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WO2019170464A1 - Microscope et procédé pour l'observation au microscope d'un échantillon pour la représentation d'images à profondeur de champ étendue ou d'images tridimensionnelles - Google Patents

Microscope et procédé pour l'observation au microscope d'un échantillon pour la représentation d'images à profondeur de champ étendue ou d'images tridimensionnelles Download PDF

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Publication number
WO2019170464A1
WO2019170464A1 PCT/EP2019/054644 EP2019054644W WO2019170464A1 WO 2019170464 A1 WO2019170464 A1 WO 2019170464A1 EP 2019054644 W EP2019054644 W EP 2019054644W WO 2019170464 A1 WO2019170464 A1 WO 2019170464A1
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WIPO (PCT)
Prior art keywords
sample
images
microscope
image
field
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.)
Ceased
Application number
PCT/EP2019/054644
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German (de)
English (en)
Inventor
Artur Degen
Alexander Gaiduk
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.)
Carl Zeiss Microscopy GmbH
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Carl Zeiss Microscopy 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 Microscopy GmbH filed Critical Carl Zeiss Microscopy GmbH
Priority to CN201980017358.6A priority Critical patent/CN111989608B/zh
Publication of WO2019170464A1 publication Critical patent/WO2019170464A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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 present invention initially relates to a method of microscopically sampling a sample, as a result of which images having an extended depth of field or three-dimensional images of the sample are displayed. Furthermore, the invention relates to a microscope with a lens for optically imaging a sample.
  • an image is taken for each one focus position and stored with the associated focus position in an image stack. From the captured images of the image stack, an image with an enhanced depth of field (EDOF) image is calculated.
  • EEOF enhanced depth of field
  • Detected pixel defects which is corrected by interpolation with adjacent pixels.
  • the corrected EDoF image is used to calculate a height map or a 3D model of the object.
  • US 2015/0185462 A1 shows a microscope with a first motorized drive in the z-direction for positioning a unit comprising a lens and a camera and with a second motorized drive in the z-direction for positioning a specimen stage for receiving a specimen.
  • the first motorized drive allows you to take pictures with an extended depth of field.
  • Image pickup device comprises a movable stage for receiving the sample and a unit for changing the focus position.
  • the image pickup device u. a. a camera and a unit for generating the images with an extended depth of field.
  • the images with extended depth of field are omnifocal
  • the microscope is designed to be a
  • US Pat. No. 7,345,816 B2 discloses an optical microscope which comprises a mirror with a controllably variable reflecting surface. By changing the surface of the mirror, images can be taken from different focal positions.
  • US 7,269,344 B2 shows an optical device with an imaging optical system having deformable mirrors and a digital zoom function. This should be the electronic magnification with high image sharpness changeable.
  • a monitoring device in which a camera is configured to capture an image of an object placed on a placement section.
  • a z-axis motion unit is for performing an automatic
  • MALS Mirror Array Lens System
  • the focus can be changed with a frequency of up to 10 kHz.
  • VHX5000 digital microscope manufactured by Keyence allows a topology of a specimen with a height of about 138 ym to be recorded through a stack of 12 images in a duration of about 9 seconds.
  • the object of the present invention is to navigate over a sample when micro-copying the sample with the aim of displaying
  • the method according to the invention is used for microscopying a sample with a microscope; especially with a digital microscope.
  • the digital microscope preferably comprises a Lens and an image sensor for converting one of the
  • the operator can move in one xy plane or in XZ / YZ planes to see more of the sample, or to see another xyz position of the sample, or to move the sample around in another
  • the extensively extended recording area of the microscope is characterized by a sample-side field of view of the microscope
  • Microscope determined. Due to their size, the sample can not be used in its entirety in a single moment
  • Acquisition area over the sample is used to explore the sample and can be thought of as navigating across the sample.
  • This navigation takes place in the lateral direction, d. H.
  • this navigation may be along a randomly generated or predefined curve in the x, y, and z directions or along a curve defined by the sample in the x, y, and z directions.
  • the microscope and the sample are moved relative to each other.
  • a lens of the microscope and the sample relative to each other emotional.
  • a stage of the microscope is moved to the arranged on the stage specimen relative to
  • the resolution in the axial and lateral direction is not decisive.
  • the resolution is preferably adapted to the speed of the navigation.
