 and 2 excites the fluorescence from two sides of the cuvette 3. In addition to the light collecting optics in the form of a first condenser lens 8, a second light collecting optic or converging lens 9 is mounted, which is offset from the first condenser lens 8 by 90 °. This type of detection of the light emanating from each particle further reduces the influence of form factors. Both beam paths for light detection are with high numerical aperture optics in
Form der Sammellinsen 8, 9, sowie mit Sperrfiltern 10, 1 1 für das anregende Laserlicht und mit Photodetektoren 7, 12 ausgestattet. Shape of the converging lenses 8, 9, and with barrier filters 10, 1 1 for the exciting laser light and photodetectors 7, 12 equipped.
Fig. 3 zeigt eine Epithelzelle, die beim Menschen bis zu 60 μιτι Durchmesser aufweisen kann. Wird diese auf der Kante be- leuchtet, erreicht das Laserlicht den Zellkern 16 nur abgeschwächt. Wird diese Zelle gleichzeitig flächig und kantig angestrahlt, heben sich die Anstrahlfehler auf. Fig. 3 shows an epithelial cell, which may have up to 60 μιτι diameter in humans. Will this be on the edge? lights, the laser light reaches the nucleus 16 only weakened. If this cell is irradiated flat and angular at the same time, the jetting errors cancel each other out.
Noch deutlicher wird dieses Problem bei der Messung von Spermien. Die zu messende Substanz, die DNS, befindet sich im Spermienkopf 17, im Mittelteil 18 befindet sich vorzugsweise RNS. Wird ein solches Objekt„kantig" angestrahlt, erreicht das Laserlicht nicht alle DNS-Anteile gleichmäßig; ein Teil des Anregungslichts wird„weggestreut" und innerhalb des Spermien- kopfes 17 kommt es zur Lichtabsorption mit der Folge, dass nicht alle DNS-Anteile gleichermaßen zur Fluoreszenz angeregt werden. This problem becomes even clearer when measuring sperm. The substance to be measured, the DNA, is located in the sperm head 17, in the middle part 18 is preferably RNA. If such an object is irradiated edgewise, the laser light does not reach all the DNA components uniformly, some of the excitation light is "scattered away", and within the sperm head 17 it absorbs light, with the result that not all DNA components contribute equally Fluorescence be excited.
Fig. 4 zeigt die Anordnung mit Laserdoppelanregung in einer räumlichen Darstellung. Rein beispielhaft ist nur eine Messoptik mit einer Sammellinse 5, einem Laserlichtsperrfilter 6 und einem Photodetektor 7 dargestellt. Die Sammellinse 5 weist eine hohe numerische Apertur auf, so dass auf der Messseite der Einfluss von Formfaktoren vermieden wird. Abweichend von diesem dargestellten Ausführungsbeispiel kann vorgesehen sein, wie beim Ausführungsbeispiel der Fig. 2 beiden Laserlichtstrahlen 1 und 2 jeweils eine eigene Messoptik zuzuordnen. 4 shows the arrangement with laser double excitation in a spatial representation. By way of example only a measuring optics with a converging lens 5, a laser light blocking filter 6 and a photodetector 7 is shown. The condenser lens 5 has a high numerical aperture, so that the influence of form factors is avoided on the measurement side. Deviating from this illustrated embodiment can be provided, as in the embodiment of FIG. 2, two laser light beams 1 and 2 each assign their own measuring optics.
In Fig. 5 ist eine weitere erfindungsgemäße Ausgestaltung der Messapparatur dargestellt: Da die zur Verfügung stehenden Laserlichtsperrfilter 6, 10, 1 1 bei manchen Wellenlängen das anregende Laserlicht nicht vollständig blockieren, wird in Fig. 5 eine Anordnung vorgesehen, die verhindert, dass der jeweilige Laserlichtstrahl 1 , 2 auf direktem Weg in die Messoptik gelangen kann. FIG. 5 shows a further embodiment of the measuring apparatus according to the invention: Since the available laser light blocking filters 6, 10, 11 do not completely block the exciting laser light at some wavelengths, an arrangement is provided in FIG. 5 which prevents the respective Laser light beam 1, 2 can go directly into the measuring optics.
