WO2003023482A1

WO2003023482A1 - Method and device for optically examining an object

Info

Publication number: WO2003023482A1
Application number: PCT/EP2002/010122
Authority: WO
Inventors: Frank Sieckmann
Original assignee: Leica Microsystems Wetzlar Gmbh
Priority date: 2001-09-11
Filing date: 2002-09-10
Publication date: 2003-03-20
Also published as: DE50212392D1; EP1436658B1; EP1436658A1; US20050031192A1; JP2005502907A; US7495778B2; WO2003023482A8

Abstract

The invention relates to a device for generating a three-dimensional image of an object (12), comprising a lens (10) and an object platform (14) for receiving the object (12). The invention is provided with a piezo-actuator (16, 22) for adjusting the distance between the lens (10) and the object (12). An image recording device records a series of individual images of the object (12) on different planes. A multifocus image is then generated from said series of individual images.

Description

Method and device for the optical examination of an object

The invention relates to a device for the optical examination of an object with a lens and an object table for receiving the object, and a method for the optical examination of an object.

When examining samples with the aid of a microscope, there is often a need to reconstruct the three-dimensional configuration of the surface of the object. For example, confocal scanning microscopy can be used for this. A sample with the focus of a light beam is scanned point by point in a plane, so that an image, albeit with a shallow depth of field, is obtained on this plane. The object can then be represented three-dimensionally by recording a plurality of different planes and by corresponding image processing. Such a confocal scanning microscopy method is known, for example, from US Pat. No. 6,128,077. However, the optical components used in confocal scanning microscopy are very expensive and, in addition to a good technical understanding, also require a lot of adjustment work.

A method for luminescence microscopy is also known from US Pat. No. 6,055,097. In this case, dyes are introduced into a sample that fluoresce under suitable lighting conditions, so that the irradiation enables the dyes to be localized in the sample. To generate a spatial image, a number of images are taken in different focal planes. Each of these images contains image information that comes directly from the focus plane, as well as image information that comes from spatial sections of the object that lie outside the focus plane. It is necessary to determine a sharp image. to eliminate the parts of the image that do not come from the focal plane. For this purpose, it is proposed to provide the microscope with an optical system that makes it possible to illuminate the sample with a special illumination field, such as a standing wave or a non-periodic excitation field.

To improve the three-dimensional reconstruction of an object with a microscope, the applicant has already proposed an improved method and an improved device in the German patent application with the application number 100 50 963.0. A series of images of an object is recorded in different z-planes by means of an image recording unit, for example a CCD camera. There is thus an image stack of plane images of the object from a multiplicity of different planes in the z direction of the object. Each of these images in the image stack contains areas of sharp image structures with a high level of detail and areas that were outside the focal plane when the image was taken and are therefore out of focus and without high level of detail in the image. An image can thus be understood as a set of sub-image areas with different, in particular high detail and low detail. With the help of image analytical methods, the partial image areas are extracted from each image of the image stack, which are present in high detail sharpness. A result image is thus determined for each image of the image stack, which now only has the image areas of high sharpness. These result images are then combined into a new, now detailed three-dimensional overall picture. This creates a new, fully detailed, three-dimensional microscope image of the object.

Since the distances and the absolute positions of the z-planes in which the images of the image stack were recorded are known, the three-dimensional microscope image of the object generated in this way can also be evaluated quantitatively. In order to record the individual images of the image stack in the different z-planes, the distance between the objective and the object has so far been changed by mechanically adjusting the microscope stage, ie the support table of the object. The high table mass and with it However, the associated inertia of the overall system forms a limit for the speed with which the individual image stacks are recorded. Because the sluggishness of the system means that a relatively long time is required to move the object into the different z-planes into the focus of the lens.

The object of the present invention is therefore to propose an apparatus for examining an object and a method for examining an object, with which it is possible to reconstruct the object quickly and in depth using conventional light microscopes.

This object is achieved by a device with the features according to claim 1 and by a method with the features according to claim 10.

With the use of a piezo-controlled device for adjusting the distance between the lens and the object, it is now possible to very precisely and quickly shift different z-planes of the object into the focus area of the lens. This considerably speeds up the possibility of taking a series of individual images in the different z-planes of the object. This also opens up the possibility of performing a fast 3D reconstruction of microscopic surfaces for the light microscopic examination of objects. This means that fast processes can be recorded three-dimensionally and in depth in different objects. At the same time, it is ensured that the object is treated gently during the examination.

