EP1886176A1 - Procede de balayage optique d'un echantillon - Google Patents

Procede de balayage optique d'un echantillon

Info

Publication number
EP1886176A1
EP1886176A1 EP05806370A EP05806370A EP1886176A1 EP 1886176 A1 EP1886176 A1 EP 1886176A1 EP 05806370 A EP05806370 A EP 05806370A EP 05806370 A EP05806370 A EP 05806370A EP 1886176 A1 EP1886176 A1 EP 1886176A1
Authority
EP
European Patent Office
Prior art keywords
image
sample
stack
images
individual
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
EP05806370A
Other languages
German (de)
English (en)
Inventor
Vasant Desai
Guido Gehrke
Ralf Lethmate
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.)
Evident Technology Center Europe GmbH
Original Assignee
Olympus Soft Imaging Solutions 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 Olympus Soft Imaging Solutions GmbH filed Critical Olympus Soft Imaging Solutions GmbH
Publication of EP1886176A1 publication Critical patent/EP1886176A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/241—Devices for focusing
    • G02B21/244—Devices for focusing using image analysis techniques
    • 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 relates to a method for optically scanning a sample by means of an adjustment unit and a scanning device, after which individual images are recorded and combined in the sense of a grid to form an overall image.
  • the sample or an object to be examined is not limited to transparent biological tissue sections or material sections. These are transilluminated with a light source, wherein the image generated in the course of these transmission measurements is taken by the scanning device and stored here or optionally in a connected control system.
  • a light source wherein the image generated in the course of these transmission measurements is taken by the scanning device and stored here or optionally in a connected control system.
  • transmission measurements and reflection measurements can be made.
  • the measurements are of course not limited to the visible wavelength range. As a rule, however, work is carried out in the visible wave range, with transmission measurements of the sample being evaluated.
  • the invention is based on the technical problem of further developing such a method so that a perfect image of the sample is generated taking into account a consistently perfect focus.
  • each frame is determined from a single image stack, wherein the frame stack composed of substantially perpendicular to the sample plane or sample individually recorded stack images.
  • the individual image stack or the stack images can also be detected perpendicular to a base plane which, for example, coincides with or clamps a sample table receiving the sample.
  • a single frame of the sample at the scanned location is explicitly captured and stored, but rather a frame stack which is composed of two, three, five or ten or even more individual stack images. From these stack images, the desired frame can now be extracted by identifying one of the stack images with the desired frame. Alternatively or additionally, it is also possible to assemble the individual image from a plurality of the stack images according to predetermined criteria.
  • the recording of the individual image stack takes place perpendicular to the sample or sample plane or the base plane.
  • the individual image stack can be arranged spatially with the aid of a reference plane independent of the base plane in a preferably three-dimensional image matrix.
  • the reference plane indicates the respective focus values or defines one (or more) focal planes. If one considers that the scanning device has an optical imaging unit in the form of mostly one or more (high-resolution) objectives, then with the aid of this reference plane the sharpness to be expected at the selected sample location can be determined. On the basis of the thus determined support point on the reference plane, the single image stack is now detected.
  • the reference plane is usually designed as a focus map and is predetermined by several focus points. If then the reference plane or focus map is defined, the necessary support point can be determined at the location of the recording of the desired frame, which specifies the range within which the individual stack images are recorded above and below the reference plane or focus map to produce the frame stack , The vertex thus reflects the expected focus at the location of the still image.
  • the reference plane or focus map is determined in time before the scanning of the individual images. This is necessary in order to be able to determine the interpolation points derived from the reference plane or focus map, by means of which the individual image stacks are recorded in the following step. It is understood that the respective individual image stack works with a predetermined number of stack images perpendicular to the sample plane above and below the aforementioned support point. Usually, one will resort here to an equal number above and below the sample plane at the location of the base, although of course also different numbers are covered by the invention.
  • the reference plane or focus map is for the most part created by determining, for example, the pixel intensity perpendicular to the sample plane in the form of image intensity values at selected pixels of the overall image or sample locations. From the intensity values or image intensity values via the vertical component thus detected (perpendicular to the sample plane or sample table plane or base plane, for example), the most intense point can now be determined and equated with the focus value at the location being examined. According to another approach, the aforementioned intensity values may also be weighted, for example, exponentially or linearly, thus leading to a calculated focus value.
  • a focus value is available as a function of an associated adjustment of the scanning unit perpendicular to the sample plane (usually in the z-direction).
