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EP3867682A1 - Method and microscope for determining a tilt of a cover slip - Google Patents

Method and microscope for determining a tilt of a cover slip

Info

Publication number
EP3867682A1
EP3867682A1 EP19797574.1A EP19797574A EP3867682A1 EP 3867682 A1 EP3867682 A1 EP 3867682A1 EP 19797574 A EP19797574 A EP 19797574A EP 3867682 A1 EP3867682 A1 EP 3867682A1
Authority
EP
European Patent Office
Prior art keywords
objective
light bundle
cover glass
measuring
measurement
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.)
Pending
Application number
EP19797574.1A
Other languages
German (de)
French (fr)
Inventor
Christian Schumann
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.)
Leica Microsystems CMS GmbH
Original Assignee
Leica Microsystems CMS GmbH
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
Application filed by Leica Microsystems CMS GmbH filed Critical Leica Microsystems CMS GmbH
Publication of EP3867682A1 publication Critical patent/EP3867682A1/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B11/00—Measuring arrangements characterised by the use of optical techniques
    • G01B11/02—Measuring arrangements characterised by the use of optical techniques for measuring length, width or thickness
    • G01B11/026—Measuring arrangements characterised by the use of optical techniques for measuring length, width or thickness by measuring distance between sensor and object
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B11/00—Measuring arrangements characterised by the use of optical techniques
    • G01B11/26—Measuring arrangements characterised by the use of optical techniques for measuring angles or tapers; for testing the alignment of axes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/241—Devices for focusing
    • G02B21/245—Devices for focusing using auxiliary sources, detectors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/0025—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for optical correction, e.g. distorsion, aberration
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B7/00—Mountings, adjusting means, or light-tight connections, for optical elements
    • G02B7/28—Systems for automatic generation of focusing signals
    • G02B7/30—Systems for automatic generation of focusing signals using parallactic triangle with a base line
    • G02B7/32—Systems for automatic generation of focusing signals using parallactic triangle with a base line using active means, e.g. light emitter
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/0088—Inverse microscopes

Definitions

  • the invention relates to a method for determining a tilt of a cover glass in a microscope, which has a cover glass facing lens.
  • the invention also relates to a microscope with a device for determining a tilting of a cover slip.
  • the quality of a light microscopic image with the aid of a lens with a high atomic aperture is strongly influenced by the position of a cover glass which covers the sample to be imaged.
  • tilting the cover glass relative to the optical axis of the objective induces aberrations. Tilting the cover glass means that the detection light used for imaging falls obliquely into the lens. This creates a coma.
  • Measuring the tilt of the cover slip can be done tactile, i.e. using a probe.
  • this is associated with a high level of procedural complexity and requires the insertion of the probe into the sample space.
  • the method according to the invention is used to determine a tilt of a cover glass in a microscope which has an objective facing the cover glass. At least three measuring points spanning a plane are defined on a surface of the cover slip. The following steps are carried out for each of the three measuring points: directing a measuring light beam through the lens onto the measuring point; Generating a reflection light bundle by at least partially reflecting the measurement light bundle at the respective measurement point; Directing the reflection light beam through the lens onto a position-sensitive sensor; Detecting the point of incidence of the reflection light beam on the position-sensitive sensor; and determining the distance of the respective measuring point from the objective along its optical axis on the basis of the detected incident location. Then, on the basis of the determined distances, a tilt of the plane spanned by the three measuring points relative to the optical axis of the objective is determined as a tilt of the surface of the cover slip.
  • the plane spanned by the at least three measuring points is coplanar with the surface of the cover slip mentioned.
  • the tilt of this plane relative to the optical axis of the lens therefore reflects the tilt of the cover slip.
  • Each of the at least three measuring points is determined by three coordinates, one of which indicates the distance of the measuring point from the lens along its optical axis, while the other two coordinates determine the position of the respective measuring point on the surface of the cover slip.
  • the method according to the invention enables the tilting of the cover slip within the microscope to be determined simply and reliably.
  • the at least three measuring points are defined by moving the cover glass and the lens relative to one another transversely to its optical axis.
  • the determination of the measuring points can be done automatically or by an operator.
  • the points can be defined while the cover slip is moved transversely to the optical axis of the objective to find a sample.
  • the cover glass is preferably moved by means of a movable microscope table relative to the objective transversely to its optical axis.
  • the measurement light bundle is directed into a partial area of an entrance pupil of the objective which is offset with respect to the center of the entrance pupil.
  • the entrance pupil of the lens is decentrally illuminated by the measuring light bundle, as a result of which the measuring light bundle is inclined to its optical axis when it exits the lens.
  • the decentralized illumination of the entrance pupil of the objective has the advantage that beam portions close to the axis are avoided, the so-called first-order reflections, which occur most strongly on the surface vertices of the lenses forming the objective, and thereby worsen the signal-to-noise ratio.
  • the reflection light bundle is preferably passed back into the lens in such a way that it penetrates another portion of the entrance pupil in the direction opposite to the direction of propagation of the measurement light bundle, which is offset from the aforementioned portion of the entrance pupil.
  • the measurement light bundle generates a measurement pattern on the surface and the measurement pattern is imaged on the position-sensitive sensor by the reflection light bundle.
  • the measurement pattern in the form of an image of a slit diaphragm, which is arranged upstream of the light source emitting the measurement light bundle.
  • the surface of the cover glass, on which the measurement light bundle is reflected to generate the reflection light bundle forms a partially reflecting interface with an adjacent optical medium.
  • the optical medium is an embedding medium that borders on the surface of the cover slip.
  • the distance measurement carried out at the respective measuring point, on which the determination of the tilt of the cover glass according to the invention is based uses a partial reflection of the measurement light beam on the surface of the cover glass.
  • This partial reflection is caused by the fact that the surface with the adjoining optical medium, which has a different refractive index than the cover glass, forms an interface at which a jump in the refractive index occurs. In this way it is possible to determine the tilting of the cover slip within the microscope in a particularly simple and reliable manner.
  • the orientation of a normal vector which is perpendicular to the plane mentioned, is determined on the basis of the at least three measuring points.
  • the tilt of the cover slip is then determined from this.
  • the angle enclosed by the normal vector and the optical axis of the objective can be determined.
  • the tilt of the plane defined by the measuring points and thus the tilt of the cover slip can be clearly quantified by this angle.
  • more than three measuring points are defined, the distances between which are determined by the objective in order to determine the tilting of the cover glass.
  • the more measuring points defined on the surface of the cover slip the more precisely the tilt of the plane defined by the measurement points and thus the tilt of the cover slip can be determined.
  • the cover slip is adjusted to compensate for the tilting determined.
  • the determined tilt can be used for the calculation of a filter function for inversion of the imaging process, for example a deconvolution or a quantitative phase reconstruction.
  • the invention further relates to a microscope which comprises a cover slip, a cover glass facing lens and a device for determining a tilt of the cover slip.
  • the device is designed to define at least three measuring points spanning a plane on a surface of the cover glass and to carry out the following steps for each of these measuring points: directing a measuring light beam through the lens onto the measuring point; Generating a reflection light bundle by at least partially reflecting the measurement light bundle at the respective measurement point; Directing the reflection light beam through the lens onto a position-sensitive sensor; Detecting the point of incidence of the reflection light beam on the position-sensitive sensor; and determining the distance of the respective measuring point from the objective along its optical axis on the basis of the detected incident location.
  • the device is also designed to determine a tilt of the plane spanned by the three measuring points relative to the optical axis of the lens as a tilt of the surface of the cover slip on the basis of the determined distances.
  • the device has an aperture diaphragm with an aperture opening which is arranged decentrally at a distance from the optical axis of the objective.
  • the device has a light source which emits the measurement light bundle in the infrared wavelength range.
  • a light source which emits the measurement light bundle in the infrared wavelength range.
  • the position-sensitive sensor is a line sensor.
  • the line sensor is preferably designed such that it can detect the intensity distribution of the reflection light beam in its entirety.
  • the position-sensitive sensor can also be designed as an area sensor, for example as a two-dimensional CCD camera.
  • the microscope preferably comprises means for correcting the determined tilting of the cover slip.
  • These means include, for example, a manually or motorized movable microscope stage.
  • the device according to the invention is also suitable for use in the microscope as an autofocus device.
  • the device offers the possibility of determining, in addition to the tilting of the cover glass, other variables influencing the light microscopic image, such as the thickness of the cover glass and / or the refractive index of an optical medium.
  • the invention is applicable to a variety of microscope types, e.g. inverse or upright transmitted light microscopes.
  • Figure 1 is a schematic representation of an inverted transmitted light microscope as the first embodiment
  • 2 shows a device for determining the tilting of the cover glass, which is part of the microscope according to FIG. 1;
  • Figure 3 is a schematic representation showing a sample space of the microscope
  • FIG. 4 shows an intensity distribution detected by a position-sensitive detector of the device according to FIG. 2;
  • Figure 5 is a schematic representation, which is determined by three measuring points
  • Figure 6 is a flowchart showing a specific embodiment of the method of the invention for determining the thickness of the cover slip
  • Figure 7 is a schematic diagram showing a movable microscope stage
  • FIG. 8 shows a schematic illustration of an upright transmitted-light microscope, which forms a second exemplary embodiment of the microscope according to the invention.
  • FIG. 1 shows a microscope 10 as the first exemplary embodiment, to which the tilt determination according to the invention is applicable.
  • the microscope 10 is designed as an inverse transmitted light microscope. Accordingly, it comprises a lens 12, which faces from below a sample space provided with the reference number 14 in FIG. 1, and a light source 16 which is directed onto the sample space 14 from above.
  • the microscope 10 also has a tube 18 with an eyepiece 20, through which an operator can view a sample captured by the lens 12.
  • a control unit 22 is provided which controls the various microscope components.
