EP3867682A1 - Procédé et système de détermination d'une inclinaison d'une lamelle couvre-objet - Google Patents

Procédé et système de détermination d'une inclinaison d'une lamelle couvre-objet

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)
English (en)
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/fr
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

L'invention concerne un procédé permettant de déterminer une inclinaison d'une lamelle couvre-objet (24) dans un microscope (10, 78) qui présente un objectif (12) tourné vers la lamelle couvre-objet (24). Au moins trois points de mesure, qui forment un plan, sont définis sur une surface supérieure (64, 68) de la lamelle couvre-objet (24) et les étapes suivantes sont mises en œuvre pour chacun des trois points de mesure : Diriger un faisceau de lumière de mesure (34) par l'objectif (12) sur le point de mesure; produire un faisceau de lumière de réflexion (54), le faisceau de lumière de mesure (34) étant réfléchi au moins en partie sur chaque point de mesure; diriger le faisceau de lumière de réflexion (54) par l'objectif (12) sur un capteur (60) sensible à la position; et déterminer une distance de chaque point de mesure à partir de l'objectif (12) le long de son axe optique (03) sur la base du point d'incidence. Un basculement du plan formé par les trois points de mesure relativement à l'axe optique (03) de l'objectif (12) est déterminé comme basculement de la lamelle couvre-objet (24), sur la base des distances déterminées.
EP19797574.1A 2018-10-19 2019-10-11 Procédé et système de détermination d'une inclinaison d'une lamelle couvre-objet Pending EP3867682A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102018125995.6A DE102018125995A1 (de) 2018-10-19 2018-10-19 Verfahren und Mikroskop zur Bestimmung einer Verkippung eines Deckglases
PCT/EP2019/077631 WO2020078854A1 (fr) 2018-10-19 2019-10-11 Procédé et système de détermination d'une inclinaison d'une lamelle couvre-objet

Publications (1)

Publication Number Publication Date
EP3867682A1 true EP3867682A1 (fr) 2021-08-25

Family

ID=68426399

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19797574.1A Pending EP3867682A1 (fr) 2018-10-19 2019-10-11 Procédé et système de détermination d'une inclinaison d'une lamelle couvre-objet

Country Status (4)

Country Link
US (1) US20210341281A1 (fr)
EP (1) EP3867682A1 (fr)
DE (1) DE102018125995A1 (fr)
WO (1) WO2020078854A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018098398A1 (fr) * 2016-11-25 2018-05-31 Glowforge Inc. Composants optiques prédéfinis dans une machine commandée numériquement par ordinateur

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19923821A1 (de) * 1999-05-19 2000-11-23 Zeiss Carl Jena Gmbh Verfahren und Anordnung zur Lageerfassung einer mit einem Laser-Scanner abzutastenden Fläche
DE102008007178A1 (de) * 2008-01-30 2009-08-06 Carl Zeiss Microimaging Gmbh Kalibriervorrichtung und Laser-Scanning-Mikroskop mit einer derartigen Kalibriervorrichtung
DE102010030430B4 (de) 2010-06-23 2015-01-29 Leica Microsystems Cms Gmbh Triangulierende Autofokuseinrichtung für Mikroskope und Verwendungen hiervon
US9575308B2 (en) * 2012-03-23 2017-02-21 Huron Technologies International Inc. Slide scanner with dynamic focus and specimen tilt and method of operation
CN104737056B (zh) * 2012-10-15 2017-04-26 索尼公司 图像获取装置及测量载片倾度的方法
US10317663B2 (en) * 2015-02-27 2019-06-11 General Electric Company Determination of deflection of a microscope slide
DE102016212019A1 (de) * 2016-07-01 2018-01-04 Carl Zeiss Microscopy Gmbh Neigungsmessung und -korrektur des Deckglases im Strahlengang eines Mikroskops

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Publication number Publication date
US20210341281A1 (en) 2021-11-04
WO2020078854A1 (fr) 2020-04-23
DE102018125995A1 (de) 2020-04-23

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