EP1886176A1

EP1886176A1 - Method for optical scanning of a sample

Info

Publication number: EP1886176A1
Application number: EP05806370A
Authority: EP
Inventors: Vasant Desai; Guido Gehrke; Ralf Lethmate
Original assignee: Olympus Soft Imaging Solutions GmbH
Current assignee: Evident Technology Center Europe GmbH
Priority date: 2005-05-25
Filing date: 2005-11-10
Publication date: 2008-02-13
Also published as: WO2006125466A1; DE102005024066A1

Abstract

The invention relates to a method for optical scanning of a sample (1) by means of a varying device (2, 3) and a scanning device (4, 5), whereupon individual images are recorded and combined to give a total image in the fashion of a raster, characterised in that each individual image is recorded from an individual image batch (7) said individual image batch (7) being composed of batch images (8) taken perpendicular to the sample.

Description

Method for optically scanning a sample

Description:

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.

A method of the embodiment described in the introduction is well known and is presented, for example, in US Pat. No. 4,760,385 or US Pat. No. 4,673,988. In addition, DE 100 23 005 A1 is relevant.

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. Of course, instead of transmission measurements and reflection measurements can be made. Incidentally, it should be emphasized that 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.

Either way, several individual images - shock to impact or overlap - are combined to form the overall image, on the basis of which the sample undergoes an examination directly on site or at a remote evaluation point. That is known. However, problems arise when the sample is inclined in relation to the recording plane of the scanning device, has a wavy or topological structure or the sample focusing problems. Likewise, the prior art is not or hardly able to To be able to scan "thick" samples sharply. Because the mostly limited depth of field of high-resolution lenses as part of the scanning device makes it usually impossible to take sharp images without having to make fundamental changes. Here, the invention aims to provide a total remedy.

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.

To solve this technical problem, a generic method according to the invention is characterized in that 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.

Thus, according to the invention, not only 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.

In any case, the recording of the individual image stack takes place perpendicular to the sample or sample plane or the base plane. Mostly you become one Detect perpendicular to a sample table or the sample table level. This is to be able to detect any inclinations of the sample compared to the sample stage level.

In this case, 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. As a rule, 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.

Thus, before the sample is scanned piece by piece by transmission and the individual images thus obtained are combined in the sense of the grid to form the overall image, a so-called advance scan for the reference plane or focus map thus takes place. In this pre-scan, the sample is usually scanned roughly, so not nationwide. Only then does the actual and previously described area-wide scanning process follow. Basically, it is also possible to summarize the actual scanning process and the so-called advance scan. Then, however, the individual focus points or focus settings for the imaging unit must be predicted or extrapolated during the actual scanning process. During the recording of the individual image stacks, a correction of the reference plane or focus map produced in this way then occurs.

In particular, for recording the focus map, a previously stated number of stack images perpendicular to the sample plane or sample table plane (Base level) above and below the (initially assumed) reference plane at the respective base recorded (advance scan). From these stack images or the individual image stack, the individual image is subsequently determined, which ideally represents a sharp image of the sample over its entire area. This process is repeated for multiple vertices so that a coarse grid of focus values is produced during the pre-scan. These focus values span the focus map or reference plane.

In this respect, 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.

It is conceivable here, for example, to identify the interpolation point with a specific position of the objective or of the optical scanning unit or imaging unit of the scanning device and then record a stacking image in the one direction every 1 mm adjustment path and then the same process in the other direction to repeat. It is understood that it is possible to work both with equidistant adjustment paths and those in which the adjustment path undergoes a change from step to step. Both can be specified from the outside and adapted to the circumstances. It is of course within the scope of the invention, alternatively or in addition to the optical scanning unit, to move the sample or a sample stage receiving it in order to receive the desired individual image stack.

As a rule, 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. In any case, at the end of the procedure for creating the reference plane or focus map, 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) corresponds to this focus value. 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. If even more focus values are used for the evaluation, then 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. Based on this focus map, 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. As a consequence of this, the individual image stacks can be detected immediately afterwards at the sample location (x, y value) and subsequently evaluated.

In order to save (computation) time in the described "advance scan" to create the reference plane or focus card, it is possible to operate the scanner or a there mostly realized (CCD) camera in the so-called binning mode. In this case, several pixels are combined, for example in groups of four. As already described above, these four (or more) pixel values are examined in such a way that at the end there is a focus value, which in this case represents, as it were, a focus value for the focus map averaged over the four or more pixels.

Subsequent to the described "advance scan", 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.

In other words, 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 Durchfokussierens" as in conventional microscopy remains so. In this case, the amounts of data are considerably increased by each recorded individual image stack, because the individual image stacks must be stored in each case.