  • cover glass 24 which covers a sample not explicitly shown in FIG. 1.
  • optical medium 26 in which the sample is embedded and which is referred to below as the embedding medium 26.
  • immersion medium 28 which borders in FIG. 1 on the lens 12 from above and on the cover glass 24 from below.
  • the microscope 10 also has a device, generally designated by the reference numeral 30 in FIG. 1, which serves to determine the tilting of the cover glass 14.
  • the device 30 is shown in more detail in FIG.
  • the device 30 has a light source 32 which emits a measurement light bundle 34 in the infrared wavelength range.
  • the light source 32 is, for example, an LED which has a slit diaphragm 33 through which the measurement light bundle 34 is directed onto an illumination optics 36.
  • the measuring light bundle 34 falls onto an aperture diaphragm 38, which is positioned centrally on the optical axis Ol of the illumination optics 36, but has an aperture 39 which is decentrally arranged at a distance from the optical axis Ol of the illumination optics 36 is.
  • the aperture 39 of the aperture diaphragm 38 limits the beam cross section of the measuring light beam 34 such that only the part of the measuring light beam 34 lying below the optical axis O1 of the illumination optics 36 in FIG. 2 passes the aperture diaphragm 38 in the direction of a deflection prism 40.
  • the measuring light bundle 34 which is limited in its beam cross section, is reflected on the deflection prism 40 in a transport optic 42, which consists of a along its optical Axis 02 displaceable focusing lens 44, a Le uchtfe Id aperture 46 and a white lens 48 is formed.
  • the measuring light bundle 34 falls onto a dichroic beam splitter 50, which reflects light in the infrared wavelength range while transmitting light in the visible range.
  • the dichroic mirror 50 the measuring light beam 34 is reflected in the direction of the lens 12 re.
  • the measuring light bundle 34 reflected on the dichroic mirror 50 runs with a parallel offset to the optical axis 03 of the objective 12.
  • the measuring light bundle 34 is guided into a partial area of an entrance pupil 52 of the objective 12, which is opposite the optical axis 03 of the objective 12 and since the entrance pupil 52 is laterally offset with respect to the center (cf. FIG. 4).
  • the entrance pupil 52 of the objective 12 is thus illuminated locally, which means that the measurement light bundle 34 is directed into the sample space 14 at an angle a obliquely to the optical axis 03.
  • the embedding medium 26 and the immersion medium 28 which adjoin the cover glass 24 from opposite sides in the sample space 14 are omitted in the illustration according to FIG.
  • the measurement light bundle 34 guided under oblique incidence into the sample space 14 is reflected on the cover glass 24, as will be explained in more detail below with reference to FIG. 4, whereby a reflection bundle 54 which is guided back into the objective 12 is produced.
  • the reflection light bundle 54 After passing through the lens 12, the reflection light bundle 54 falls onto the dichroic mirror 50, which directs the reflection light bundle 54 into the transport optics 42. After passing through the transport optics 42, the reflection light bundle 54 falls onto the order deflecting prism 40, which reflects the reflection light bundle 54 onto a detector optics 56.
  • the detector optics 56 directs the reflection light bundle 54 to a spectral filter 58 which is only permeable to light in the infrared wavelength range and blocks stray light outside this wavelength range.
  • the trans- by the spectral filter 58 Centered reflection light bundle 54 finally falls on a position sensitive detector 60, which is able to detect the intensity of the reflection light bundle 54 in a spatially resolved manner.
  • FIG. 2 also illustrates the coupling of the tube 18 to the device 30, which is implemented via the dichroic mirror 50.
  • the dichroic mirror 50 in the present exemplary embodiment is also used for the actual microscopic imaging, visible detection light 62, which guides the lens 12 from the sample space 14 in the direction of the dichroic mirror 50, to be transmitted to the tube 18 by transmission.
  • a distance measurement at three measuring points PI, P2 and P3 causes the cover glass 24 to tilt relative to the optical axis 03 of the objective 12 is determined.
  • FIG. 3 it is initially illustrated how the reflection light bundle 54 is generated for each of the measurement points PI to P3 by reflection of the measurement light bundle 34, which bundle is used according to the invention to determine the distance of the respective measurement point from the objective 12.
  • the measuring pupil 34 which decentrally illuminates the entrance pupil 52 of the objective 12, is directed through the objective 12 at an angle a obliquely to the optical axis 03 on the objective 12, designated in FIG. 3 by 64 in front of the surface of the cover glass 24. Since the cover glass 24 and the immersion medium 28 bordering its front surface 64 have different refractive indices, the front surface 64 of the cover glass 24 and the adjoining always sions medium 28 form an interface on which the incident measurement light bundle 34 is partially is reflected. The part of the measuring light bundle 34 reflected at this interface generates the reflection light bundle 54, which is directed back into the objective 12.
  • FIG. 4 shows an intensity distribution V that the reflection light beam 54 generates on the position-sensitive detector 60.
  • the abscissa of the diagram according to FIG. 4 represents the point of incidence on the detector 60 and the ordinate represents the intensity measured at the respective point of incidence.
  • the intensity distribution V according to FIG. 4 shows a peak P, the position Xi of which, which can be determined on the position-sensitive detector 60 with respect to a reference position Xref, is a measure of the distance zi that the surface 64 of the cover glass 24 along the optical axis 03 of the lens 12.
  • the coordinates xi, yi are predefined, while the coordinate zi, which specifies the distance of the associated measurement point Pi from the objective, represents the variable to be determined according to the invention.
  • the optical axis 03 of the objective 12 is aligned with the measuring point PI, which means that in this example the distance of the point PI is determined. If the distance determinations are to be carried out for the other two measuring points P2 and P3, they should approach these measuring points accordingly.
  • the cover glass 24 and the lens 12 are moved relative to one another transversely to its optical axis 03 until the optical axis 03 is set to the desired measuring point P2 or P3.
  • FIG. 5 also shows a normal vector N which is perpendicular to the plane defined by the measurement points PI, P2 and P3.
  • the normal vector N includes an angle ⁇ with the optical axis 03 of the objective, which indicates the tilt of the plane determined by the measurement points PI, P2 and P3 and thus the front surface 64 of the cover glass 24.
  • the flow chart according to FIG. 6 shows an example of how the method for determining the tilting of the cover glass 24 can be implemented.
  • a first step S1 the three measuring points PI, P2, P3 on the surface 64 of the cover slip 24 are defined such that a point representing the surface 64 according to FIG. 5 is defined by the points PI, P2 and P3.
  • step S2 the first measurement point PI, if it has not already been set anyway, is approached such that the optical axis 03 of the objective 12 is aligned with the first measurement point PI. Then, as described above with reference to FIGS. 3 and 4, the distance z1 of the first measuring point PI from the objective 12 is measured along its optical axis 03.
  • step S3 the second measuring point P2 is then approached by aligning the optical axis 03 of the objective 12 with the measuring point P2, and the distance z2, which the second measuring point has from the objective 12, in the same way as for the first measuring point PI determined.
  • step S4 the third measuring point P3 is approached by aligning the optical axis 03 of the objective 12 with the third measuring point. Then the distance z3, which the third measuring point P3 has from the objective 12, is determined in the same way as for the measuring points PI and P2.
  • step S5 on the basis of the distance measurements carried out in steps S2, S3 and S4, the normal vector N is determined, which is perpendicular to the plane defined by the three measuring points PI, P2 and P3, and the angle ⁇ determined by the normal vector N with the optical axis 03 of the lens 12 includes.
  • the tilt of the surface 64 of the cover glass 12 is finally determined.
  • the various measuring points PI, P2 and P3 can be approached, for example, by means of a microscope table 86 shown purely schematically in FIG. This can be adjusted transversely to the optical axis of the objective 12 in order to carry out the desired distance measurements.
  • the objective 12 is arranged above the sample space 18, while the light source 16 is located below the sample space 18. Accordingly, the immersion medium 28, which on the one hand borders on the objective 12 and on the other hand on the cover glass 24, is located above the cover glass 24, while the embedding medium 26 is arranged below the cover glass 24.
  • the determination of the tilting of the cover glass 24 according to the invention takes place in the microscope 78 according to FIG. 8 in the same way as in the microscope 10 shown in FIG. 1.
  • the measurement light bundle 34 is partially reflected by the interface which is formed by the front surface 64 of the cover glass 24 and the immersion medium 28 adjoining it.
  • the measurement light bundle 34 is partially reflected by an interface which is formed by a rear surface 68 of the cover glass 24 facing away from the lens 12 and the embedding medium 26 adjoining it.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Microscoopes, Condenser (AREA)
  • Automatic Focus Adjustment (AREA)

Abstract

The invention relates to a method for determining a tilt of a cover slip (24) in a microscope (10, 78) which has a lens (12) facing the cover slip (24). At least three measurement points which span a plane are defined on a surface (64, 68) of the cover slip, and the following steps are performed for each of the three measurement points: directing a measurement light beam (34) through the lens (12) onto the measurement point; generating a reflection light beam (54) in that at least part of the measurement light beam (54) is reflected at the measurement point; directing the reflection light beam (54) through the lens (12) onto a position-sensitive sensor (60); detecting the incidence site of the reflection light beam (54) on the position-sensitive sensor (60); and determining the distance of the measurement point from the lens (12) along the optical axis (O3) thereof on the basis of the detected incidence site. A tilt of the plane spanned by the three measurement points relative to the optical axis (O3) of the lens (12) is determined as a tilt of the cover slip (24) on the basis of the determined distances.