Dazu wird die optische Achse der Sammellinse 5 gegenüber den Strahlrichtungen der beiden Anregungslaserlichtstrahlen 1 , 2 in deren Winkelhalbierender angeordnet, also in einem Winkel von jeweils 45° bzw. 135°. Eine besondere Formgebung der Messküvette 19 mit einer als Schrägfläche 20 bezeichneten fünften Fläche im Querschnitt ist dafür vorgesehen. Diese fünfte Fläche erlaubt die Anbringung einer Messoptik mit hoher numerischer Apertur, ohne dass Laser-Anregungslicht in den Messstrahlengang gelangt. Die parallele Ausrichtung der Frontfläche der Sammellinse 5 zu der Schrägfläche 20 stellt sicher, dass das Fluoreszenzlicht verlustarm aus der Messküvette 19 in die Sammellinse 5 einstrahlt. For this purpose, the optical axis of the converging lens 5 with respect to the beam directions of the two excitation laser light beams 1, 2 arranged in the bisector, ie at an angle of 45 ° or 135 °. A special shape of the Measuring cuvette 19 with a designated as an inclined surface 20 fifth surface in cross section is provided. This fifth surface allows the attachment of a measuring optics with high numerical aperture, without laser excitation light enters the measuring beam path. The parallel alignment of the front surface of the converging lens 5 to the inclined surface 20 ensures that the fluorescent light from the measuring cuvette 19 into the converging lens 5 with low loss.
Fig. 6 zeigt eine Messküvette, die im wesentlichen wie die der Fig. 5 ausgestaltet ist und daher ebenfalls mit 19 gekennzeichnet ist. Aufgrund ihrer beiden im Winkel von 90° zueinander ausgerichteten, an die Schrägfläche 20 angrenzenden Flächen kann zusätzlich zu der Sammellinse 5, welche der Schrägfläche 20 zugeordnet ist, auch die Anordnung zweier Sammellinsen 8, 9 im Winkel von 90° zueinander vorgesehen sein, ähnlich wie dies in Fig. 2 dargestellt ist. Mit dieser Anordnung sind besonders präzise Messergebnisse erzielbar: FIG. 6 shows a measuring cuvette which is designed essentially like that of FIG. 5 and is therefore likewise designated by 19. Due to their two aligned at an angle of 90 ° to each other, adjacent to the inclined surface 20 surfaces in addition to the converging lens 5, which is associated with the inclined surface 20, the arrangement of two converging lenses 8, 9 are provided at an angle of 90 ° to each other, similar this is shown in Fig. 2. With this arrangement, particularly precise measurement results can be achieved:
Mittels der Photodetektoren 7, die den Sammellinsen 8 und 9 nachgeschaltet sind, wird nicht das Fluoreszenzlicht ausgewertet, sondern Vorwärtsstreulicht bzw. negatives Absorptionslicht, welches sich nach dem Auftreffen des Anregungslichts, also der Laserlichtstrahlen 1 und 2, auf die jeweilige Zelle ergibt. Sperrfilter 21 und 22 sind zwischen der Messküvette 19 und den Sammellinsen 8 und 9 vorgesehen. Sie blockieren die unerwünschten Lichtanteile und sind möglichst nur für das Streulicht durchlässig. Da nur Streulichtanteile erfasst werden sollen, also Lichtanteile, die in einem Winkel von mehr als 0° einfallen, wird das direkt einfallende Laserlicht mit so genannten Laserstops 24 blockiert. By means of the photodetectors 7, which are connected downstream of the collecting lenses 8 and 9, not the fluorescent light is evaluated, but forward scattered light or negative absorption light, which results after the impingement of the excitation light, ie the laser light beams 1 and 2, on the respective cell. Notch filters 21 and 22 are provided between the measuring cuvette 19 and the converging lenses 8 and 9. They block the unwanted light components and are only permeable to stray light if possible. Since only scattered light components are to be detected, ie light components that are incident at an angle of more than 0 °, the directly incident laser light is blocked with so-called laser stops 24.