  • the lateral and axial resolution are adjusted so that no artifacts are present in the microscopic frames.
  • the resolution is preferably increased, which corresponds to a natural visual process.
  • the microscopic frames of each of the areas of the sample are mixed with several different ones
  • Focus positions recorded For each of the areas microscopically moved during the movement, a plurality of the individual images are recorded which differ with respect to their focal positions set for recording.
  • the images of the microscopic individual images of each of these regions take place with different focus positions, so that details of the respective region are sharply imaged in at least one of the microscopic individual images.
  • the different focus positions are particularly preferred by different controls of a
  • Microsystem with movable mirrors causes.
  • Different focus positions are alternatively preferred by different activations of a deformable optical lens or a mechanical, piezoelectric or hybrid actuator for positioning a
  • the Different focus positions are alternatively preferably formed by different dimensions of a distance between the sample and the lens of the microscope. Alternatively, a position of a focusing optical element in an intermediate image plane or a rear intermediate film plane may be changed. The distance between the sample and the
  • Lens of the microscope can also be described as a z-coordinate.
  • the microscopic frames of each of the areas of the sample microscoped during movement of the receiving area form a stack.
  • the images of each of the stacks differ in the z coordinate of theirs
  • the microscopic frames of each of the microscopic portions of the sample moved during the motion are thus each processed into a microscopic image with extended depth of field.
  • the microscopic image to be calculated images the sample with extended depth of field.
  • the microscopic frames of each of the microscopically-scanned regions become one at a time
  • the three-dimensional images comprise information about the respective region of the sample to be displayed for each point in space. For example, in one
  • orthogonal coordinate system with x, y and z axis for each point in space intensity values and or color values can be specified.
  • Three-dimensional images can be determined in which, for example, only one z-value is known for each pair of x and y coordinates. This is
  • the images are displayed with extended depth of field or the three-dimensional image
  • This presentation is preferably carried out in synchronism with the moving of the recording area over the regions of the sample in the sense that between moving the recording region over the regions of the sample and displaying the individual regions of the sample, only one user does not disturb the region
  • Delay occurs. This delay may be due to the calculations required to determine the images with
  • extended depth of field or the three-dimensional images are not zero, however, it is preferably so small that they are not perceived by the operator or at least not disturb the operator.
  • the necessary calculations for determining the images with extended depth of field or the three-dimensional images be temporarily suspended because the resolution is no longer visually perceived.
  • the calculation of a 3D model for the three-dimensional images can be done with a delay, if sufficient again
  • Computing power is available. Displaying the images with extended depth of field or the three-dimensional
  • Images are taken at a frame rate which is preferably greater than or equal to at least 10 frames / s and more preferably at least
  • Focus positions recorded Thus, more frames are taken with different focus positions for this area over which the recording area rests than for
  • the further captured frames are used to increase an axial resolution and a lateral resolution of the currently displayed extended depth of field image or the currently displayed three-dimensional image.
  • the increased axial resolution is due to increased depth resolution, i. H. formed in the z-direction, which corresponds to a smaller voxie size.
  • the increased lateral resolution is due to an increase in
  • Resolution in the lateral direction ie formed in the x and / or y direction, which corresponds to finer pixels.
  • the increase in the resolution leads in any case to an increase in the quality of the currently displayed image with extended depth of field or the currently displayed three-dimensional image.
  • the increased quality can be achieved for example by an increased dynamic range, by a reduction of Reflections or characterized by an improvement in color reproduction.
  • a particular advantage of the method according to the invention is that the operator, while navigating over the sample, the images with extended depth of field or the three-dimensional images with a frame rate and a
  • Depth of field or the subsequent three-dimensional images leads, as the number of microscopic frames with different focus positions for the individual areas decreases again.
  • a fast electromechanical microsystem with movable mirrors is preferably used.
  • piezo actuators or other actuators for improving the positioning of the recording area or for scanning in the z direction and / or xy direction used.
  • a method of shifting the pixels is used to increase the lateral resolution.
  • Such a method for shifting the pixels is also referred to as pixel shifting.
  • a structured illumination of the sample takes place, wherein the structured illumination is preferably refined in order to increase the axial resolution and / or the lateral resolution of the currently displayed image.
  • a method for reducing reflections in the image on the basis of a segmented illumination of the sample is used or applied to a greater extent in order to increase the axial resolution and / or the lateral resolution of the currently displayed image.