  • the corresponding sample location ie its position in the x and y direction
  • the focus value (or the corresponding x, y and z value) is now written into the focus map to be created. If you repeat this process at least twice, there are already three focus values that span a unique plane, namely the reference plane or focus map.
  • the respectively closest three focus values can define a triangle in the sense of a triangulation, whereby the focus map is in the end composed of a multiplicity of such triangles.
  • the desired interpolation points for the subsequent scanning process or the recording of the individual images can now be determined. This is done, for example, by setting three focus values to a triangle and placing the vertex on this triangle. This results in an expected z value or value for the adjustment of the scanning unit at the interpolation point.
  • the individual image stacks can be detected immediately afterwards at the sample location (x, y value) and subsequently evaluated.
  • the individual image stacks are detected completely over the entire surface of the sample, wherein the support points or the focus map or reference plane is used to preset the scanning device at the respective sample location accordingly. Otherwise, it is practically not possible with thick samples, for example, with the subsequently recorded single image stack and taking into account the limited depth of field of high-resolution lenses to get a sharp pile picture at all.
  • the described focus map or reference plane therefore ensures that at least one sharp stack image is present in the subsequent area-wide scan in each individual image stack, which is used for the subsequent evaluations.
  • the focus map allows a virtually virtual image of the sample such that it can be focused in the Z-direction (focus value) as in the conventional microscope, whereby at the same time navigation in the X- / Y-direction becomes possible (sample location).
  • the impression of the so-called "manual fürfokussierens" as in conventional microscopy remains so.
  • the amounts of data are considerably increased by each recorded individual image stack, because the individual image stacks must be stored in each case.
  • the corresponding stack images can be joined to each other when the individual image stacks have been recorded with overlap relative to each other. How the overlap of the stack images of the individual image stacks is used in each case for their mutual alignment and their connection will be explained in more detail with reference to the subsequent description of the figures.
  • the sample can be displayed as a total three-dimensional object. It is conceivable here to reproduce this three-dimensional representation with the aid of a 3D bhIIe or an SD screen. That is, a viewer receives not only a sample information in a plane in which the sharp stack images or frames have been assembled to the overall image, but also more information about the topography of the sample, ie a true three-dimensional representation and a corresponding impression.
  • the single-image stack can be stored (in the mostly compulsory control unit) and the desired single image can be extracted in a subsequent evaluation process.
  • a real-time evaluation is conceivable and is included.
  • the invention is basically applicable to the entire electromagnetic spectrum. So also applications in the IR, UV and X-ray range are covered.
  • a novel optical sample scanning method which provides a heretofore unknown sharp image of the sample practically over the entire frame of the individual images.
  • the sample examination is significantly improved compared to previous procedures and can be originally blurred. easy to evaluate rich.
  • an image of the sample is made available, which then enables a "sharp" recording of the individual images.
  • the generated individual images are added to the overall image with overlap.
  • the overlap is analyzed in each case in order to be able to align the individual images or the individual interconnected stack images with one another.
  • FIG. 1 shows a device for optical scanning of a sample, which operates according to the described method
  • Fig. 7 the composite of the individual images overall picture.
  • a device for optical scanning of a sample 1 is shown.
  • This sample 1 is not limited to one Section through a biological tissue or a material.
  • the cut is carried out so that the sample 1 is transilluminated by a white light source W and its image can be recorded by transmission measurement.
  • the device has in its basic structure an adjusting unit 2, 3 and a scanning device 4, 5.
  • the adjusting unit 2, 3 in the embodiment of two spindle drives 2 as a drive device 2 and a sample table 3 is composed.
  • the white light source W is arranged, so that the image of the sample 1 can be picked up by the scanning device 4, 5 located above it.
  • the scanning device 4, 5 combines a plurality of imaging units or scanning units or microscope objectives 4 and a recording unit or a surface sensor or a CCD chip 5 together.
  • the sample 1 with respect to the scanning device 4, 5 is moved.
  • the sample 1 which is generally larger by a multiple than the detectable angle range of the scanning device 4, 5, can be moved relative to the scanning device 4, 5.
  • Each movement step is usually associated with the recording of a single image E, which are combined to form an overall image.
  • 3 selected sample locations are approached in the x / y direction.
  • the adjusting unit 2, 3 acted upon by a control system or control unit 6, which controls the adjustment 2, 3 accordingly and the individual images E edited if necessary and composed butt to joint or with predetermined overlap to the overall picture.
  • the control unit 6 serves to create and store a reference plane 10 which will be explained in more detail below.