Aus den beiden Vorwärtsstreulicht-Signalen kann die Ausrichtung der Zelle innerhalb der Messküvette 19 berechnet werden, so dass beispielsweise die Messergebnisse von Zellen, die unvorteilhaft ausgerichtet sind, für die weitere Untersuchung nicht berücksichtigt werden, oder so dass den Messergebnissen des Fluoreszenzlichts bestimmten Korrekturfaktoren - je nach Ausrichtung der gemessenen Zelle - zugeordnet werden können. From the two forward scattered light signals, the alignment of the cell within the measuring cuvette 19 can be calculated so that, for example, the measurement results of cells that are unfavorably aligned are not sufficient for further investigation be taken into account, or so that the measurement results of the fluorescent light certain correction factors - depending on the orientation of the measured cell - can be assigned.
Mittels der Sammellinse 5 wird bei der Messanordnung gemäß Fig. 6 ausschließlich das von der untersuchten Zelle ausgesandte Fluoreszenzlicht erfasst, da die Laserlichtstrahlen 1 und 2 nicht auf die Sammellinse 5 gerichtet sind. Mit dieser Anordnung wird daher ein besonders reines Messsignal erhalten, welches nicht von Laserlichtanteilen kontaminiert ist. Aus diesem Grund ist rein beispielhaft kein Laserlichtsperrfilter zwischen der Schrägfläche 20 und dem ihr zugeordneten Photodetektor 7 vorgesehen. Abweichend von dieser Darstellung kann an dieser Stelle jedoch ein Laserlichtsperrfilter 6 wie in Fig. 5 vorgesehen sein, um unerwünschte Streulichtanteile auszuschließen. By means of the converging lens 5, only the fluorescence light emitted by the cell under examination is detected in the measuring arrangement according to FIG. 6, since the laser light beams 1 and 2 are not directed onto the condenser lens 5. With this arrangement, therefore, a particularly pure measurement signal is obtained, which is not contaminated by laser light components. For this reason, by way of example, no laser light blocking filter is provided between the inclined surface 20 and the photodetector 7 associated therewith. Notwithstanding this illustration, however, a laser light blocking filter 6 may be provided at this point as shown in Fig. 5, to exclude unwanted stray light components.
Abweichend von den dargestellten Ausführungsbeispielen kann vorgesehen sein, das Licht von der Messküvette in Richtung zum Photodetektor durch ein optisches Element zu leiten, welches als Zylinder mit einer zylindrischen Reflexionsfläche ausgestaltet ist. Dabei kann der Zylinder als Hohlzylinder ausgestaltet sein, dessen innere Oberfläche die zylindrische Reflexionsfläche bildet, oder als massiver, lichtdurchlässiger Zylinder ausgestaltet ist, dessen äußere Oberfläche die zylindrische Reflexionsfläche bildet. Notwithstanding the illustrated embodiments may be provided to direct the light from the measuring cuvette in the direction of the photodetector by an optical element which is designed as a cylinder with a cylindrical reflection surface. In this case, the cylinder may be designed as a hollow cylinder whose inner surface forms the cylindrical reflection surface, or is designed as a solid, translucent cylinder whose outer surface forms the cylindrical reflection surface.

Claims

Anordnung zur Messung optischer Eigenschaften von Partikeln einer fließfähigen Dispersion Arrangement for measuring optical properties of particles of a flowable dispersion
unter Verwendung einer Messküvette, deren zentraler Innenraum von der Dispersion durchflössen und von einem Laserlichtstrahl durchleuchtet wird,  using a measuring cuvette whose central interior is traversed by the dispersion and is illuminated by a laser light beam,
wobei das aus der Messküvette ausgetretene Licht auf einen lichtempfindlichen, als Photodetektor bezeichneten Sensor geführt wird,  wherein the leaked light from the cuvette is passed to a photosensitive sensor called a photodetector,
dadurch gekennzeichnet,  characterized,
dass der Innenraum der Küvette (3, 19) von zwei Laserlichtstrahlen (1 , 2) beleuchtet wird,  the interior of the cuvette (3, 19) is illuminated by two laser light beams (1, 2),
welche mit einem Versatz von 90° zueinander ausgerichtet sind.  which are aligned with an offset of 90 ° to each other.
Anordnung nach Anspruch 1 , Arrangement according to claim 1,
dadurch gekennzeichnet,  characterized,
dass ein gemeinsamer Laser vorgesehen ist, dessen Strahl mit optischen Mitteln aufgeteilt ist, derart, dass die beiden Laserstrahlen (1 , 2) von dem selben Laser erzeugt sind.  a common laser is provided, the beam of which is split by optical means, such that the two laser beams (1, 2) are generated by the same laser.
