WO2012016753A1 - Système autofocus - Google Patents

Système autofocus Download PDF

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Publication number
WO2012016753A1
WO2012016753A1 PCT/EP2011/060081 EP2011060081W WO2012016753A1 WO 2012016753 A1 WO2012016753 A1 WO 2012016753A1 EP 2011060081 W EP2011060081 W EP 2011060081W WO 2012016753 A1 WO2012016753 A1 WO 2012016753A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample substance
sample
interferometer
substance
focal plane
Prior art date
Application number
PCT/EP2011/060081
Other languages
German (de)
English (en)
Inventor
Markus Sticker
Original Assignee
Carl Zeiss Microimaging 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 Carl Zeiss Microimaging Gmbh filed Critical Carl Zeiss Microimaging Gmbh
Priority to EP11733817.8A priority Critical patent/EP2601551A1/fr
Publication of WO2012016753A1 publication Critical patent/WO2012016753A1/fr

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Classifications

    • 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
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison

Definitions

  • the invention relates to a method and a device for determining the Z position in the direction of the optical axis of a microscopic imaging system, in which a sample substance to be imaged is located, as well as for automatically focusing the imaging system on this Z position.
  • Microscopic imaging systems are often used to digitally record a sample substance, for example a tissue section, which is stored on a microscope slide protected by a cover glass. If the lateral extent of the sample substance to be recorded is greater than the field of view of the imaging system or greater than a range which can be directly absorbed by a camera, then it is necessary to first consecutively extend several times, for example 220 ⁇ m x 165, perpendicular to the optical axis of the imaging system ⁇ record large areas of a sample substance. In the prior art, this is done with the aid of a so-called digital "slide scanner.” The images of the individual regions are then combined to form a so-called "tiled image" as an overall image.
  • the problem here is that the depth of field of the optical imaging system decreases with increasing lateral optical resolution. If the sample is focused at a location with respect to a first region to be recorded, it is highly probable that the focus position after shifting the sample to accommodate an adjacent region is different, ie the sample is defocused there, unless an adjustment of the depth position or a renewed focus is made.
  • Causes for the change in the focus position may be, for example, a non-perpendicular orientation of the slide to the optical axis, thickness variations in the slide or in the cover glass, deflections of the slide or thickness variations of the embedding between cover glass and sample substance. It is also possible that the distance between the sample substance to be recorded and the slide varies with lateral displacement.
  • the focus position is determined by means of a software autofocus, whereby several images are recorded at different depths of the sample and, for example iteratively, the best Focus position determined on the basis of the image contrast.
  • Another known procedure provides to determine the focus position at predetermined time intervals during the scanning with the aid of a respective software autofocus and thereby correct any deviations ascertained. Both methods have the disadvantage that they take undesirably much time.
  • Another known method for focusing the sample is the so-called hardware autofocus, in which the Z-positions of reflective interfaces are determined, such as those of the cover glass top or the slide bottom.
  • depth positions for example the position of the sample substance to be imaged
  • the information about the position of, for example, the cover glass top or the slide bottom is often insufficient as a prerequisite for focusing the sample substance, since the distance of the sample substance to these interfaces is not constant.
  • the invention has the object, a method and a device of the type mentioned in such a way that with higher efficiency than in the prior art, a determination of the Z-position of the sample substance is ensured with subsequent automatic focusing on this Z-position.
  • the Z-position of a sample substance to be imaged is determined in a microscopic imaging system and the automatic focusing on the sample substance is carried out in the following method steps:
  • the focal plane and the sample substance to be imaged are moved in the Z-direction relative to each other until the sample substance of the focal plane is, and then the sample substance is imaged.
  • the sample substance is recorded digitally.
  • the interferometer used is preferably a short-coherence interferometer.
  • Short-coherence interferometry is a relatively young, high-precision optical method for examining surfaces and thin layers and scattering media, such as the retina of the eye, skin, tissue, etc. It is currently still in the medical and industrial development phase applications.
  • the underlying measuring principle is based for example on a fiber optic Michelson interferometer; the essential components are a short-coherent light source, a seroptic beam splitter and a detector.
  • a sample beam emerging from the beam splitter passes to the measurement object, and the detector detects the interference of the light of the sample beam reflected or scattered by the measurement object with the light from the reference arm.
  • By shifting a mirror inserted into the optical path different optical path lengths result, and as a function of this, interferences in the form of different intensities are displayed on the detector.
  • the detected signal is analyzed for depth and reflectance or backscatter potential information of a reflective or backscattering interface or structure in the sample.
  • White light sources or superluminescent diodes are particularly suitable as light sources.
  • the wavelength ranges are typically in the range from 400 nm to 1600 nm.
  • the temporal coherence of the light source is related to the spectral power density via a Fourier transformation, which leads to a short coherence length for a large spectral bandwidth.
  • the size of the possible path length differences of reference and object light, which still reveal an interference, depends on the coherence length. Thus, with a short coherence length, high axial resolutions can be achieved even with a large depth of field of optics.
  • the sample beam of the short-coherence interferometer is preferably coupled parallel to the beam path for image acquisition into the microscopic imaging system, so that it is directed through the microscope objective to the sample substance. It is advantageous if the sample beam uses only a small portion of the aperture of the objective, so that the lateral extent and the depth of focus of the focus in the sample substance are as large as possible. An increase in the depth of focus can additionally be achieved with an axial lens.
  • the interference signals are analyzed with respect to the various interfaces and backscattering structures as well as the depth position of the sample substance, and the Z-position of the sample substance is determined based on the analysis result.
  • the focus position in the sample can be adjusted so that the sample substance or certain structures of the sample substance are in the focal plane.
  • the focus position can also be determined very quickly during the scanning. A prior generation of a focus map is no longer necessary.
  • the time for the determination of the focus position as well as the subsequent focusing can be restricted in time to the duration of the image acquisition, if the invention is advantageously used, as will be explained in more detail below.
  • the invention can be configured with further method steps to the effect that even after the alignment of the focus position on the male sample substance from this outgoing optical information is used to control the correct focus and maintain.