  • a method of increasing the dynamic range of the image is used or applied to a greater extent to the axial
  • a Fourier Ptychography method is used to increase the axial resolution and / or the lateral resolution of the currently displayed image.
  • Preferred is a method for
  • Frames z. B. increased by the image processing hardware to the axial resolution and / or the lateral
  • parameters of algorithms used for the processing of the individual images are changed in order to increase the axial resolution and / or the lateral resolution of the currently displayed image.
  • Measures for increasing the axial resolution and / or the lateral resolution of the currently displayed image are preferably also combined.
  • the method will be the axial resolution and the lateral resolution
  • Resolution of the currently displayed image and possibly also other aspects of the quality of the image for example, a 2, 5-dimensional image increased.
  • This increase in the resolution is preferably effected as a function of the movement of the
  • binning ie, combining adjacent pixels of the individual images; or binning takes place to a greater extent.
  • Reflections in the individual images made by applying a segmented illumination of the sample, wherein during the movement of the receiving area, d. H. during the
  • Navigation preferably a reduced number, in particular a small or smallest number of segments of the lighting is activated in order to achieve the highest possible speed of image acquisition. This allows for an increased
  • a dynamic range of image pickup and image processing is increased, wherein during movement of the pickup area, i. H. while navigating the rate of images and / or a cost for post-processing of the images is reduced.
  • Changes in the image and / or in the settings of the hardware are present, ie as soon as the movement of the recording area is stopped, the rate of images and / or the cost of post-processing to increase the dynamic range is increased again, the lateral resolution of the current to increase the displayed image with extended depth of field or the currently displayed three-dimensional image, in particular in order to obtain optimum or maximum image quality.
  • a fourth preferred embodiment takes place during the movement of the receiving area, ie
  • the cited preferred embodiments or a selection of the cited preferred embodiments are preferably also used in combination.
  • the number of images during a navigation may be predefined and constant or may be selected dynamically depending on the speed of the movement made by the operator over the sample.
  • Speed is preferably estimated on the basis of the contents of the captured frames or measured with a sensor.
  • the sensor can for example by a
  • Microsystem with movable mirrors or by deforming a deformable optical lens of the lens Microsystem with movable mirrors or by deforming a deformable optical lens of the lens.
  • Frame images are not constant, but in particular can be changed continuously, so that, for example, an actuator for changing the height of a specimen-carrying object table between the shots of the individual images does not have to be stopped.
  • an actuator is preferably placed on an active optical element of the microscope.
  • the mechanical actuator can be designed, for example, to form the active optical element, which is controlled by a flexible lens, a lens controllable by mechanical vibrations, a liquid lens or a diffractive lens for measuring a depth information of the sample may be formed to deform.
  • the actuator is controlled by a microsystem for mechanically moving
  • the actuator is preferably a focus actuator and / or an aberration actuator.
  • the actuator is preferably operated at a frequency of at least 1 kHz and more preferably of at least 10 kHz.
  • the optical detector in an advantageous embodiment, the optical detector
  • Micro mirrors designed to accommodate an extended depth of field.
  • MALS module as an optical actuator
  • a MALS module can be designed, for example, as a Fresnel lens, as described, for example, in WO 2005/119331 A1.
  • This Fresnel lens is formed of a plurality of micromirrors. By changing the position of the micromirrors, the focal length of the Fresnel lens can be changed very quickly. This fast change of the focal length allows a very fast adjustment of the focus plane to be imaged, d. H. the
  • the different regions of the sample are each recorded with a predetermined number of different focus positions, while the recording area of the microscope is moved over the different areas of the sample. This number is smaller than the number of Focusing positions, with which the microscopic frames of the area, on which the movement of the receiving area of the microscope rests, are recorded.
  • Method be moved while the recording area of the microscope over the different areas of the sample, the different areas each with the
  • the minimum image rate is preferably 10 fps, and more preferably 25 fps, where the images are the images with extended depth of field or the three-dimensional images.
  • the predetermined quality level may be defined by a resolution and / or a severity measure. If the operator rests for a long time over the navigation system
  • the quality is not increased to an unnecessarily high level.
  • Embodiments of the method according to the invention the focus positions of the further recorded after stopping the movement of the recording area frames for a
  • focus positions are selected which lie between the focus positions of the previously recorded individual images.