Anordnung nach Anspruch 1 oder 2, Arrangement according to claim 1 or 2,
dadurch gekennzeichnet,  characterized,
dass beide Laserstrahlen (1 , 2) die gleiche Strahlenenergie und Strahlengeometrie aufweisen.  that both laser beams (1, 2) have the same beam energy and beam geometry.
Anordnung nach Anspruch 3, Arrangement according to claim 3,
dadurch gekennzeichnet,  characterized,
dass eine Sammellinse (5, 8, 9) mit hoher numerischer Apertur zwischen der Messküvette (3, 19) und dem Photodetektor (7) angeordnet ist.  a collecting lens (5, 8, 9) with a high numerical aperture is arranged between the measuring cuvette (3, 19) and the photodetector (7).
5. Anordnung nach Anspruch 4, 5. Arrangement according to claim 4,
dadurch gekennzeichnet,  characterized,
dass zwei Sammellinsen (8, 9) mit hoher numerischer Apertur jeweils zwischen der Messküvette (19) und einem Photodetektor (7) angeordnet sind, that two converging lenses (8, 9) with high numerical Aperture are each arranged between the measuring cuvette (19) and a photodetector (7),
wobei die optischen Achsen der beiden Sammellinsen (8, 9) um 90° zueinander versetzt sind. wherein the optical axes of the two converging lenses (8, 9) are offset by 90 ° to each other.
Anordnung nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, Arrangement according to one of the preceding claims, characterized
dass der Querschnitt der Messküvette (19) eine Schrägfläche (20) aufweist, die in einem von 90° abweichenden Winkel zu jeder der beiden Einstrahlrichtungen der Laserstrahlen (1 , 2) ausgerichtet ist. the cross-section of the measuring cuvette (19) has an oblique surface (20) which is oriented at an angle deviating from 90 ° to each of the two directions of irradiation of the laser beams (1, 2).
Anordnung nach Anspruch 6, Arrangement according to claim 6,
dadurch gekennzeichnet, characterized,
dass die Schrägfläche (20) im Winkel von 45° zu jedem der beiden Laserstrahlen (1 , 2) ausgerichtet ist. in that the oblique surface (20) is oriented at an angle of 45 ° to each of the two laser beams (1, 2).
Anordnung nach Anspruch 6 oder 7, Arrangement according to claim 6 or 7,
dadurch gekennzeichnet, characterized,
dass die Frontfläche der Sammellinse (5) parallel zu der Schrägfläche (20) verläuft in that the front face of the condenser lens (5) runs parallel to the oblique surface (20)
Anordnung nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, Arrangement according to one of the preceding claims, characterized
dass in direkter Richtung der beiden Laserstrahlen (1 , 2) Sammellinsen (8, 9) zwischen der Messküvette (3, 19) und dem Photodetektor (7) angeordnet sind, welche zur Erfassung der Vorwärtsstreulichts bzw. negativen Absorptionslichtes ausgestaltet sind. that in the direct direction of the two laser beams (1, 2) collecting lenses (8, 9) between the measuring cuvette (3, 19) and the photodetector (7) are arranged, which are designed to detect the forward scattered light or negative absorption light.
Anordnung nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, Arrangement according to one of the preceding claims, characterized
dass zwischen der Messküvette (3, 19) und dem Photodetektor (7) ein Lasersperrfilter (6, 10, 1 1 ) angeordnet ist, welcher das Licht der anregenden Laserstrahlen (1 , 2) sperrend ausgestaltet ist. that between the measuring cuvette (3, 19) and the photodetector (7) a laser blocking filter (6, 10, 1 1) is arranged, which detects the light of the exciting laser beams (1, 2) is configured blocking.
Anordnung nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, Arrangement according to one of the preceding claims, characterized
dass der Partikelmessanordnung eine Vorrichtung zum Sortieren von Partikeln nachgeschaltet ist. that the particle measuring arrangement is followed by a device for sorting particles.
EP11782374A 2010-05-18 2011-05-13 Arrangement for measuring the optical properties of particles of a dispersion Withdrawn EP2572182A2 (en)

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CA2784073A1 (en) 2011-05-24
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