  • either the specimen or the objective can be moved, or internal focusing can be used, changing the beam divergence in front of the objective.
  • 2D sensors Tile Scan
  • TDI sensors Line Scan
  • 1 D sensors Line Scan
  • Single sensors Point Scan
  • contrast method bright field, fluorescence, phase contrast, DIC and dark field can be used in connection with the method according to the invention.
  • the invention further relates to a device for automatic displacement of the focal plane of a microscopic imaging system in a Z-position, in which there is a sample substance to be imaged.
  • a sample beam emanating from an interferometer is directed in the Z direction through the sample substance,
  • an evaluation device which is designed to determine the Z position of the sample substance from the set of determined Z positions,
  • an adjusting device which is designed to shift the focal plane in the Z direction up to the Z position of the sample substance to be imaged; a control unit connected to the evaluation device and the adjusting device, which is designed to generate setting commands for automatically shifting the focal plane to the specific Z position, and a camera connected to the drive unit, designed to hold the sample substance after the automatic focusing.
  • the illumination and imaging beam path of the imaging system pass through the objective of the imaging system together with the beam paths of the interferometer.
  • a beam splitter for coupling and decoupling the interferometer beam paths from the illumination and imaging beam path is provided on the image side of the objective.
  • the coupling in and out of the interferometer beam paths can also be carried out by means of mirrors.
  • a smaller proportion of the aperture of the objective is provided for the interferometer beam paths than for the illumination and imaging beam path, which advantageously has the consequence that the lateral extent of the focal plane and the depth of focus of the focus range are relatively large.
  • an axicon lens can be arranged in the illumination and imaging beam path. In this case, the axicon lens is arranged outside the beam path provided for image recording.
  • a short-coherence interferometer with a super-luminescence diode as the light source is provided as an interferometer.
  • the wavelength of the light emitted by the light source is in the range of 750 nm to 1600 nm.
  • the Fourier domain method is preferably used, the sampling or repetition rate of the depth scan being, for example, 20 kHz.
  • the sample substance to be imaged and the cover glass surface are of interest with regard to the determination of the Z positions in the depth of the sample.
  • the sample substance to be imaged and the cover glass surface are of interest with regard to the determination of the Z positions in the depth of the sample.
  • an overall image of the sample substance is obtained by adding several recorded adjacent to the optical axis of the imaging system adjacent areas of the sample substance and then assembled into an overall image.
  • the image of the sample substance or of the individual regions of the sample substance advantageously takes place on a spatially resolving digital image sensor.
  • a sample substance 1 for example a tissue section, is deposited on a microscope slide 2 and covered with a cover glass 3.
  • the sample substance 1 extends in a plane X, Y perpendicular to the optical axis 4 of the illumination and imaging beam path 5 of a microscopic imaging system not shown in detail.
  • the sample substance 1 is located in the focal plane of an objective 6, which is part of the imaging system.
  • the illumination for the microscopic image acquisition takes place in the example chosen here according to the method of fluorescence microscopy in reflected light. Deviating from this, however, the application of bright field microscopy is conceivable, in which case the illumination takes place in transmitted light in the visible wavelength range.
  • FIG. 1 omits the representation of the light source and of a beam splitter for coupling the illumination light into the illumination and imaging beam path 5.
  • the procedure for coupling is known from the prior art and therefore need not be explained in detail here.
  • a short-coherence interferometer 9 is provided according to the invention, from which a sample beam 10 originates and is coupled into the illumination and imaging beam path 5 via a beam splitter 11 so that it passes through in the Z direction as well as the illumination light the objective 6 is directed onto the cover glass 3, the sample substance 1 and the slide 2.
  • the short-coherence interferometer 9 is provided to carry out a depth scan in time before the taking of the sample substance 1 or the taking of a partial image of the sample substance 1, which serves to exactly determine the Z-position of the sample substance 1 or a region of the sample substance 1 to be recorded in order then to be able to align the focal plane accordingly.
  • FIG. 2 An example of the interference signals that result is shown in FIG. It can be seen from FIG. 2 that the interferences caused by boundary layers or structures differ with regard to their amplitudes and are assigned to different depths in the sample in the Z direction.
  • an interference 12 caused by the slide top it corresponds to the depth position Z1.
  • an interference 13 is caused, which corresponds to the depth position Z2.
  • the amplitude also gives information about the optical properties of the relevant reflecting or backscattering interface or structure. However, these should not be considered here.
  • a related information via a drive unit is passed to actuators, either by movement of the lens 6 or the slide 2 to cause a shift of the focal plane in the Z direction to the extent that this is in the depth position Z2 and thus in the plane X, Y, in which the sample substance 1 to be recorded extends.
  • a triggering command is transmitted to the camera by the drive unit and an image of the sample substance 1 is taken.
  • a sample substance 1 is to be taken whose lateral extent is greater than the image field of the imaging system or larger than an area that can be directly absorbed by a camera image, then it is necessary to have several perpendicular to the optical axis 4 of FIG Record imaging system extending areas n, n + 1, n + 2, etc. of the sample substance 1 and then join the shots of these individual areas to form an overall picture.
  • a very advantageous application of the method according to the invention and the device according to the invention is to carry out the depth scan in an area n + 1 by means of the sample beam 10 of the short coherence interferometer 9, the Z position of the sample substance 1 in this area n + 1 and store this Z position assigned to the area n + 1 while the camera is still focused on the previous area n and recording the area n.
  • the sample substance 1 is moved continuously under the objective 6 in the X and / or Y direction, with a speed corresponding to the camera image repetition rate, so that in the next image acquisition the region n + 1 is in the image field of the camera, and the The Z-position of the sample substance 1 assigned to this region n + 1 has already been approached while the depth scan in the region n + 2 to be subsequently recorded already determines the Z-position of the sample substance 1 in the region n + 2, the region n + 2 stored and provided for automatic focusing in the subsequent recording of the area n + 2. This process continues until all areas of the sample substance have been collected.
  • the microscopic device according to the invention is designed such that the sample beam 10 of the short-coherence interferometer 9 is directed outside of the image field of the camera onto the sample substance 1. This is achieved by coupling the sample beam 10 at an angle into the objective 6.
  • an autofocus system is provided which, in connection with a microscopic device for imaging in particular of tissue sections, ensures fast and highly accurate focusing.