Since a lens is used for the proposed examination of the object and the object usually lies on an object table, it is necessary to shift different z-planes of the object into the focus area of the lens. According to the invention, there are now basically several options for shifting the desired z-plane of the object into the focus area of the lens. It is possible to use a piezo controlled lens. The lens itself uses a piezo actuator provided, which can be controlled with the aid of a suitable control unit so that it causes a shift of the lens and thus a shift of the focal plane of the lens.

In a further embodiment of the invention, instead of moving the objective, it is possible to move the object on the table or the table with the object with the aid of a piezo-controlled actuator. The piezo-controlled actuator is connected to the stage and coupled to a control device. The control device is able to control the piezo actuator in such a way that the object is displaced on the object table in the z direction and thus the displacement of a new z plane of the object into the focus area of the objective.

In a further embodiment of the invention, both the objective and the object table can be provided with a piezo actuator, both being controlled via a control device, so that both the objective and the object table or the object lying on the object table are in z. Direction can be shifted. The path length by which the object can be shifted in the z-direction into the different focal planes can thus be increased in a simple manner. In a further embodiment of the invention, both the objective and the object table can be provided with a piezo actuator and the object table can also have a conventional controllable adjustment unit (for example an electromechanical), with all units (objective piezo, table piezo and electromechanical table adjustment unit) then having one Control device are controlled so that both the lens and the stage or the object lying on the stage can be moved in the z direction. The path length by which the object can be shifted in the z direction into the different focal planes can thus be increased even further in a simple manner.

In all the exemplary embodiments of the invention described, it is expedient to record the individual images of the image stack, which are recorded in the different z planes, in z planes which are equidistant from one another. On the one hand, this facilitates image reconstruction and, on the other hand, enables with the smallest possible z-plane spacing, an extremely precise detection of the object in the z-direction.

An essential point of the method in question here is to separate the sharp from the blurred image areas in the individual layers. It has been found here that a significant improvement in the image quality can be generated if each individual plane image is arithmetically deployed with an individual apparatus profile. The apparatus profile means the imaging properties of the entire structure, which are described mathematically by the point transfer function (English: point spread function, PSF) or the optical transfer function (English: optical transfer function, OTF) of the entire apparatus optics. Details of the point transfer function are known to the person skilled in the art from relevant publications, for example from: Joseph W. Goodman, "Introduction to Fourier Optics", Mc Graw Hill, New York, 1968.

The deconvolution has the effect that the imaging errors resulting from the imaging system used are removed from each plane image, i.e. can be deducted. Details of the mathematical unfolding are also known to the person skilled in the art from specialist books. Advantageously, each level image is unfolded before the calculation process for determining and separating the sharp image areas begins. This means that each level becomes sharper and the sharp image areas of each level are easier to determine. To increase the calculation speed, the deconvolution can only be carried out on the multifocus image. However, the result of the unfolding can be worse than if all the individual images were unfolded and the multifocus image was calculated.

With the device proposed according to the invention and the method according to the invention, there is now the possibility of overcoming the light microscopic limitation of a relatively shallow depth of field of the image and at the same time a three-dimensional surface reconstruction of an object at high speed by using piezo-controlled actuators to position the focal planes in the object. The microscopic image quality is thus improved and there is also the possibility of rapid changes in microscopic surface topologies using image analysis and evaluation. This also enables real-time measurements of surface structures to be achieved while eliminating the limitation of image quality due to insufficient depth of field.

Further advantages and advantageous embodiments of the invention are the subject of the following schematic figures and their descriptions, the representation of which has not been drawn to scale in the interests of clarity.

They show in detail:

1: a detail from a device for examining an object with a piezo-controlled lens;

2: the basic procedure of the examination of an object with a piezo-controlled lens;

3 shows a process sequence of an improved method for examining an object with a piezo-controlled lens;

4: a process sequence of a further improved method for examining an object with a piezo-controlled lens;

5: a detail from a device for examining an object with a piezo-controlled object table;

6: the basic procedure of the examination of an object with a piezo-controlled object table; 7: a process sequence of an improved method for examining an object with a piezo-controlled object table;

8: a process sequence of a further improved method for examining an object with a piezo-controlled object table;

9 shows a detail from a device for examining an object with a piezo-controlled object table and a piezo-controlled lens;