Description

Verfahren und Mikroskop zur Bestimmung einer Verkippung  Method and microscope for determining tilt
eines Deckglases  a cover slip
Die Erfindung betrifft ein Verfahren zur Bestimmung einer Verkippung eines Deckgla ses in einem Mikroskop, das ein dem Deckglas zugewandtes Objektiv aufweist. Die Er findung betrifft ferner ein Mikroskop mit einer Einrichtung zur Bestimmung einer Ver kippung eines Deckglases. The invention relates to a method for determining a tilt of a cover glass in a microscope, which has a cover glass facing lens. The invention also relates to a microscope with a device for determining a tilting of a cover slip.
Die Qualität einer lichtmikroskopischen Abbildung mit Hilfe eines Objektivs hoher nu merischer Apertur wird stark durch die Lage eines Deckglases beeinflusst, das die abzu bildende Probe bedeckt. Beispielsweise werden durch eine Verkippung des Deckglases relativ zur optischen Achse des Objektives Abbildungsfehler induziert. Dabei führt eine Verkippung des Deckglases dazu, dass das der Abbildung dienende Detektionslicht schräg in das Objektiv fällt. Hierdurch entsteht eine Koma. Um eine wirksame Korrek tion der Koma, die durch eine Verkippung des Deckglases verursacht wird, zu ermögli chen, ist es wichtig, die Verkippung möglichst genau zu kennen. The quality of a light microscopic image with the aid of a lens with a high atomic aperture is strongly influenced by the position of a cover glass which covers the sample to be imaged. For example, tilting the cover glass relative to the optical axis of the objective induces aberrations. Tilting the cover glass means that the detection light used for imaging falls obliquely into the lens. This creates a coma. In order to enable effective coma correction caused by tilting the cover slip, it is important to know the tilt as precisely as possible.
Eine Messung der Verkippung des Deckglases kann taktil, d.h. mithilfe eines Messtas ters erfolgen. Dies ist jedoch mit einem hohen verfahrenstechnischen Aufwand ver bunden und erfordert das Einbringen des Messtasters in den Probenraum. Measuring the tilt of the cover slip can be done tactile, i.e. using a probe. However, this is associated with a high level of procedural complexity and requires the insertion of the probe into the sample space.
Zum Stand der Technik wird ferner auf DE 10 2010 030 430 Al verwiesen, worin eine triangulierende Autofokuseinrichtung für ein Mikroskop offenbart ist. Diese Autofokus einrichtung erzeugt ein Spaltbild auf der Probe, das auf einen positionssensitiven De tektor abgebildet wird. Über die durch den Detektor erfasste Einfallsposition wird der Autofokus gesteuert. With regard to the prior art, reference is also made to DE 10 2010 030 430 A1, in which a triangulating autofocus device for a microscope is disclosed. This autofocus device creates a slit image on the sample, which is imaged on a position-sensitive detector. The autofocus is controlled via the incidence position detected by the detector.
Es ist Aufgabe der Erfindung, ein Verfahren und ein Mikroskop anzugebenen, die eine einfache und präzise Bestimmung einer Verkippung eines Deckglases ermöglicht. Diese Aufgabe wird durch ein Verfahren mit den Merkmalen des Anspruchs 1 und durch ein Mikroskop mit den Merkmalen des Anspruchs 12 gelöst. Vorteilhafte Weiter bildungen finden sich in den abhängigen Ansprüchen. It is an object of the invention to provide a method and a microscope which enables a tilting of a cover slip to be determined simply and precisely. This object is achieved by a method with the features of claim 1 and by a microscope with the features of claim 12. Advantageous further training can be found in the dependent claims.
Das erfindungsgemäße Verfahren dient der Bestimmung einer Verkippung eines Deck glases in einem Mikroskop, das ein dem Deckglas zugewandtes Objektiv aufweist. Auf einer Oberfläche des Deckglases werden mindestens drei Messpunkte, die eine Ebene aufspannen, definiert. Es werden für jeden der drei Messpunkte folgende Schritte durchgeführt: Lenken eines Messlichtbündels durch das Objektiv auf den Messpunkt; Erzeugen eines Reflexionslichtbündels, indem das Messlichtbündel zumindest zum Teil an dem jeweiligen Messpunkt reflektiert wird; Lenken des Reflexionslichtbündels durch das Objektiv auf einen positionssensitiven Sensor; Erfassen des Einfallsortes des Reflexionslichtbündels auf dem positionssensitiven Sensor; und Ermitteln des Abstands des jeweiligen Messpunktes von dem Objektiv längs dessen optischer Achse auf Grundlage des erfassten Einfallsortes. Anschließend wird auf Grundlage der ermittel ten Abstände eine Verkippung der durch die drei Messpunkte aufgespannten Ebene relativ zur optischen Achse des Objektivs als Verkippung der Oberfläche des Deckglases bestimmt. The method according to the invention is used to determine a tilt of a cover glass in a microscope which has an objective facing the cover glass. At least three measuring points spanning a plane are defined on a surface of the cover slip. The following steps are carried out for each of the three measuring points: directing a measuring light beam through the lens onto the measuring point; Generating a reflection light bundle by at least partially reflecting the measurement light bundle at the respective measurement point; Directing the reflection light beam through the lens onto a position-sensitive sensor; Detecting the point of incidence of the reflection light beam on the position-sensitive sensor; and determining the distance of the respective measuring point from the objective along its optical axis on the basis of the detected incident location. Then, on the basis of the determined distances, a tilt of the plane spanned by the three measuring points relative to the optical axis of the objective is determined as a tilt of the surface of the cover slip.
Erfindungsgemäß wird angenommen, dass die durch die mindestens drei Messpunkte aufgespannte Ebene koplanar zur genannten Oberfläche des Deckglases ist. Die Verkip pung dieser Ebene relativ zur optischen Achse des Objektivs spiegelt daher die Verkip pung des Deckglases wider. Jeder der mindestens drei Messpunkte ist jeweils durch drei Koordinaten bestimmt, von denen eine den zu bestimmenden Abstand des Mess punktes von dem Objektiv längs dessen optischer Achse angibt, während die beiden anderen Koordinaten die Lage des jeweiligen Messpunktes auf der Oberfläche des Deckglases festlegen. According to the invention, it is assumed that the plane spanned by the at least three measuring points is coplanar with the surface of the cover slip mentioned. The tilt of this plane relative to the optical axis of the lens therefore reflects the tilt of the cover slip. Each of the at least three measuring points is determined by three coordinates, one of which indicates the distance of the measuring point from the lens along its optical axis, while the other two coordinates determine the position of the respective measuring point on the surface of the cover slip.
Das erfindungsgemäße Verfahren ermöglicht es, die Verkippung des Deckglases inner halb des Mikroskops einfach und zuverlässig zu bestimmen. In einer bevorzugten Ausführungsform werden die mindestens drei Messpunkte defi niert, indem das Deckglas und das Objektiv quer zu dessen optischer Achse relativ zuei nander bewegt werden. Die Festlegung der Messpunkte kann nach Vorgabe einer Be dienperson oder automatisiert erfolgen. Beispielsweise können die Punkte definiert werden, während das Deckglas zum Suchen einer Probe quer zur optischen Achse des Objektivs bewegt wird. The method according to the invention enables the tilting of the cover slip within the microscope to be determined simply and reliably. In a preferred embodiment, the at least three measuring points are defined by moving the cover glass and the lens relative to one another transversely to its optical axis. The determination of the measuring points can be done automatically or by an operator. For example, the points can be defined while the cover slip is moved transversely to the optical axis of the objective to find a sample.
Vorzugsweise wird das Deckglas mittels eines verfahrbaren Mikroskoptisches relativ zu dem Objektiv quer zu dessen optischer Achse bewegt. The cover glass is preferably moved by means of a movable microscope table relative to the objective transversely to its optical axis.
In einer besonders bevorzugten Ausführungsform wird das Messlichtbündel in einen Teilbereich einer Eintrittspupille des Objektivs geleitet, der gegenüber der Mitte der Eintrittspupille versetzt ist. Auf diese Weise wird die Eintrittspupille des Objektivs durch das Messlichtbündel dezentral unterleuchtet, wodurch das Messlichtbündel beim Austritt aus dem Objektiv schräg zu dessen optischer Achse gestellt wird. Die de zentrale Unterleuchtung der Eintrittspupille des Objektivs hat den Vorteil, dass achs- nahe Strahlanteile vermieden werden, die sogenannten Reflexe erster Ordnung, die am stärksten an den Flächenscheiteln der das Objektiv bildenden Linsen entstehen, verursachen und dadurch das Signal-Rausch-Verhältnis verschlechtern. Das Reflexions lichtbündel wird vorzugsweise so zurück in das Objektiv geleitet, dass es in der der Ausbreitungsrichtung des Messlichtbündels entgegengesetzten Richtung einen ande ren Teilbereich der Eintrittspupille durchsetzt, der gegenüber dem vorgenannten Teil bereich der Eintrittspupille versetzt ist. In a particularly preferred embodiment, the measurement light bundle is directed into a partial area of an entrance pupil of the objective which is offset with respect to the center of the entrance pupil. In this way, the entrance pupil of the lens is decentrally illuminated by the measuring light bundle, as a result of which the measuring light bundle is inclined to its optical axis when it exits the lens. The decentralized illumination of the entrance pupil of the objective has the advantage that beam portions close to the axis are avoided, the so-called first-order reflections, which occur most strongly on the surface vertices of the lenses forming the objective, and thereby worsen the signal-to-noise ratio. The reflection light bundle is preferably passed back into the lens in such a way that it penetrates another portion of the entrance pupil in the direction opposite to the direction of propagation of the measurement light bundle, which is offset from the aforementioned portion of the entrance pupil.