If, during recording, the individual image stacks are operated with the same or reciprocally adjustable setting of the scanning device, 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.

Instead of storing all the individual image stacks described, it is also possible to use only the reference plane or focus map and additionally the

Save texture image, which reflects the intensities or contrast values of each sharp stack image at the associated X- / Y-position (sample location). This can greatly reduce the amount of data. This procedure simulates the so-called manual focussing. In this case, all pixels or pixels of the same Z-value (Focus value) is in focus and all other Z values are out of focus. Here you can work with different blur values, the blur is set as a function of the distance to the focal plane, for example, becomes larger with increasing distance. As a result, depth-of-focus ranges in the sense of virtual microscopy can be predefined practically in terms of software.

Characterized in that the focus map is present at the end of the described process and incidentally at each point of this focus map a frame stack and at least one sharp stack image from this frame stack, 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. Of course, a real-time evaluation is conceivable and is included. In addition, the invention is basically applicable to the entire electromagnetic spectrum. So also applications in the IR, UV and X-ray range are covered.

As a result, a novel optical sample scanning method is provided, which provides a heretofore unknown sharp image of the sample practically over the entire frame of the individual images. As a result, the sample examination is significantly improved compared to previous procedures and can be originally blurred. easy to evaluate rich. Because with the help of the reference plane or focus map determined at the outset, 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 same applies alternatively or additionally also for the individual stack images of the individual image stack. In this case, 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. Here are the main benefits.

In the following the invention will be explained in more detail with reference to a drawing showing only one exemplary embodiment; show it:

1 shows a device for optical scanning of a sample, which operates according to the described method,

2 is a schematic representation of the focus determination,

3 determined in the context of FIG. 2 Foki,

4 possible weights of the Foki of Fig. 3,

5 shows the three-dimensional frame image matrix,

Fig. 6, the generated focus map with the individual image stacks and

Fig. 7, the composite of the individual images overall picture.

In the figures, 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. In this case, 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. For this purpose, the device has in its basic structure an adjusting unit 2, 3 and a scanning device 4, 5.

In detail, 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. Below the sample stage 3, 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.

By means of the adjusting unit 2, 3, the sample 1 with respect to the scanning device 4, 5 is moved. In this way, 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. With the help of the adjusting unit 2, 3 selected sample locations are approached in the x / y direction.

For this purpose, 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. In addition, the control unit 6 serves to create and store a reference plane 10 which will be explained in more detail below. In the example shown, 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. - Of course, of course, instead of the transmission of the sample 1 and their reflection behavior can be detected with a comparably constructed device.

The present invention now takes into account the fact that the sample 1 may under certain circumstances have a topological structure and / or inclinations relative to the surface sensor or CCD chip 5. Without appropriate precautionary measures, this leads to the captured individual images E and of course also the overall image formed therefrom in the sense of a grid having blurred areas or areas of different depth of field. To counteract this phenomenon, 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. If a sample misalignment is to be expected, 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).

With reference to FIG. 1, it can be seen further that 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.

To achieve this in detail, first of all 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. Ideally, 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.

If one then plots the "depth" of the respectively sharply set planes of the imaging unit 4 in the z-direction on the x-axis with respect to the intensity determined on the pixel output side on the y-axis, the result is a diagram corresponding to FIG the intensity of the light incident on the pixel 12 is plotted as a function of the z-direction and consequently of the adjustment of the imaging unit 4. On the basis of this intensity curve according to FIG. 3, an optimal position of the image input for the pixel 12 can be determined. unit 4, which in the example case corresponds to the z-value 3, if one only looks for the intensity maximum.

Alternatively, however, 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. Then the individual intensity maxima are weighted beforehand, for example, by exponential functions. In any case, 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.

Either way, the evaluation of the intensity maxima according to FIG. 3 or FIG. 4 leads to a focus value 11 associated with the pixel 12, as is entered in FIG. 5. The focus value 11 includes a sample location (x / y value) which specifies the position of the examined pixel 12. Thus, the focus value 11 is ultimately represented by a three-dimensional value x, y and z. In this case, 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. It is understood that 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. In any case, the size of the work area specifies the number of focus values 11 necessary for the creation of the focus map 10. Of further importance is 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. In any case, the focus values 11 are determined by sampling the sample 1 coarse and not nationwide in a so-called advance scan.

If now the individual focus values 11 have been detected with the procedure described, 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. In this way, 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. On the basis of 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.

For example, one could use the depth of focus of the objective or imaging unit 4 used to set the step size s, which can be calculated from the numerical aperture and the wavelength used. In any case, to each approached x- / y-value (sample location) or

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. In this way, 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.

In general, however, a contrast measurement is made in the individual stack images 8. In this case, 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.