  • Frames are selected on the basis of an analysis of the previously recorded frames of this area. Sharpness and / or spatial spectra are preferred in this analysis Range or subareas of the range.
  • the depth of the images is extended in depth, which corresponds for example to several EDoF images, so that an extended EDoF image can be determined.
  • Method is the stopping of the movement of the recording area of the microscope over the areas of the sample recognized by the fact that the contents in the last recorded individual images have a same local position. Stopping the movement of the receiving area of the microscope over the However, regions of the sample can also be recognized, for example, by triggering an object table of the microscope to cause the object table to stop. The detection of the stopping of the movement of the recording area of the microscope over the areas of the sample is preferably carried out while maintaining a debounce time, so that very short
  • an aperture of a diaphragm is changed in order to move the diaphragm
  • the aperture is located in
  • the opening of the aperture is in particular reduced to the depth of field of the microscope.
  • the frame rate at high speeds of moving the recording area can not fall below a minimum value of the frame rate, thereby limiting the movement of the frame
  • Recording area can be carried out in large dimensions in the lateral direction and also in the depth, but a simultaneous decrease in the resolution in the three spatial directions is accepted.
  • a correction of an inclination which the sample has with respect to the microscope is undertaken. This correction takes place while the receiving area of the microscope is moved laterally over the different areas of the sample.
  • an analysis of the individual images with different focus positions preferably takes place in order to determine an alignment and / or a location of individual tiles of the respective area.
  • the sharpness in the individual images is preferably determined.
  • the other focus positions are selected according to the analysis of the individual images, so that the correction of the inclination can be made quickly and accurately.
  • a prediction is made which can lead further focus positions to individual images which cause an improvement in the resolution of the displayed image with extended depth of field or an improvement in the resolution of the illustrated three-dimensional image.
  • the microscope according to the invention is preferably digital and initially comprises an objective for the enlarged optical
  • the objective includes optical components for increased optical imaging of the sample in the image plane.
  • the optical components are
  • optical lenses formed in particular by optical lenses and possibly also by one or more mirrors, diaphragms and filters.
  • the microscope preferably further comprises an image sensor for converting the image imaged directly or indirectly on the image sensor by the objective into an electrical signal.
  • the microscope according to the invention comprises at least one
  • the Actuator for changing a focus position of the microscope.
  • the Actuator is preferably formed in simple embodiments, a distance between a sample and the
  • the actuator preferably comprises an electric motor for moving a sample table carrying the sample or for moving the objective.
  • the actuator is preferably designed to change a focal length of the objective.
  • the actuator is preferably used for actuating an active optical element, with which the focus is adjustable.
  • the actuator of the active optical element is formed by a microsystem for mechanical movement of micro mirrors and / or microlenses.
  • the micromirrors preferably form a lens, in particular a Fresnel lens. By changing the position of the micromirrors, the focal length of the Fresnel lens can be changed very quickly. This fast change of the focal length allows a very fast adjustment of the
  • Focus position Alternatively preferred is the actuator for
  • the microscope according to the invention further comprises a control and imaging unit, which for controlling the actuator, for
  • Image unit is for carrying out the invention
  • microscope according to the invention preferably also has features that are associated with the method according to the invention and its preferred
  • FIG. 4 shows a graphical user interface for carrying out a preferred embodiment of the invention
  • Fig. 1 shows a symbolic representation of microscopic images in a first phase of a preferred embodiment form of a method according to the invention.
  • this first phase an operator moves an exemplified sample 01 relative to a microscope (not shown) to explore different regions of the sample 01, which may also be referred to as navigating across the sample 01, so that the first phase is also called the navigation phase can be designated.
  • this navigation phase of the method according to the invention are each several microscopic frames of
  • Focus positions 02 are represented by dashed lines.
  • the recorded microscopic individual images of the individual regions of the sample 01 are used to calculate an image with extended depth of field or a three-dimensional image of the respective region of the sample 01.
  • the number of focus positions 02 is minimized in the navigation phase, so that for each of the areas of the sample 01, an extended depth of field image or a three-dimensional image can be calculated and displayed in a short period of time, thereby giving the operator a continuous moving image during navigation ,
  • the small number of focus positions 02 can cause not all
  • Areas of the respective image with extended depth of field or the respective three-dimensional image are sharp and / or the resolution in depth is low.