Es ist vorteilhaft, wenn das Messlichtbündel an der Oberfläche ein Messmuster erzeugt wird und das Messmuster durch das Reflexionslichtbündel auf den positionssensitiven Sensor abgebildet wird. So ist es beispielsweise möglich, das Messmuster in Form ei nes Bildes einer Spaltblende zu generieren, die der das Messlichtbündel emittierenden Lichtquelle vorgeordnet ist. In einer bevorzugten Ausführungsform bildet die Oberfläche des Deckglases, an der das Messlichtbündel zu Erzeugung des Reflexionslichtbündels reflektiert wird, mit ei nem angrenzenden optischen Medium eine teilreflektierende Grenzfläche. Insbeson dere ist das optische Medium ein Einbettmedium, das an die genannte Oberfläche des Deckglases grenzt. It is advantageous if the measurement light bundle generates a measurement pattern on the surface and the measurement pattern is imaged on the position-sensitive sensor by the reflection light bundle. For example, it is possible to generate the measurement pattern in the form of an image of a slit diaphragm, which is arranged upstream of the light source emitting the measurement light bundle. In a preferred embodiment, the surface of the cover glass, on which the measurement light bundle is reflected to generate the reflection light bundle, forms a partially reflecting interface with an adjacent optical medium. In particular, the optical medium is an embedding medium that borders on the surface of the cover slip.
In dieser Ausführungsform nutzt die an dem jeweiligen Messpunkt vorgenommene Ab standsmessung, auf der die erfindungsgemäße Ermittlung der Verkippung des Deckgla ses basiert, eine Teilreflexion des Messlichtbündels an der Oberfläche des Deckglases. Diese Teilreflektion wird dadurch verursacht, dass die Oberfläche mit dem an sie gren zenden optischen Medium, das einen anderen Brechungsindex als das Deckglas hat, eine Grenzfläche bildet, an der ein Sprung im Brechungsindex auftritt. Auf diese Weise ist es möglich, die Verkippung des Deckglases innerhalb des Mikroskops besonders ein fach und zuverlässig zu bestimmen. In this embodiment, the distance measurement carried out at the respective measuring point, on which the determination of the tilt of the cover glass according to the invention is based, uses a partial reflection of the measurement light beam on the surface of the cover glass. This partial reflection is caused by the fact that the surface with the adjoining optical medium, which has a different refractive index than the cover glass, forms an interface at which a jump in the refractive index occurs. In this way it is possible to determine the tilting of the cover slip within the microscope in a particularly simple and reliable manner.
In einer besonders bevorzugten Ausführungsform wird auf Grundlage der mindestens drei Messpunkte die Ausrichtung eines Normalenvektors, der senkrecht zu der genann ten Ebene liegt, ermittelt. Daraus wird dann die Verkippung des Deckglases bestimmt. Insbesondere kann der durch den Normalenvektor und die optische Achse des Objek tivs eingeschlossene Winkel bestimmt werden. Durch diesen Winkel ist die Verkippung der durch die Messpunkte definierten Ebene und damit die Verkippung des Deckglases eindeutig quantifizierbar. In a particularly preferred embodiment, the orientation of a normal vector, which is perpendicular to the plane mentioned, is determined on the basis of the at least three measuring points. The tilt of the cover slip is then determined from this. In particular, the angle enclosed by the normal vector and the optical axis of the objective can be determined. The tilt of the plane defined by the measuring points and thus the tilt of the cover slip can be clearly quantified by this angle.
Vorzugsweise werden mehr als drei Messpunkte definiert, deren Abstände von dem Objektiv zur Bestimmung der Verkippung des Deckglases ermittelt werden. Je mehr Messpunkte auf der Oberfläche des Deckglases definiert werden, desto genauer kann die Verkippung der durch die Messpunkte festgelegten Ebene und damit die Verkip pung des Deckglases bestimmt werden. In einer bevorzugten Ausführungsform wird das Deckglas zum Ausgleichen der ermit telten Verkippung verstellt. Alternativ kann die ermittelte Verkippung für die Berech nung einer Filterfunktion zur Inversion des Abbildungsvorganges verwendet werden, beispielsweise einer Dekonvolution oder einer quantitativen Phasenrekonstruktion. Preferably, more than three measuring points are defined, the distances between which are determined by the objective in order to determine the tilting of the cover glass. The more measuring points defined on the surface of the cover slip, the more precisely the tilt of the plane defined by the measurement points and thus the tilt of the cover slip can be determined. In a preferred embodiment, the cover slip is adjusted to compensate for the tilting determined. Alternatively, the determined tilt can be used for the calculation of a filter function for inversion of the imaging process, for example a deconvolution or a quantitative phase reconstruction.
Die Erfindung betrifft ferner ein Mikroskop, das ein Deckglas, ein dem Deckglas zuge wandtes Objektiv und eine Einrichtung zum Bestimmen einer Verkippung des Deckgla ses umfasst. Die Einrichtung ist ausgebildet, auf einer Oberfläche des Deckglases min destens drei Messpunkte, die eine Ebene aufspannen, zu definieren und für jeden die ser Messpunkte folgende Schritte durchzuführen: Lenken eines Messlichtbündels durch das Objektiv auf den Messpunkt; Erzeugen eines Reflexionslichtbündels, indem das Messlichtbündel zumindest zum Teil an dem jeweiligen Messpunkt reflektiert wird; Lenken des Reflexionslichtbündels durch das Objektiv auf einen positionssensitiven Sensor; Erfassen des Einfallsortes des Reflexionslichtbündels auf dem positionssensiti ven Sensor; und Ermitteln des Abstands des jeweiligen Messpunktes von dem Objektiv längs dessen optischer Achse auf Grundlage des erfassten Einfallsortes. Die Einrichtung ist ferner ausgebildet auf Grundlage der ermittelten Abstände eine Verkippung der durch die drei Messpunkte aufgespannten Ebene relativ zur optischen Achse des Ob jektivs als Verkippung der Oberfläche des Deckglases zu bestimmen. The invention further relates to a microscope which comprises a cover slip, a cover glass facing lens and a device for determining a tilt of the cover slip. The device is designed to define at least three measuring points spanning a plane on a surface of the cover glass and to carry out the following steps for each of these measuring points: directing a measuring light beam through the lens onto the measuring point; Generating a reflection light bundle by at least partially reflecting the measurement light bundle at the respective measurement point; Directing the reflection light beam through the lens onto a position-sensitive sensor; Detecting the point of incidence of the reflection light beam on the position-sensitive sensor; and determining the distance of the respective measuring point from the objective along its optical axis on the basis of the detected incident location. The device is also designed to determine a tilt of the plane spanned by the three measuring points relative to the optical axis of the lens as a tilt of the surface of the cover slip on the basis of the determined distances.
In einer bevorzugten Ausführungsform weist die Einrichtung eine Aperturblende mit einer Blendenöffnung auf, die dezentriert mit Abstand zur optischen Achse des Objek tivs angeordnet ist. In a preferred embodiment, the device has an aperture diaphragm with an aperture opening which is arranged decentrally at a distance from the optical axis of the objective.
In einer speziellen Ausgestaltung hat die Einrichtung eine Lichtquelle, die das Mess lichtbündel im Infrarot-Wellenlängenbereich emittiert. Dies hat den Vorteil, dass das durch das Messlichtbündel an dem Deckglas erzeugte Messmuster für das menschliche Auge nicht sichtbar ist und somit die Beobachtung der Probe durch das Mikroskop nicht stört. Es ist jedoch ebenso möglich, ein Messlichtbündel im sichtbaren Wellenlän genbereich einzusetzen. In einer bevorzugten Ausführungsform ist der positionssensitive Sensor ein Zeilen sensor ist. Der Zeilensensor ist vorzugsweise derart ausgebildet, dass er die Intensitäts verteilung des Reflexionslichtbündels in seiner Gesamtheit erfassen kann. Alternativ kann der positionssensitive Sensor auch als Flächensensor, z.B. als zweidimensionale CCD-Kamera ausgeführt sein. In a special embodiment, the device has a light source which emits the measurement light bundle in the infrared wavelength range. This has the advantage that the measurement pattern generated by the measurement light bundle on the cover glass is not visible to the human eye and thus does not interfere with the observation of the sample by the microscope. However, it is also possible to use a measuring light beam in the visible wavelength range. In a preferred embodiment, the position-sensitive sensor is a line sensor. The line sensor is preferably designed such that it can detect the intensity distribution of the reflection light beam in its entirety. Alternatively, the position-sensitive sensor can also be designed as an area sensor, for example as a two-dimensional CCD camera.
Vorzugsweise umfasst das Mikroskop Mittel zum Korrigieren der ermittelten Verkippung des Deckglases. Diese Mittel umfassen beispielsweise einen manuell oder motorisiert bewegbaren Mikroskoptisch. The microscope preferably comprises means for correcting the determined tilting of the cover slip. These means include, for example, a manually or motorized movable microscope stage.
Die erfindungsgemäße Einrichtung ist aufgrund ihrer vorliegend beschriebenen struk turellen und funktionellen Eigenschaften auch dazu geeignet, in dem Mikroskop als Au tofokuseinrichtung genutzt zu werden. Zudem bietet die Einrichtung aufgrund ihrer Ei genschaften die Möglichkeit, neben der Verkippung des Deckglases andere, die licht mikroskopische Abbildung beeinflussende Größen zu bestimmen, wie etwa die Dicke des Deckglases und/oder den Brechungsindex eines optischen Mediums. Because of the structural and functional properties described here, the device according to the invention is also suitable for use in the microscope as an autofocus device. In addition, due to its properties, the device offers the possibility of determining, in addition to the tilting of the cover glass, other variables influencing the light microscopic image, such as the thickness of the cover glass and / or the refractive index of an optical medium.
Die Erfindung ist auf eine Vielzahl von Mikroskoptypen anwendbar, z.B. inverse oder aufrechte Durchlichtmikroskope. The invention is applicable to a variety of microscope types, e.g. inverse or upright transmitted light microscopes.