If one evaluates the intensities of adjacent pixels of the stack images 8 by Fourier transforming the intensity of the respective pixel or the intensities of respectively adjacent pixels, then 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.

Basically, however, 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.

Not shown is the ability to create the focus map 10 using an external device. It is conceivable here, in addition to the scanning device 4, 5 to realize an external second optical scanning device. This can, for example, resort to a laser beam which is directed onto the sample 1 and whose focus and consequently image size-independently of the scanning device 4, 5 -is determined by corresponding parallel adjustment of the second scanning device. Finally, it should be pointed out that the described process of individual image generation can of course be used to generate further focus values 11 of the focus map 10 and thus to define the focus map 10 with an increasingly dense network of focus values 11.

Overall, it is understood that 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. Likewise, 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. Here is the overlap 14i ₂ , 14-ι _{3 of} adjacent individual images Ei, E ₂ and E ₃ shown. In fact, 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

To be able to assemble the individual images E ₁ , E ₂ and E ₃ to the desired overall image distortion-free, it is necessary to analyze the respective overlap, for example 14-ι ₂ . Reference is made to the already mentioned Fig. 7. The respective overlap 14-ι ₂ and 14- | ₃ in the example case each amount to 10% of the area.

By now the respective overlap 14 ₁₂ and 14-ι _{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. For this purpose, the respective (strip-shaped) overlap 14 ₁₂ or 14 _{13 is} subdivided into respective squares 15 in the example case. Of course, the overlap 14 or 14- | ₂ or 14i _{3 are} also divided into rectangular or other sections 15. In any case, 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.

In fact, 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. In this context, 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.

Now, this Fourier image, for each partial region 15, on the one hand in the example of the overlap 14-ι 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

For example, the single image E _{2 has} to be compared to the single image

Egg (in the control system 6) are moved or rotated to align the structures against each other or to achieve a maximum match of the respective textures in the sub-area 15. This frame comparison now takes place in the sense of a cross-correlation.

In this cross-correlation, the previously determined respective Fourier images or Fourier coefficients per pixel are subjected to a mathematical operation which leads to a result, which is the greater, the greater the match of the respective Fourier coordinates. The cross-correlation is similar to the convolution. Details on

Fourier transformation and convolution as well as cross-correlation can be found in the book "Bildbearbeitung für Einsteiger" by B. Neumann, Springer Verlag, 2004, to which reference should be made expressly.

By now examining the subregions 15 of the respective overlap 14 ₁₂ and 14i ₃ with regard to the coincidence of their respective textures, and if the associated individual images E ₂ and E ₃ experience a shift and rotation with respect to the individual image E ₁ , an overall image is formed from the individual images E ₁ , E ₂ , E ₃ , etc., which virtually compensates for any mechanical inaccuracies in the recording of the individual images E by electronic means. In principle, 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.

Claims

claims:

1. A method for optically scanning a sample (1) by means of an adjusting unit (2, 3) and a scanning device (4, 5), after which individual images (E) are recorded and combined in the sense of a grid to form an overall image, characterized in that each Single image (E) is determined from a single image stack (7), wherein the individual image stack (7) composed of individually recorded stack images (8).

2. The method according to claim 1, characterized in that the single image (E) corresponds to one of the stack images (8) or is composed of a plurality of stack images (8).

3. The method according to claim 1 or 2, characterized in that the individual image stack (7) spatially by means of a reference plane (10) in a preferably three-dimensional image frame by means of a on the reference plane (10) located support point (13) is arranged.

4. The method according to any one of claims 1 to 3, characterized in that a work area is set, within which the overall picture is taken.

5. The method according to any one of claims 1 to 4, characterized in that the reference plane (10) is designed as a focus card (10) and is spanned by at least three focus points (11).

6. The method according to any one of claims 1 to 5, characterized in that the reference plane (10) is determined in time before the scanning of the individual images (E).

7. The method according to any one of claims 1 to 6, characterized in that the respective individual image stack (7) with a predetermined number of stack images (8) is taken perpendicular to the sample plane above and below the associated support point (13).

8. The method according to any one of claims 1 to 7, characterized in that the reference plane (10) is created by using the basis of selected pixels of the overall image, for example, the pixel intensity is determined perpendicular to the sample plane in the form of image intensity values.

9. The method according to any one of claims 1 to 8, characterized in that the image intensity values are weighted.

10. The method according to any one of claims 1 to 9, characterized in that at least one image intensity value is extracted as focus value per selected pixel.

11. The method according to any one of claims 1 to 10, characterized in that the generated individual images (E) and / or stack images (8) are recorded with overlap (14-12, 14-13), wherein the overlap (14 ₁₂ , 14 ₁₃ ) is analyzed for mutual alignment.