  • Fig. 2 shows a representation of recordings with others
  • Fig. 1 illustrated embodiment of the method according to the invention.
  • This second phase is characterized in that the operator has interrupted or adjusted the movement of the sample 01 relative to the microscope (not shown), so that the sample 01 has reached a rest position with respect to the microscope (not shown).
  • This second phase can
  • the second phase represents one Beginning of a picture improvement phase.
  • Image enhancement phase will be more microscopic
  • the other focus positions 02 may also be random or as a result of an analysis of the previously recorded microscopic
  • Focusing positions 02 taken microscopic frames are used to increase the resolution and the quality of the currently displayed image with extended depth of field or the currently displayed three-dimensional image.
  • Fig. 3 shows a representation of recordings with others
  • Fig. 1 and Fig. 2 illustrated embodiment of the method according to the invention.
  • This third phase represents a continuation of the second phase
  • the sample 01 rests with respect to the microscope (not shown) for a further period of time.
  • the enhancement of the resolution and quality of the currently displayed image with extended depth of field or the currently displayed three-dimensional image is continued. There are more microscopic frames of the currently microscopically
  • Focusing positions 02 compared to that in FIG. 2 illustrated second phase is further increased.
  • Fig. 4 shows a graphical user interface for
  • the graphical user interface allows the selection of parameters for different modes when microscopically using a microscope.
  • Autofocus mode automatically creates focused microscopic images
  • Navigation mode refers to navigation while creating images with extended depth of field or three-dimensional images.
  • the "Documentation” mode refers to a recording, while Advanced
  • Depth of field or three-dimensional images are generated.
  • the operator can specify how many z positions to select at the beginning in a "Start" input field
  • the operator can specify in an input field "End” how many z positions are selected at the end.
  • the operator can enter in an input field
  • Step specifies how fine the step size should be by specifying the number of steps for that step size, or the graphical user interface may be configured to allow the operator to enter these specifications in an absolute form in a metric unit Operator acknowledges his input by clicking on the appropriate "Apply” button.
  • An algorithm button allows the operator to select an algorithm for incrementing and a type of increments of focus positions at the end of the motion change. LIST OF REFERENCE NUMBERS

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  • Physics & Mathematics (AREA)
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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Length Measuring Devices By Optical Means (AREA)

Abstract

La présente invention concerne un procédé pour l'observation au microscope d'un échantillon (01) au moyen d'un microscope. Dans une étape du procédé, des images microscopiques individuelles de différentes zones de l'échantillon (01) sont prises pendant qu'une zone d'enregistrement du microscope est déplacée au dessus des zones de l'échantillon (01). Les images individuelles de chaque zone de l'échantillon (01) sont prises avec plusieurs positions de mise au point (02) différentes. Des images à profondeur de champ étendue ou des images tridimensionnelles sont déterminées pour chacune des zones de l'échantillon (01) à partir des images individuelles et représentées pour un opérateur. Dès que le mouvement de la zone d'enregistrement au-dessus d'une des zones de l'échantillon (01) s'arrête, des images microscopiques individuelles supplémentaires de cette zone de l'échantillon (01) sont prises selon l'invention avec différentes positions de mise au point (02). Les images individuelles supplémentaires sont utilisées pour augmenter une résolution axiale ou latérale de l'image à profondeur de champ étendue présentée actuellement ou de l'image tridimensionnelle présentée actuellement. L'invention concerne en outre un microscope comprenant un objectif pour la représentation d'un échantillon (01).
PCT/EP2019/054644 2018-03-08 2019-02-26 Microscope et procédé pour l'observation au microscope d'un échantillon pour la représentation d'images à profondeur de champ étendue ou d'images tridimensionnelles Ceased WO2019170464A1 (fr)

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CN201980017358.6A CN111989608B (zh) 2018-03-08 2019-02-26 对样品进行显微观察以呈现具有扩展景深的图像或三维图像的显微镜和方法

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DE102018105308.8 2018-03-08
DE102018105308.8A DE102018105308A1 (de) 2018-03-08 2018-03-08 Mikroskop und Verfahren zum Mikroskopieren einer Probe für die Darstellung von Bildern mit erweiterter Schärfentiefe oder dreidimensionalen Bildern

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