Weitere Merkmale und Vorteile der Erfindung ergeben sich aus der folgenden Be schreibung, die die Erfindung anhand von Ausführungsbeispielen im Zusammenhang mit den beigefügten Figuren näher erläutert. Further features and advantages of the invention will become apparent from the following description, which explains the invention in more detail using exemplary embodiments in conjunction with the accompanying figures.
Es zeigen: Show it:
Figur 1 eine schematische Darstellung eines inversen Durchlichtmikroskops als ers tes Ausführungsbeispiel; Figur 2 eine zur Bestimmung der Verkippung des Deckglases bestimmte Einrich tung, die Teil des Mikroskops nach Figur 1 ist; Figure 1 is a schematic representation of an inverted transmitted light microscope as the first embodiment; 2 shows a device for determining the tilting of the cover glass, which is part of the microscope according to FIG. 1;
Figur 3 eine schematische Darstellung, die einen Probenraum des Mikroskops nach Figure 3 is a schematic representation showing a sample space of the microscope
Figur 1 zeigt;  Figure 1 shows;
Figur 4 eine durch einen positionssensitiven Detektor der Einrichtung nach Figur 2 erfasste Intensitätsverteilung; FIG. 4 shows an intensity distribution detected by a position-sensitive detector of the device according to FIG. 2;
Figur 5 eine schematische Darstellung, die eine durch drei Messpunkte festgelegte Figure 5 is a schematic representation, which is determined by three measuring points
Ebene zeigt;  Plane shows;
Figur 6 ein Flussdiagramm, das eine spezielle Ausführungsform des erfindungsge mäßen Verfahrens zur Bestimmung der Dicke des Deckglases zeigt; Figure 6 is a flowchart showing a specific embodiment of the method of the invention for determining the thickness of the cover slip;
Figur 7 eine schematische Darstellung, die einen verschiebbaren Mikroskoptisch zeigt; und Figure 7 is a schematic diagram showing a movable microscope stage; and
Figur 8 eine schematische Darstellung eines aufrechten Durchlichtmikroskop, das ein zweites Ausführungsbeispiel des erfindungsgemäßen Mikroskops bil det. FIG. 8 shows a schematic illustration of an upright transmitted-light microscope, which forms a second exemplary embodiment of the microscope according to the invention.
Figur 1 zeigt ein Mikroskop 10 als erstes Ausführungsbeispiel, auf das die erfindungsge mäße Verkippungsbestimmung anwendbar ist. FIG. 1 shows a microscope 10 as the first exemplary embodiment, to which the tilt determination according to the invention is applicable.
Das Mikroskop 10 ist als inverses Durchlichtmikroskop ausgeführt. Es umfasst demnach ein Objektiv 12, das von unten einem in Figur 1 mit dem Bezugszeichen 14 versehenen Probenraum zugewandt ist, sowie eine Lichtquelle 16, die von oben auf den Proben raum 14 gerichtet ist. Das Mikroskop 10 weist ferner einen Tubus 18 mit einem Okular 20 auf, durch das eine Bedienperson ein durch das Objektiv 12 eingefangenes Proben bild betrachten kann. Zudem ist eine Steuereinheit 22 vorgesehen, welche die ver schiedenen Mikroskopkomponenten ansteuert. The microscope 10 is designed as an inverse transmitted light microscope. Accordingly, it comprises a lens 12, which faces from below a sample space provided with the reference number 14 in FIG. 1, and a light source 16 which is directed onto the sample space 14 from above. The microscope 10 also has a tube 18 with an eyepiece 20, through which an operator can view a sample captured by the lens 12. In addition, a control unit 22 is provided which controls the various microscope components.
In dem Probenraum 14 des Mikroskops 10 befindet sich ein Deckglas 24, das eine in Fi gur 1 nicht explizit dargestellten Probe bedeckt. Auf dem Deckglas 24 befindet sich ein optisches Medium 26, in das die Probe eingebettet ist und das im Weiteren als Einbett medium 26 bezeichnet wird. In dem Probenraum 14 ist ferner ein Immersionsmedium 28 angeordnet, das in Figur 1 von oben an das Objektiv 12 und von unten an das Deck glas 24 grenzt. In the sample space 14 of the microscope 10 there is a cover glass 24 which covers a sample not explicitly shown in FIG. 1. On the cover glass 24 there is an optical medium 26 in which the sample is embedded and which is referred to below as the embedding medium 26. In the sample space 14 there is also an immersion medium 28 which borders in FIG. 1 on the lens 12 from above and on the cover glass 24 from below.
Das Mikroskop 10 weist ferner eine in Figur 1 allgemein mit dem Bezugszeichen 30 be- zeichnete Einrichtung auf, die dazu dient, die Verkippung des Deckglases 14 zu ermit teln. Die Einrichtung 30 ist in Figur 2 genauer dargestellt. The microscope 10 also has a device, generally designated by the reference numeral 30 in FIG. 1, which serves to determine the tilting of the cover glass 14. The device 30 is shown in more detail in FIG.
Wie in Figur 2 gezeigt, weist die Einrichtung 30 eine Lichtquelle 32 auf, die ein Mess lichtbündel 34 im Infrarot-Wellenlängenbereich emittiert. Die Lichtquelle 32 ist bei spielweise eine LED, die eine Spaltblende 33 aufweist, durch die das Messlichtbündel 34 auf eine Beleuchtungsoptik 36 gerichtet wird. Nach Durchtritt durch die Beleuch tungsoptik 36 fällt das Messlichtbündel 34 auf eine Aperturblende 38, die zwar mittig auf der optischen Achse Ol der Beleuchtungsoptik 36 positioniert ist, jedoch eine Blen denöffnung 39 aufweist, die dezentriert mit Abstand zur optischen Achse Ol der Be leuchtungsoptik 36 angeordnet ist. Die Blendenöffnung 39 der Aperturblende 38 be grenzt den Strahlquerschnitt des Messlichtbündels 34 derart, dass nur der in Figur 2 unterhalb der optischen Achse Ol der Beleuchtungsoptik 36 liegende Teil des Mess lichtbündels 34 die Aperturblende 38 in Richtung eines Umlenkprismas 40 passiert. As shown in FIG. 2, the device 30 has a light source 32 which emits a measurement light bundle 34 in the infrared wavelength range. The light source 32 is, for example, an LED which has a slit diaphragm 33 through which the measurement light bundle 34 is directed onto an illumination optics 36. After passing through the lighting optics 36, the measuring light bundle 34 falls onto an aperture diaphragm 38, which is positioned centrally on the optical axis Ol of the illumination optics 36, but has an aperture 39 which is decentrally arranged at a distance from the optical axis Ol of the illumination optics 36 is. The aperture 39 of the aperture diaphragm 38 limits the beam cross section of the measuring light beam 34 such that only the part of the measuring light beam 34 lying below the optical axis O1 of the illumination optics 36 in FIG. 2 passes the aperture diaphragm 38 in the direction of a deflection prism 40.
Das in seinem Strahlquerschnitt begrenzte Messlichtbündel 34 wird an dem Umlenk prisma 40 in eine Transportoptik 42 reflektiert, die aus einer längs ihrer optischen Achse 02 verschiebbaren Fokussierlinse 44, einer Le uchtfe Id blende 46 und einer wei teren Linse 48 gebildet ist. Nach Durchtritt durch die Transportoptik 42 fällt das Mess lichtbündel 34 auf einen dichroitischen Strahlteiler 50, der Licht im Infrarot-Wellenlän genbereich reflektiert, während er Licht im sichtbaren Bereich transmittiert. Durch den dichroitischen Spiegel 50 wird das Messlichtbündel 34 in Richtung des Objektivs 12 re flektiert. Das an dem dichroitischen Spiegel 50 reflektierte Messlichtbündel 34 verläuft dabei mit einem Parallelversatz zur optischen Achse 03 des Objektivs 12. Auf diese Weise wird das Messlichtbündel 34 in einen Teilbereich einer Eintrittspupille 52 des Objektivs 12 geleitet, der gegenüber der optischen Achse 03 des Objektivs 12 und da mit gegenüber der Mitte der Eintrittspupille 52 seitlich versetzt ist (vgl. Figur 4). Die Eintrittspupille 52 des Objektivs 12 wird somit dezentral unterleuchtet, was dazu führt, dass das Messlichtbündel 34 unter einem Winkel a schräg zur optischen Achse 03 in den Probenraum 14 gelenkt wird. The measuring light bundle 34, which is limited in its beam cross section, is reflected on the deflection prism 40 in a transport optic 42, which consists of a along its optical Axis 02 displaceable focusing lens 44, a Le uchtfe Id aperture 46 and a white lens 48 is formed. After passing through the transport optics 42, the measuring light bundle 34 falls onto a dichroic beam splitter 50, which reflects light in the infrared wavelength range while transmitting light in the visible range. By the dichroic mirror 50, the measuring light beam 34 is reflected in the direction of the lens 12 re. The measuring light bundle 34 reflected on the dichroic mirror 50 runs with a parallel offset to the optical axis 03 of the objective 12. In this way, the measuring light bundle 34 is guided into a partial area of an entrance pupil 52 of the objective 12, which is opposite the optical axis 03 of the objective 12 and since the entrance pupil 52 is laterally offset with respect to the center (cf. FIG. 4). The entrance pupil 52 of the objective 12 is thus illuminated locally, which means that the measurement light bundle 34 is directed into the sample space 14 at an angle a obliquely to the optical axis 03.