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  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un dispositif microscopique pour la représentation d'une substance d'un échantillon (1), comprenant : - un système d'imagerie microscopique, et - des moyens pour le déplacement automatique du plan de focalisation le long de l'axe optique (4) du système d'imagerie dans une position Z, dans laquelle se trouve la substance d'échantillon (1) à représenter, caractérisé par - un faisceau d'échantillonnage (10) partant d'un interféromètre, orienté à travers la substance d'échantillon (1) dans la direction Z, les signaux d'interférence formés par l'interférence de la lumière réfléchie ou renvoyée du faisceau d'échantillonnage (10) par l'interférence de surfaces limites et/ou de structures optiquement actives avec le faisceau de référence de l'interféromètre servant à la détermination des positions Z des surfaces limites et/ou des structures de l'échantillon, - un dispositif d'évaluation pour la détermination de la position Z de la substance d'échantillon (1), - un dispositif de réglage, réalisé pour le déplacement du plan de focalisation et de la substance d'échantillon (1) l'un par rapport à l'autre, jusqu'à ce que la substance d'échantillon (1) se trouve dans le plan de focalisation, et - une unité de commande reliée au dispositif d'évaluation et de réglage pour la génération de commandes de position pour le déplacement automatique du plan de focalisation dans la position Z déterminée.
PCT/EP2011/060081 2010-08-03 2011-06-17 Système autofocus WO2012016753A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP11733817.8A EP2601551A1 (fr) 2010-08-03 2011-06-17 Système autofocus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102010033249.6 2010-08-03
DE201010033249 DE102010033249A1 (de) 2010-08-03 2010-08-03 Autofokussystem