  • the sample table 3 is only in the x and y directions moved, although the adjustment 2, 3 in principle could also be adjusted in addition in height or z-direction, which is not shown.
  • the transmission of the sample 1 and their reflection behavior can be detected with a comparably constructed device.
  • each frame E is determined or extracted from a frame stack 7, as shown in the side view in FIG.
  • This frame stack 7 is composed of several stack images 8, in the embodiment of ten stack images 8, which is not mandatory. These individual stack images 8 are taken up substantially perpendicular to the sample plane P, which consequently represents a base plane P.
  • the stack images 8 are recorded perpendicular to the sample table plane spanned by the sample table 3 or the relevant base plane P, which is congruent with the x- / y-plane (see Fig. 1).
  • the sample 1 is received by a slide 9. If the sample 1 is plane and the slide 9 and the sample 1 are coplanar with each other, an image that is always sharp is also produced on the surface sensor 5 in the ideal case. However, this does not correspond to reality because, for example, the sample 1 can be arranged inclined relative to the slide 9, has a curvature etc. For this reason, the individual image stacks 7 are arranged perpendicular to the sample stage or respectively determines x- / y-base plane P and extracted from the stack images 8 respectively desired single image E, wherein the individual images are assembled in the sense of a grid to the overall image.
  • a reference plane or focus map 10 is created with the aid of the control unit 6, which is shown schematically in FIGS. 5, 6.
  • the reference plane or focus map 10 is an image of the sample surface, so that ripples, inclinations, etc. of the sample 1 find an entry into the reference plane or focus map 10, which is spanned by at least three focus points 11.
  • the procedure is as follows. First, a pixel or pixel 12 of the surface sensor 5 is at the same position in the x and y direction, d. H. recorded at a specific sample location using various settings of the imaging unit 4. In other words, the sample image is recorded at the respective x / y position and the associated pixel 12, respectively, for different positions of the imaging unit 4 and consequently different foci. In the simplest case, the imaging unit 4 is adjusted for this purpose so that the plane of their focus in the z-direction, d. H. their focal plane changes. This is indicated by the different indicated planes in FIG. 2, the pixel 12 being examined being highlighted.
  • the intensity values of the pixel 12 detected to determine the focus points 11 may also be weighted, as shown in FIG. 4.
  • One possibility is to connect the maxima of the intensity values according to FIG. 3 with the aid of a compensation function and, taking into account the areas thus generated, derive a moving average value thereof as the focus value.
  • Other weighting functions such. B. exponential and not linear as shown, are conceivable.
  • the individual intensity maxima are weighted beforehand, for example, by exponential functions.
  • a focus value and consequently the focal point 11 can be defined for the selected pixel 12. This corresponds - as explained - to a specific adjustment of the imaging unit 4, in the z direction, in order to produce a sharp image at the examined x / y location.
  • the focus value 11 includes a sample location (x / y value) which specifies the position of the examined pixel 12.
  • the focus value 11 is ultimately represented by a three-dimensional value x, y and z.
  • the above-described procedure can be repeated for a given number of pixels 12 or pixels.
  • the number of focus values 11 essentially depends on two specifications. First of all, the working area plays a role, ie the area specified by the operator within which the sample 1 or parts thereof are to be scanned.
  • the working area can be determined or limited by an upstream scanning so that the sample outline is first determined, the work area is the smallest possible rectangle, which encloses the sample outline as a whole.
  • the size of the work area specifies the number of focus values 11 necessary for the creation of the focus map 10.
  • the expected sample topology With a wavy sample 1, one will work at this point with a higher number of focus values 11 than when a smooth sample is to be expected. Both can optionally be set and specified by the user.
  • the focus values 11 are determined by sampling the sample 1 coarse and not nationwide in a so-called advance scan.
  • the reference plane or focus map 10 is generated or stored in the control system 6. For this purpose, a so-called triangulation takes place in which three closest focus values 11 are connected to one another in a triangle. This process is repeated for all focus values 11, as indicated in FIGS. 5, 6. This ensures that the regions of the sample 1 indicated in FIG. 5 below the focus map 10 in any case undergo a sharp image on the area sensor 5.
  • the intermediate areas are filled by extrapolation, in that the area of the triangle formed therefrom and its position in space is determined between the respective existing focus values 11.
  • support points 13 can be set on the surface of the triangles thus generated.
  • the interpolation points 13 are extrapolated three-dimensional points on the focus map 10. That is, the interpolation points 13 include a sample location (x / y value) and an in-focus for the scanning device 4, 5 or the imaging unit 4 (z value) at the sample site.
  • the position of the support point 13 on the defined triangle can therefore be set to the support point 13, a z-value, which the adjustment or "Focusing" the imaging unit 4 at base 13 and the associated expected focus value 1 1 documented.