Der Einfachheit halber sind in der Darstellung nach Figur 2 das Einbettmedium 26 und das Immersionsmedium 28, die in dem Probenraum 14 von entgegengesetzten Seiten her an das Deckglas 24 grenzen, weggelassen. Das unter schrägem Einfall in den Pro benraum 14 geleitete Messlichtbündel 34 wird, wie weiter unten unter Bezugnahme auf Figur 4 genauer erläutert ist, an dem Deckglas 24 reflektiert, wodurch ein in das Objektiv 12 zurückgeleitetes Reflexionsbündel 54 entsteht. For the sake of simplicity, the embedding medium 26 and the immersion medium 28 which adjoin the cover glass 24 from opposite sides in the sample space 14 are omitted in the illustration according to FIG. The measurement light bundle 34 guided under oblique incidence into the sample space 14 is reflected on the cover glass 24, as will be explained in more detail below with reference to FIG. 4, whereby a reflection bundle 54 which is guided back into the objective 12 is produced.
Nach Durchtritt durch das Objektiv 12 fällt das Reflexionslichtbündel 54 auf den dich roitischen Spiegel 50, der das Reflexionslichtbündel 54 in die Transportoptik 42 lenkt. Nach Passieren der Transportoptik 42 fällt das Reflexionslichtbündel 54 auf das Um lenkprisma 40, welches das Reflexionslichtbündel 54 auf eine Detektoroptik 56 reflek tiert. Die Detektoroptik 56 richtet das Reflexionslichtbündel 54 auf ein Spektralfilter 58, das nur für Licht im Infrarot-Wellenlängenbereich durchlässig ist und Streulicht au ßerhalb dieses Wellenlängenbereichs blockiert. Das durch das Spektralfilter 58 trans- mittierte Reflexionslichtbündel 54 fällt schließlich auf einen positionssensitiven Detek tor 60, der im Stande ist, die Intensität des Reflexionslichtbündels 54 ortsaufgelöst zu erfassen. After passing through the lens 12, the reflection light bundle 54 falls onto the dichroic mirror 50, which directs the reflection light bundle 54 into the transport optics 42. After passing through the transport optics 42, the reflection light bundle 54 falls onto the order deflecting prism 40, which reflects the reflection light bundle 54 onto a detector optics 56. The detector optics 56 directs the reflection light bundle 54 to a spectral filter 58 which is only permeable to light in the infrared wavelength range and blocks stray light outside this wavelength range. The trans- by the spectral filter 58 Centered reflection light bundle 54 finally falls on a position sensitive detector 60, which is able to detect the intensity of the reflection light bundle 54 in a spatially resolved manner.
In der Figur 2 ist der Vollständigkeit halber auch die über den dichroitischen Spiegel 50 realisierte Ankopplung des Tubus 18 an die Einrichtung 30 veranschaulicht. Demnach dient der dichroitische Spiegel 50 im vorliegenden Ausführungsbeispiel auch dazu, für die eigentliche mikroskopische Bildgebung genutztes, sichtbares Detektionslicht 62, welches das Objektiv 12 aus dem Probenraum 14 in Richtung des dichroitischen Spie gels 50 leitet, durch Transmission dem Tubus 18 zuzuführen. For the sake of completeness, FIG. 2 also illustrates the coupling of the tube 18 to the device 30, which is implemented via the dichroic mirror 50. Accordingly, the dichroic mirror 50 in the present exemplary embodiment is also used for the actual microscopic imaging, visible detection light 62, which guides the lens 12 from the sample space 14 in the direction of the dichroic mirror 50, to be transmitted to the tube 18 by transmission.
Im Weiteren wird unter Bezugnahme auf die Figuren 3 bis 6 erläutert, wie nach dem erfindungsgemäßen Verfahren im Wege einer Abstandsmessung an drei Messpunkten PI, P2 und P3 (vgl. Figur 5) eine Verkippung des Deckglases 24 gegenüber der opti schen Achse 03 des Objektivs 12 ermittelt wird. Furthermore, with reference to FIGS. 3 to 6, it is explained how, according to the method according to the invention, a distance measurement at three measuring points PI, P2 and P3 (see FIG. 5) causes the cover glass 24 to tilt relative to the optical axis 03 of the objective 12 is determined.
In Figur 3 ist zunächst veranschaulicht, wie für jeden der Messpunkte PI bis P3 durch Reflexion des Messlichtbündels 34 das Reflexionslichtbündel 54 generiert wird, das er findungsgemäß dazu genutzt wird, den Abstand des jeweiligen Messpunktes von dem Objektiv 12 zu ermitteln. In Figur 3 ist der betrachtete Messpunkt mit Pi und der zuge hörige Abstand längs der optischen Achse 03 des Objektivs 12 mit zi bezeichnet (i = 1,2,3). In FIG. 3 it is initially illustrated how the reflection light bundle 54 is generated for each of the measurement points PI to P3 by reflection of the measurement light bundle 34, which bundle is used according to the invention to determine the distance of the respective measurement point from the objective 12. In Figure 3, the measurement point under consideration is designated with Pi and the associated distance along the optical axis 03 of the objective 12 with zi (i = 1,2,3).
Gemäß Figur 3 wird das die Eintrittspupille 52 des Objektivs 12 dezentral unterleuch tende Messlichtbündel 34 durch das Objektiv 12 unter einem Winkel a schräg zur opti schen Achse 03 auf dem Objektiv 12 zugewandte, in Figur 3 mit 64 bezeichnete Vor derfläche des Deckglases 24 gelenkt. Da das Deckglas 24 und das an dessen Vorderflä che 64 grenzende Immersionsmedium 28 unterschiedliche Brechungsindizes aufwei sen, bilden die Vorderfläche 64 des Deckglases 24 und das daran angrenzende Immer sionsmedium 28 eine Grenzfläche, an der das einfallende Messlichtbündel 34 zum Teil reflektiert wird. Der an dieser Grenzfläche reflektierte Teil des Messlichtbündels 34 er zeugt das Reflexionslichtbündel 54, das zurück in das Objektiv 12 geleitet wird. According to FIG. 3, the measuring pupil 34, which decentrally illuminates the entrance pupil 52 of the objective 12, is directed through the objective 12 at an angle a obliquely to the optical axis 03 on the objective 12, designated in FIG. 3 by 64 in front of the surface of the cover glass 24. Since the cover glass 24 and the immersion medium 28 bordering its front surface 64 have different refractive indices, the front surface 64 of the cover glass 24 and the adjoining always sions medium 28 form an interface on which the incident measurement light bundle 34 is partially is reflected. The part of the measuring light bundle 34 reflected at this interface generates the reflection light bundle 54, which is directed back into the objective 12.
Figur 4 zeigt eine Intensitätsverteilung V, die das Reflexionslichtbündel 54 auf dem po sitionssensitiven Detektor 60 erzeugt. Dabei gibt die Abszisse des Diagramms nach Fi gur 4 den Einfallsort auf dem Detektor 60 und die Ordinate die an dem jeweiligen Ein fallsort gemessene Intensität wieder. Die Intensitätsverteilung V nach Figur 4 zeigt ei nen Peak P, dessen Lage Xi, die auf dem positionssensitiven Detektor 60 bezüglich ei ner Referenzlage Xref bestimmbar ist, ein Maß für den Abstand zi ist, den die Oberflä che 64 des Deckglases 24 längs der optischen Achse 03 von dem Objektiv 12 aufweist. FIG. 4 shows an intensity distribution V that the reflection light beam 54 generates on the position-sensitive detector 60. The abscissa of the diagram according to FIG. 4 represents the point of incidence on the detector 60 and the ordinate represents the intensity measured at the respective point of incidence. The intensity distribution V according to FIG. 4 shows a peak P, the position Xi of which, which can be determined on the position-sensitive detector 60 with respect to a reference position Xref, is a measure of the distance zi that the surface 64 of the cover glass 24 along the optical axis 03 of the lens 12.
In der schematischen Darstellung nach Figur 5 ist veranschaulicht, dass die drei Mess punkte PI, P2 und P3 erfindungsgemäß so definiert werden, dass durch sie eine Ebene aufgespannt wird, deren Verkippung die zu ermittelnde Verkippung der Vorderfläche 64 gegenüber der optischen Achse 03 des Objektivs 12 widerspiegelt. Jeder der Mess punkte PI, P2 und P3 ist durch drei Koordinaten (xi, yi, zi) definiert (i=l,2,3). Dabei sind die Koordinaten xi, yi vorgegeben, während die Koordinate zi, die den Abstand des zu gehörigen Messpunktes Pi von dem Objektiv angibt, die erfindungsgemäß zu ermit telnde Größe darstellt. In dem Beispiel nach Figur 5 ist die optische Achse 03 des Ob jektivs 12 auf den Messpunkt PI ausgerichtet, was bedeutet, dass in diesem Beispiel der Abstand des Punktes PI bestimmt wird. Sollen die Abstandsbestimmungen für die beiden anderen Messpunkte P2 und P3 durchgeführt werden, so sie diese Messpunkte entsprechend anzufahren. Hierzu werden beispielsweise das Deckglas 24 und das Ob jektiv 12 quer zu dessen optischer Achse 03 gegeneinander bewegt, bis die optische Achse 03 auf den gewünschten Messpunkt P2 bzw. P3 eingestellt ist. In Figur 5 ist fer ner ein Normalenvektor N gezeigt, der senkrecht auf der durch die Messpunkte PI, P2 und P3 festgelegten Ebene steht. Der Normalenvektor N schließt mit der optischen Achse 03 des Objektivs einen Winkel ß ein, der die Verkippung der durch die Mess punkte PI, P2 und P3 festgelegten Ebene und damit der Vorderfläche 64 des Deckgla ses 24 angibt. Das Flussdiagramm nach Figur 6 zeigt ein Beispiel, wie das Verfahren zur Ermittlung der Verkippung des Deckglases 24 realisiert werden kann. 5 shows that the three measuring points PI, P2 and P3 are defined according to the invention in such a way that a plane is spanned by them, the tilt of which tilts the tilt of the front surface 64 to be determined relative to the optical axis 03 of the objective 12 reflects. Each of the measuring points PI, P2 and P3 is defined by three coordinates (xi, yi, zi) (i = l, 2,3). The coordinates xi, yi are predefined, while the coordinate zi, which specifies the distance of the associated measurement point Pi from the objective, represents the variable to be determined according to the invention. In the example according to FIG. 5, the optical axis 03 of the objective 12 is aligned with the measuring point PI, which means that in this example the distance of the point PI is determined. If the distance determinations are to be carried out for the other two measuring points P2 and P3, they should approach these measuring points accordingly. For this purpose, for example, the cover glass 24 and the lens 12 are moved relative to one another transversely to its optical axis 03 until the optical axis 03 is set to the desired measuring point P2 or P3. FIG. 5 also shows a normal vector N which is perpendicular to the plane defined by the measurement points PI, P2 and P3. The normal vector N includes an angle β with the optical axis 03 of the objective, which indicates the tilt of the plane determined by the measurement points PI, P2 and P3 and thus the front surface 64 of the cover glass 24. The flow chart according to FIG. 6 shows an example of how the method for determining the tilting of the cover glass 24 can be implemented.