Publications (1)

Publication Number Publication Date
WO2012016753A1 true WO2012016753A1 (fr) 2012-02-09

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Application Number Title Priority Date Filing Date
PCT/EP2011/060081 WO2012016753A1 (fr) 2010-08-03 2011-06-17 Système autofocus

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EP (1) EP2601551A1 (fr)
DE (1) DE102010033249A1 (fr)
WO (1) WO2012016753A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016029913A1 (fr) 2014-08-29 2016-03-03 Fraunhofer-Gesellschaft Zur Foerderungder Angewandten Forschung E.V. Procédé et dispositif d'imagerie en microscopie
US9869852B2 (en) 2015-01-26 2018-01-16 Thorlabs, Inc. Microscopy system with auto-focus adjustment by low-coherence interferometry
CN109737969A (zh) * 2019-03-21 2019-05-10 孔祥明 一种物联网定位信息系统及方法

Families Citing this family (3)

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Publication number Priority date Publication date Assignee Title
DE102014002584A1 (de) 2014-01-23 2015-07-23 Euroimmun Medizinische Labordiagnostika Ag Verfahren zum Abbilden eines Obiektes und Optikvorrichtung
US10598915B2 (en) * 2014-06-23 2020-03-24 Perkinelmer Cellular Technologies Germany Gmbh Method for autofocusing a microscope at a correct autofocus position in a sample
DE102015117824B4 (de) * 2015-10-20 2017-06-29 Carl Zeiss Meditec Ag Vorrichtung und Verfahren zum Einstellen eines Fokusabstands in einem optischen Beobachtungsgerät

Citations (3)

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Publication number Priority date Publication date Assignee Title
US5122648A (en) * 1990-06-01 1992-06-16 Wyko Corporation Apparatus and method for automatically focusing an interference microscope
WO2001084209A2 (fr) * 2000-05-03 2001-11-08 Dirk Soenksen Numeriseur de lames de microscope rapide et entierement automatique
WO2003012528A2 (fr) * 2001-07-27 2003-02-13 Isis Innovation Limited Procede et appareil de production de faisceau lumineux

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US6927860B2 (en) * 2003-05-19 2005-08-09 Oti Ophthalmic Technologies Inc. Optical mapping apparatus with optimized OCT configuration
GB2411066B (en) * 2004-02-14 2009-04-29 Oti Ophthalmic Technologies Compact high resolution imaging apparatus
EP1962079B1 (fr) * 2007-02-21 2016-06-01 Agfa HealthCare N.V. Système et procédé destinés à la tomographie de cohérence optique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5122648A (en) * 1990-06-01 1992-06-16 Wyko Corporation Apparatus and method for automatically focusing an interference microscope
WO2001084209A2 (fr) * 2000-05-03 2001-11-08 Dirk Soenksen Numeriseur de lames de microscope rapide et entierement automatique
WO2003012528A2 (fr) * 2001-07-27 2003-02-13 Isis Innovation Limited Procede et appareil de production de faisceau lumineux

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016029913A1 (fr) 2014-08-29 2016-03-03 Fraunhofer-Gesellschaft Zur Foerderungder Angewandten Forschung E.V. Procédé et dispositif d'imagerie en microscopie
DE102014217328A1 (de) 2014-08-29 2016-03-03 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und eine Vorrichtung zur Bildgebung in der Mikroskopie
US9869852B2 (en) 2015-01-26 2018-01-16 Thorlabs, Inc. Microscopy system with auto-focus adjustment by low-coherence interferometry
US9989749B2 (en) 2015-01-26 2018-06-05 Thorlabs, Inc. Microscopy system with auto-focus adjustment by low-coherence interferometry
EP3250956A4 (fr) * 2015-01-26 2019-03-06 Thorlabs, Inc. Système de microscopie avec réglage de mise au point automatique par interférométrie à faible cohérence
CN109737969A (zh) * 2019-03-21 2019-05-10 孔祥明 一种物联网定位信息系统及方法
CN109737969B (zh) * 2019-03-21 2023-07-21 孔祥明 一种物联网定位信息系统及方法

Also Published As

Publication number Publication date
EP2601551A1 (fr) 2013-06-12
DE102010033249A1 (de) 2012-02-09

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