  • This interpolation point 13 is now the starting point for the subsequent scanning process or the area-wide recording of the individual images and their combination with the overall image. Because the base 13 is - as described - the expected focus value at the x- / y-location (sample location) of the base 13, so that the imaging unit 4 with the aid of the control system 6 when approaching the base 13 in x-, y- and The respective individual image stack 7 is then determined at this interpolation point 13. For this purpose, the imaging unit 4 is moved in one and in the other z-direction so that a previously stated number of stack images 8 perpendicular to Level of the sample 1 or of the sample stage 3 (x- / y-plane) respectively of the base plane P is received above and below the specified support point 13. Of course, the step size s (see FIG if necessary.
  • Base 13 a plurality of stack images 8 taken in the form of the image frame stack 7, wherein the starting point for this particular frame stack 7 of the focus map 10 derived base 13 is.
  • the individual image stack 7 undergoes a spatial classification in the sense of the three-dimensional individual image matrix which is shown as a partial approach in FIG. 6.
  • the respective stack images 8 can now be evaluated in such a way that the desired depth-sharp single image is determined pixel by pixel in the sense that that in turn each individual pixel 12 of the stack image 8 undergoes an evaluation in the sense of its intensity maximum.
  • a contrast measurement is made in the individual stack images 8.
  • the pixels or pixels of the individual stack images 7 are examined in terms of how strong the light-dark differences are in the neighboring area. Because blurred images usually have a low contrast, ie low light-dark differences in closely adjacent image zones.
  • large light-dark differences correspond to frequency spectra, which in particular include high frequencies.
  • Adjacent pixels with large light-dark differences have more or less steep transition edges in their intensity or step transitions, which correspond to high frequencies in the Fourier space. These high frequencies can be filtered out and are a measure for a high-contrast reproduction.
  • Each individual stack image 8 can now be evaluated in terms of how high its high-frequency components are in the Fourier transformation of the intensities recorded by the pixels. The higher the frequency components in the stack image 8, the more contrasts are present and the “sharper" the stacking image 8 is designed. On the basis of this criterion, the desired (“sharpest") stack image 8 can then be extracted from the individual image stack 7.
  • the invention also opens up the possibility of selecting the individual image E of the individual image stack 7 in sections from the most contrast-rich the stack images 8 compose and not rely on a single stack image 8, but rather the individual image E from the most contrastive portions of the respective stack images 8 of the image stack 7 compose.
  • the creation of the focus map 10 can take place virtually at the same time as the actual scanning process for detecting the individual image stacks 7. This means that the described advance scan can be omitted.
  • the stack images 8 can be evaluated in real time for determining the individual image at the location being examined, so that the scanning process is ultimately determined "only" by the duration of the mechanical process of the adjustment unit 2, 3.
  • the stack images 8 of the individual stacks 7 or the desired individual images E are regularly recorded with an overlap and then combined to form the overall image, as shown schematically in FIG.
  • the overlap between the frames is egg and E 3 labeled with 14i 3, while the overlap between the individual images Ei and E 2 is designated 14i. 2
  • the respective overlap 14 12 and 14- ⁇ 3 is analyzed, for example, the images E 2 and E 3 relative to the single image E 1 are aligned in the common coordinate system of the overall picture.
  • the respective (strip-shaped) overlap 14 12 or 14 13 is subdivided into respective squares 15 in the example case.
  • 2 or 14i 3 are also divided into rectangular or other sections 15.
  • these subareas 15 are examined by performing a Fourier transformation of the associated image values or pixel values of each associated individual image E, that is to say in the example case of the individual images Ei, E 2 and E 3 , in the region of the subarea 15.
  • such a subregion 15 corresponds, for example, to a square of 100 ⁇ 100 pixels. If the associated image values of each individual image E are then Fourier-transformed in the control unit 6 in the relevant subarea 15, then conclusions can be drawn about the respective texture, ie the local distribution and variation of the gray values in the associated image area.
  • the invention makes use of the fact that a regular texture, for example, corresponds to a periodic (two-dimensional) gray value distribution which can be approximated by means of sine and cosine functions. From these functions it is possible to derive (two-dimensional) Fourier coefficients, which are a Fourier image produce. For example, a uniform pattern of vertical stripes corresponds to horizontally arranged dots in the Fourier image.
  • this Fourier image for each partial region 15, on the one hand in the example of the overlap 14- ⁇ 2 from the individual image E 1 and the other generated by the individual image E. 2 It is important to compare the two Fourier images of the individual images E 1 and E 2 - in the sub-area 15 - with each other. As a result of this
  • the single image E 2 has to be compared to the single image
  • the described cross-correlation can also be carried out in the single image comparison in such a way that individual images E are first recorded with a specific (coarse) resolution and aligned with one another in the manner described. Then the respective individual images E are examined again with increased resolution and in turn subjected to the described cross-correlation.