In einem ersten Schritt S1 werden die drei Messpunkte PI, P2, P3 auf der Oberfläche 64 des Deckglases 24 so definiert, dass durch die Punkte PI, P2 und P3 eine die Ober fläche 64 repräsentierende Ebene gemäß Figur 5 festgelegt ist. In a first step S1, the three measuring points PI, P2, P3 on the surface 64 of the cover slip 24 are defined such that a point representing the surface 64 according to FIG. 5 is defined by the points PI, P2 and P3.
In Schritt S2 wird der erste Messpunkt PI, sofern er nicht schon ohnehin eingestellt ist, so angefahren, dass die optische Achse 03 des Objektivs 12 auf den ersten Messpunkt PI ausgerichtet ist. Anschließend wird, wie oben unter Bezugnahme auf die Figuren 3 und 4 beschrieben, der Abstand zl des ersten Messpunktes PI von dem Objektiv 12 längs dessen optischer Achse 03 gemessen. In step S2, the first measurement point PI, if it has not already been set anyway, is approached such that the optical axis 03 of the objective 12 is aligned with the first measurement point PI. Then, as described above with reference to FIGS. 3 and 4, the distance z1 of the first measuring point PI from the objective 12 is measured along its optical axis 03.
In Schritt S3 wird dann der zweite Messpunkt P2 angefahren, indem die optische Achse 03 des Objektivs 12 auf den Messpunkt P2 ausgerichtet wird, und der Abstand z2, den der zweite Messpunkt von dem Objektiv 12 aufweist, in gleicher Weise wie für den ers ten Messpunkt PI bestimmt. In step S3, the second measuring point P2 is then approached by aligning the optical axis 03 of the objective 12 with the measuring point P2, and the distance z2, which the second measuring point has from the objective 12, in the same way as for the first measuring point PI determined.
In Schritt S4 wird der dritte Messpunkt P3 angefahren, indem die optische Achse 03 des Objektivs 12 auf den dritten Messpunkt ausgerichtet wird. Dann wird der Abstand z3, den der dritte Messpunkt P3 von dem Objektiv 12 aufweist, in gleicher Weise wie für die Messpunkte PI und P2 bestimmt. In step S4, the third measuring point P3 is approached by aligning the optical axis 03 of the objective 12 with the third measuring point. Then the distance z3, which the third measuring point P3 has from the objective 12, is determined in the same way as for the measuring points PI and P2.
In Schritt S5 wird dann auf Basis der in den Schritten S2, S3 und S4 durchgeführten Ab standsmessungen der Normalenvektor N bestimmt, der senkrecht auf der durch die drei Messpunkte PI, P2 und P3 festgelegten Ebene steht, und der Winkel ß bestimmt, den der Normalenvektor N mit der optischen Achse 03 des Objektivs 12 einschließt. Anhand des Winkels ß wird schließlich die Verkippung der Oberfläche 64 des Deckgla ses 12 ermittelt. Das Anfahren der verschiedenen Messpunkte PI, P2 und P3 kann beispielsweise mit tels eines rein schematisch in Figur 7 gezeigten Mikroskoptisches 86 erfolgen. Dieser lässt sich quer zur optischen Achse des Objektivs 12 verstellen, um die gewünschten Abstandsmessungen vorzunehmen. In step S5, on the basis of the distance measurements carried out in steps S2, S3 and S4, the normal vector N is determined, which is perpendicular to the plane defined by the three measuring points PI, P2 and P3, and the angle β determined by the normal vector N with the optical axis 03 of the lens 12 includes. On the basis of the angle β, the tilt of the surface 64 of the cover glass 12 is finally determined. The various measuring points PI, P2 and P3 can be approached, for example, by means of a microscope table 86 shown purely schematically in FIG. This can be adjusted transversely to the optical axis of the objective 12 in order to carry out the desired distance measurements.
Im Unterschied zur Ausführungsform nach Figur 1 ist bei dem in Figur 8 dargestellten Mikroskop 78 das Objektiv 12 oberhalb des Probenraums 18 angeordnet, während sich die Lichtquelle 16 unterhalb des Probenraums 18 befindet. Dementsprechend befindet sich das Immersionsmedium 28, das zum einen an das Objektiv 12 und zum anderen an das Deckglas 24 grenzt, oberhalb des Deckglases 24, während sich das Einbettmedium 26 unterhalb des Deckglases 24 angeordnet ist. In contrast to the embodiment according to FIG. 1, in the microscope 78 shown in FIG. 8, the objective 12 is arranged above the sample space 18, while the light source 16 is located below the sample space 18. Accordingly, the immersion medium 28, which on the one hand borders on the objective 12 and on the other hand on the cover glass 24, is located above the cover glass 24, while the embedding medium 26 is arranged below the cover glass 24.
Die erfindungsgemäße Bestimmung der Verkippung des Deckglases 24 erfolgt bei dem Mikroskop 78 nach Figur 8 in gleicher Weise wie bei dem in Figur 1 gezeigten Mikro skop 10. The determination of the tilting of the cover glass 24 according to the invention takes place in the microscope 78 according to FIG. 8 in the same way as in the microscope 10 shown in FIG. 1.
Die Erfindung wurde vorstehend anhand spezieller Ausführungsbeispiele erläutert. Es versteht sich von selbst, dass die Erfindung nicht auf diese Ausführungsbeispiele be schränkt ist und eine Reihe von Abwandlungen möglich sind. The invention has been explained above on the basis of special exemplary embodiments. It goes without saying that the invention is not limited to these exemplary embodiments and that a number of modifications are possible.
So wird in dem Beispiel nach Figur 3 das Messlichtbündel 34 durch die Grenzfläche teil reflektiert, die durch die Vorderfläche 64 des Deckglases 24 und das daran angren zende Immersionsmedium 28 gebildet ist. Es ist jedoch ebenso möglich, dass das Messlichtbündel 34 durch eine Grenzfläche teilreflektiert wird, die durch eine dem Ob jektiv 12 abgewandte rückseitige Fläche 68 des Deckglases 24 und das daran angren zende Einbettmedium 26 gebildet ist. Bezugszeichenliste Thus, in the example according to FIG. 3, the measurement light bundle 34 is partially reflected by the interface which is formed by the front surface 64 of the cover glass 24 and the immersion medium 28 adjoining it. However, it is also possible that the measurement light bundle 34 is partially reflected by an interface which is formed by a rear surface 68 of the cover glass 24 facing away from the lens 12 and the embedding medium 26 adjoining it. Reference list
10 Mikroskop 10 microscope
12 Objektiv  12 lens
14 Probenraum  14 rehearsal room
16 Lichtquelle  16 light source
18 Tubus  18 tube
20 Okular  20 eyepiece
22 Steuereinheit  22 control unit
24 Deckglas  24 coverslip
26, 28 optisches Medium 30 Einrichtung  26, 28 optical medium 30 device
32 Lichtquelle  32 light source
34 Messlichtbündel 36 Beleuchtungsoptik 38 Aperturblende  34 measurement light bundles 36 illumination optics 38 aperture diaphragm
40 Umlenkprisma  40 deflection prism
42 Transportoptik  42 Transport optics
44 Fokussierlinse  44 focusing lens
46 Leuchtfeldblende 50 Strahlteiler  46 light field diaphragm 50 beam splitters
52 Eintrittspupille  52 entrance pupil
54 Reflexionslichtbündel 56 Detektoroptik  54 reflected light bundles 56 detector optics
58 Spektralfilter  58 spectral filters
60 Detektor  60 detector
62 Abbildungsstrahlengang 62 imaging beam path
64, 68 Oberfläche 64, 68 surface
80, 82, 84 Messpunkt  80, 82, 84 measuring point
N Normalenvektor 01, 02, 03 optische Achse VI, V2 Vektor a, ß Winkel N normal vector 01, 02, 03 optical axis VI, V2 vector a, ß angle

Claims

Ansprüche Expectations
1. Verfahren zur Bestimmung einer Verkippung eines Deckglases (24) in einem Mikroskop (10, 78), das ein dem Deckglas (24) zugewandtes Objektiv (12) auf weist, 1. Method for determining a tilt of a cover slip (24) in a microscope (10, 78), which has a lens (12) facing the cover slip (24),
dadurch gekennzeichnet, dass auf einer Oberfläche (64, 68) des Deckglases (24) mindestens drei Messpunkte (80, 82, 84), die eine Ebene aufspannen, definiert werden und für jeden der drei Messpunkte (80, 82, 84) folgende Schritte durch geführt werden:  characterized in that at least three measuring points (80, 82, 84) spanning a plane are defined on a surface (64, 68) of the cover glass (24) and the following steps for each of the three measuring points (80, 82, 84) be performed:
Lenken eines Messlichtbündels (34) durch das Objektiv (12) auf den Mess- punkt,  Directing a measuring light bundle (34) through the objective (12) onto the measuring point,
Erzeugen eines Reflexionslichtbündels (54), indem das Messlichtbündel (34) zumindest zum Teil an dem jeweiligen Messpunkt reflektiert wird, Generating a reflection light bundle (54) by at least partially reflecting the measurement light bundle (34) at the respective measurement point,
Lenken des Reflexionslichtbündels (54) durch das Objektiv (12) auf einen positionssensitiven Sensor (60), Directing the reflection light bundle (54) through the objective (12) onto a position-sensitive sensor (60),
Erfassen des Einfallsortes des Reflexionslichtbündels (54) auf dem positions- sensitiven Sensor (60), und  Detecting the point of incidence of the reflection light bundle (54) on the position-sensitive sensor (60), and
Ermitteln des Abstands des jeweiligen Messpunktes von dem Objektiv (12) längs dessen optischer Achse (03) auf Grundlage des erfassten Einfallsortes, und dass auf Grundlage der ermittelten Abstände eine Verkippung der durch die drei Messpunkte (80, 82, 84) aufgespannten Ebene relativ zur optischen Achse (03) des Objektivs (12) als Verkippung der Oberfläche (64, 68) des Deck glases (24) bestimmt wird.  Determining the distance of the respective measuring point from the objective (12) along its optical axis (03) on the basis of the detected incident location, and that on the basis of the determined distances a tilt of the plane spanned by the three measuring points (80, 82, 84) relative to the optical axis (03) of the lens (12) as tilting of the surface (64, 68) of the cover glass (24) is determined.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass die mindestens drei Messpunkte (80, 82, 84) definiert werden, indem das Deckglas (24) und das Ob jektiv (12) quer zu dessen optischer Achse (03) relativ zueinander bewegt wer den. 2. The method according to claim 1, characterized in that the at least three measuring points (80, 82, 84) are defined by the cover glass (24) and the lens (12) which is moved transversely to its optical axis (03) relative to one another the.