10: the basic procedure of the examination of an object with a piezo-controlled object table and a piezo-controlled lens;

11: a process sequence of an improved method for examining an object with a piezo-controlled object stage and a piezo-controlled lens;

12: a process sequence of a further improved method for examining an object with a piezo-controlled object table and a piezo-controlled lens;

13: a section of a device for examining an object with an electromechanically controlled object table and a piezo-controlled lens;

14: the basic procedure of the examination of an object with an electromechanically controlled object table and a piezo-controlled lens;

15: a process sequence of an improved method for examining an object with an electromechanically controlled object table and a piezo-controlled lens; 16: a process sequence of a further improved method for examining an object with an electromechanically controlled object table and a piezo-controlled objective;

17: a process sequence of a further improved method for examining an object with an electromechanically controlled object table and a piezo-controlled lens;

18: a process sequence of a further improved method for examining an object with an electromechanically controlled object table and a piezo-controlled lens;

19: a process sequence of a further improved method for examining an object with an electro-mechanically controlled object table and a piezo-controlled lens;

20: a detail from a device for examining an object with a piezo- and electromechanically controlled object table and a piezo-controlled lens;

21: the basic procedure of the examination of an object with a piezo- and electromechanically controlled object table and a piezo-controlled lens;

22: a process sequence of a further improved method for examining an object with a piezo- and electromechanically controlled object table and a piezo-controlled lens;

23: a process sequence of a further improved method driving to examine an object with a piezo and electromechanically controlled stage and a piezo controlled lens.

Fig. 1 shows a schematic representation of a section of a device for examining an object with a piezo-controlled lens. A lens 10 is used to examine an object 12 that is attached to an object table 14. In order to generate a sharp image and a 3-dimensional surface profile of this object, a series of individual images is recorded according to the invention, each of these individual images lying in different z-planes of the sample. For this purpose, the focus of the objective 10 must be positioned in the sample such that it lies in the desired plane. This is achieved by setting the distance d of the lens from the object. For this purpose, a piezo actuator 16 is provided on the objective, which is connected to a control device 18. The piezo actuator 16 can be controlled via the control device 18 and thus a displacement of the objective 10 can be achieved, which is indicated by the double arrow. Thus, the setting of the focus within the object 12 can be achieved with the piezo-controlled lens 10. With the piezo actuator, the lens can be shifted extremely precisely and in very small shifting distances Δz, which are preferably in the range of the resolution of the observation light used.

After a single image has been recorded in each desired z-plane, the series of individual images is available for further image processing. The z-planes are preferably equidistant from one another.

2 shows the basic procedure of the examination of an object with a piezo-controlled lens, the method preferably being implemented via software control. After a rough presetting of the lens distance from the object 12 has been carried out, the further workflow of the device can proceed automatically. For this purpose, after the start 30 of the method in step 32, the lens 10 is moved into the starting position by means of the piezo actuator 16. Then in step 34 checked whether the end position entered or automatically determined by the user has already been reached. If this is not the case, the image of this focus plane is recorded and stored in step 36 with the aid of an analog or digital camera, preferably a CCD camera. Thereafter, in step 38, the piezo actuator 16 is controlled via the control device 18 in such a way that the objective 10 is moved in such a way that the next z plane in which the object is to be recorded is in the focus of the objective 10. This loop is repeated until it is determined in step 34 that the end position has been reached. Then, in step 40, a multifocus image and then a 3-dimensional surface profile is generated from the series of individual images. This image is stored in step 42 and the method is ended in step 44.

3 shows the procedure for a method for examining an object, in which the image quality achieved is improved. Compared to the procedure according to 2, an additional step 41 is inserted between steps 40 and 42, in which the multifocus image obtained is unfolded with the apparatus profile of the microscope. It has been found that a significant improvement in the image quality can be achieved if the multifocus image obtained has an individual apparatus profile, taking into account the apparatus 'own optical transfer function (OTF = optical transfer function) or the apparatus' own point transfer function (PSF = point spread function) is developed. This deconvolution means that the blurred pixels originating from the imaging system used are removed from each plane image, i.e. can be calculated.

4 shows the process sequence for a method for examining an object with a piezo-controlled lens and an additional step for further improvement of the image quality. As soon as step 34 determines that the end position has been reached, each of the stored individual images is unfolded with the respective apparatus function in additional step 39. The unfolding of each individual image with subsequent multifocus image generation leads to a more precise multifocus image; because through the folds, the blurring resulting from the optics is already eliminated in each individual image, so that the overall image, which represents an overlay of the individual images, no longer has such blurring.