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  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Multimedia (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un procédé de balayage optique d'un échantillon (1) au moyen d'une unité ajustable (2, 3) et d'un dispositif de balayage (4, 5), des images individuelles étant enregistrées et réunies de façon à produire une image globale sous forme de trame. L'invention se caractérise en ce que chaque image individuelle est déterminée à partir d'une pile d'images individuelles (7), cette pile d'images individuelles (7) étant constituée d'images en pile (8) enregistrées sensiblement perpendiculairement à l'échantillon.
EP05806370A 2005-05-25 2005-11-10 Procede de balayage optique d'un echantillon Ceased EP1886176A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200510024066 DE102005024066A1 (de) 2005-05-25 2005-05-25 Verfahren und Vorrichtung zur optischen Abtastung einer Probe
PCT/EP2005/012014 WO2006125466A1 (fr) 2005-05-25 2005-11-10 Procede de balayage optique d'un echantillon

Publications (1)

Publication Number Publication Date
EP1886176A1 true EP1886176A1 (fr) 2008-02-13

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EP05806370A Ceased EP1886176A1 (fr) 2005-05-25 2005-11-10 Procede de balayage optique d'un echantillon

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EP (1) EP1886176A1 (fr)
DE (1) DE102005024066A1 (fr)
WO (1) WO2006125466A1 (fr)

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DE102009054704A1 (de) * 2009-12-15 2011-06-16 Carl Zeiss Imaging Solutions Gmbh Mikroskop zur Aufnahme eines Mosaikbildes sowie Aufnahmeverfahren für ein solches Mikroskop
DE102011084347B4 (de) * 2011-10-12 2022-07-21 Carl Zeiss Microscopy Gmbh Mikroskop und Mikroskopierverfahren zur Durchführung eines Multipositionsexperimentes
DE102011116734B4 (de) 2011-10-19 2013-06-06 Carl Zeiss Industrielle Messtechnik Gmbh Verfahren zum Ermitteln eines fokussierten Bildabstands eines optischen Sensors eines Koordinatenmessgeräts
DE102012021726A1 (de) * 2012-11-06 2014-05-08 Celltool Gmbh Mikroskopsystem und Verfahren zur Datenerfassung
DE102014107044B4 (de) 2014-05-19 2016-01-14 Carl Zeiss Industrielle Messtechnik Gmbh Verbesserte Autofokusverfahren für ein Koordinatenmessgerät sowie Koordinatenmessgerät
DE102015117756A1 (de) 2015-10-19 2017-04-20 Carl Zeiss Industrielle Messtechnik Gmbh Verfahren zum Ermitteln eines fokussierten Bildabstands eines optischen Sensors eines Koordinatenmessgeräts
DE102023101782B3 (de) 2023-01-25 2024-06-13 Leica Microsystems Cms Gmbh Vorrichtung und Verfahren zum Erzeugen eines zusammengesetzten Bildes einer Probe

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EP0743784A2 (fr) * 1995-05-17 1996-11-20 Sharp Kabushiki Kaisha Caméra pour images fixes
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EP1415187A4 (fr) * 2001-08-06 2008-03-26 Bioview Ltd Mise au point d'images dans un appareil d'imagerie par fluorescence
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WO2006125466A1 (fr) 2006-11-30
DE102005024066A1 (de) 2006-12-07

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