3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, dass das Deckglas (24) mittels eines verfahrbaren Mikroskoptisches relativ zu dem Objektiv (12) quer zu dessen optischer Achse (03) bewegt wird. 3. The method according to claim 2, characterized in that the cover glass (24) is moved by means of a movable microscope table relative to the objective (12) transversely to its optical axis (03).
4. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass das Messlichtbündel (40) in einen Teilbereich einer Eintrittspupille des Objektivs (12) geleitet wird, der gegenüber der Mitte der Eintrittspupille versetzt ist. 4. The method according to any one of claims 1 to 3, characterized in that the measuring light bundle (40) is directed into a partial area of an entrance pupil of the objective (12) which is offset with respect to the center of the entrance pupil.
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass durch das Messlichtbündel (34) an der Oberfläche (64, 68) ein Messmuster er zeugt wird und dass das Messmuster durch das Reflexionslichtbündel (54) auf den positionssensitiven Sensor (60) abgebildet wird. 5. The method according to any one of claims 1 to 4, characterized in that a measurement pattern is generated by the measurement light bundle (34) on the surface (64, 68) and that the measurement pattern by the reflection light bundle (54) on the position-sensitive sensor (60 ) is mapped.
6. Verfahren nach Anspruch 5, dadurch gekennzeichnet, dass das auf den positi onssensitiven Sensor (60) abgebildete Messmuster in Form einer räumlichen Intensitätsverteilung erfasst wird, aus der Einfallort des Reflexionslichtbündels (54) bestimmt wird. 6. The method according to claim 5, characterized in that the measurement pattern imaged on the position-sensitive sensor (60) is recorded in the form of a spatial intensity distribution, from the point of incidence of the reflection light bundle (54) is determined.
7. Verfahren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, dass die Oberfläche (64, 68) des Deckglases (24), an der das Messlichtbündel (34) zu Er zeugung des Reflexionslichtbündels (54) reflektiert wird, mit einem angrenzen den optischen Medium (26, 28) eine teilreflektierende Grenzfläche bildet. 7. The method according to any one of claims 1 to 6, characterized in that the surface (64, 68) of the cover glass (24) on which the measurement light beam (34) for generating the reflection light beam (54) is reflected with an adjoining the optical medium (26, 28) forms a partially reflecting interface.
8. Verfahren nach Anspruch 7, dadurch gekennzeichnet, dass das optische Me dium ein Einbettmedium (26) ist, das an die genannte Oberfläche (64, 68) des Deckglases (24) grenzt. 8. The method according to claim 7, characterized in that the optical medium is an embedding medium (26) which borders on said surface (64, 68) of the cover slip (24).
9. Verfahren nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, dass auf Grundlage der mindestens drei Messpunkte (PI, P2, P3) die Ausrichtung eines Normalenvektors (N), der senkrecht zu der genannten Ebene liegt, ermittelt und daraus die Verkippung des Deckglases (24) bestimmt wird. 9. The method according to any one of claims 1 to 8, characterized in that on the basis of the at least three measuring points (PI, P2, P3) the alignment of a Normal vector (N), which is perpendicular to said plane, is determined and the tilting of the cover slip (24) is determined therefrom.
10. Verfahren nach einem der Ansprüche 1 bis 9, dadurch gekennzeichnet, dass mehr als drei Messpunkte (80, 82, 84) definiert werden, deren Abstände von dem Objektiv (12) zur Bestimmung der Verkippung des Deckglases (24) ermit telt werden. 10. The method according to any one of claims 1 to 9, characterized in that more than three measuring points (80, 82, 84) are defined, the distances from the lens (12) for determining the tilt of the cover glass (24) are determined.
11. Verfahren nach einem der Ansprüche 1 bis 10, dadurch gekennzeichnet, dass das Deckglas (24) zum Ausgleichen der ermittelten Verkippung verstellt wird. 11. The method according to any one of claims 1 to 10, characterized in that the cover glass (24) is adjusted to compensate for the determined tilt.
12 Mikroskop (10, 78), umfassend: 12 microscope (10, 78) comprising:
ein Deckglas (24),  a cover slip (24),
ein dem Deckglas (24) zugewandtes Objektiv (12), und  a cover glass (24) facing lens (12), and
eine Einrichtung (30) zum Bestimmen einer Verkippung des Deckglases (24), dadurch gekennzeichnet, dass die Einrichtung (30) ausgebildet ist, auf einer Oberfläche (64, 68) des Deckglases (24) mindestens drei Messpunkte (PI, P2, P3), die eine Ebene aufspannen, zu definieren und für jeden dieser Messpunkte (PI, P2, P3) folgende Schritte durchzuführen:  A device (30) for determining a tilt of the cover glass (24), characterized in that the device (30) is designed to have at least three measurement points (PI, P2, P3) on a surface (64, 68) of the cover glass (24). that define a plane and define the following steps for each of these measuring points (PI, P2, P3):
Lenken eines Messlichtbündels (34) durch das Objektiv (12) auf den Mess- punkt,  Directing a measuring light bundle (34) through the objective (12) onto the measuring point,
Erzeugen eines Reflexionslichtbündels (54), indem das Messlichtbündel (54) zumindest zum Teil an dem jeweiligen Messpunkt (80, 82, 84) reflektiert wird,  Generating a reflection light bundle (54) by at least partially reflecting the measurement light bundle (54) at the respective measuring point (80, 82, 84),
Lenken des Reflexionslichtbündels (54) durch das Objektiv (12) auf einen positionssensitiven Sensor (60),  Directing the reflection light bundle (54) through the objective (12) onto a position-sensitive sensor (60),
Erfassen des Einfallsortes des Reflexionslichtbündels (54) auf dem positions- sensitiven Sensor (60),  Detecting the point of incidence of the reflection light bundle (54) on the position-sensitive sensor (60),
und Ermitteln des Abstands des j eweiligen Messpunktes (P 1 , P2, P3) von dem Objektiv (12) längs dessen optischer Achse (03) auf Grundlage des erfassten Einfallsortes, und dass die Einrichtung (30) ausgebildet ist, auf Grundlage der ermittelten Ab stände eine Verkippung der durch die drei Messpunkte (PI, P2, P3) aufge spannten Ebene relativ zur optischen Achse (03) des Objektivs (12) als Verkip pung der Oberfläche (64, 68) des Deckglases (24) zu bestimmen. and determining the distance of the respective measurement point (P 1, P2, P3) from the objective (12) along its optical axis (03) on the basis of the detected point of incidence, and that the device (30) is designed, on the basis of the determined distances, a tilting of the plane spanned by the three measuring points (PI, P2, P3) relative to the optical axis (03) of the objective (12) as a tilting of the surface (64, 68) of the cover slip (24).
13. Mikroskop (10, 78) nach Anspruch 12, dadurch gekennzeichnet, dass die Einrichtung (30) eine Aperturblende (38) mit einer Blendenöffnung aufweist, die dezentriert mit Abstand zur optischen Achse (03) des Objektivs (12) angeordnet ist. 13. Microscope (10, 78) according to claim 12, characterized in that the device (30) has an aperture diaphragm (38) with an aperture opening which is arranged decentrally at a distance from the optical axis (03) of the objective (12).
14. Mikroskop (10, 78) nach Anspruch 12 oder 13, dadurch gekennzeichnet, dass die Einrichtung (30) eine Lichtquelle (18) hat, die das Messlichtbündel (34) im Infrarot-Wellenlängenbereich emittiert. 14. Microscope (10, 78) according to claim 12 or 13, characterized in that the device (30) has a light source (18) which emits the measuring light beam (34) in the infrared wavelength range.
15. Mikroskop (10, 78) nach einem der Ansprüche 12 bis 14, dadurch gekennzeichnet, dass der positionssensitive Sensor (60) ein Zeilensensor ist. 15. microscope (10, 78) according to one of claims 12 to 14, characterized in that the position-sensitive sensor (60) is a line sensor.
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