The methods shown in FIGS. 2 to 4 thus differ in the sharpness and accuracy of the multifocus image determined. It should be taken into account that the increased accuracy requires more computing effort. It takes more time to create the multifocus image. The user is therefore given various options, from which he can choose freely. If a less sharp image is sufficient for him, he can proceed according to the method described in FIG. 2 and very quickly obtain an image of the object to be examined. The method sequence according to FIG. 3 lends itself for higher accuracies, which, however, entails more computational effort and therefore more time for creating the multifocus image. Extremely precise and sharp images can be achieved with the aid of the method shown in FIG. 3. However, since it is necessary to unfold each of the captured individual images, the computing and time expenditure is also greatest here.

In the method shown in FIG. 4, it is alternatively also possible to proceed in such a way that the recorded image is developed immediately before it is stored, that is to say as part of step 36, and is only stored after it has been developed.

5 shows a further embodiment of the invention. A section of a device for examining an object 12 with a piezo-controlled object table 14 is shown schematically.

The objective 10 is used to examine the object 12 which is applied to the object table 14. For positioning the focus of the lens 10 in the sample 12 is the distance d of the objective from the object then adjusted by a piezo actuator 22 is provided on the object table, which is connected to a control device ^• 20th This stage piezo actuator 22 can on the side facing the lens onto the surface 15 of the stage 14 mounted or mounted on the surface 17 of the table 14 (not shown) facing away from the lens. Mounting on the surface 15 of the table 14 facing the lens 12 is, however, advantageous because fewer masses have to be moved to generate the z movement. The stage table can be

Piezo actuator 22 are controlled and thus a displacement of the object 10, which is applied to the piezo actuator 22 on the object table 14, can be achieved in the z direction by Δz. This movement is indicated by the double arrow. Thus, the setting of the focus within the object 12 can be achieved with the piezo-controlled object table 14.

After a single image has been recorded in each desired z plane, the series of individual images is again available for further image processing.

FIG. 6 now shows the basic process sequence of examining an object with a piezo-controlled object table, the method preferably being implemented via software control. This method essentially corresponds to that already shown in FIG. 2. However, the focal plane of the objective is set in this method with method step 37, in which the piezo control 19 is addressed in such a way that a change in thickness in the z direction of the piezo actuator 22 is achieved by an amount Δz. Since the object 12 is applied to the piezo actuator 22, the focus plane can thus be shifted in the object.

An improvement in the image quality can also be achieved by using an object stage piezo actuator 22 for adjusting the distance of the objective from the sample. For this purpose, in accordance with the method described in FIG. 3, by inserting step 41, the multifocus image is unfolded with the apparatus function. This process is shown in FIG. 7.

Finally, FIG. 8 shows the further improvement by unfolding the individual images in step 39, which corresponds to the method used in the method. 4 can also be used when using a stage piezo actuator with the advantages already described.

9 shows a further embodiment of the invention. A section of a device for examining an object 12 with a piezo-controlled object table 14 and a piezo-controlled lens 10 is shown schematically. In this embodiment of the invention, a lens piezo actuator 16 is provided on the lens 10 and is connected to the controller 18. Furthermore, an object table piezo actuator 22 is provided on the object table 14, which can be controlled via a control device 20. As already described in connection with FIG. 5, the object stage piezo actuator can be mounted both on the side of the surface 15 of the table 14 facing the objective or also on the surface 17 of the table 14 (not shown) facing away from the objective. Because of the usually small maximum adjustment paths that can be achieved with the piezo actuators, this embodiment offers the possibility of scanning a substantially larger z range of the object 12 with high resolution, since both the objective piezo actuator 16 by Δz and the object stage piezo actuator 22 can be shifted by Δz ₂ .

With the aid of these two piezo actuators, a number of procedures can now be implemented, one of which is shown by way of example in FIG. 10. After a rough presetting of the lens distance from the object, the further workflow of the device can proceed automatically. For this purpose, after the start 30 of the method in step 32, the objective 10 is moved into the starting position by means of the objective piezo actuator and the objective piezo actuator. It is then checked in step 34 whether the end position of the table pie entered or automatically determined by the user has already been reached. If this is not the case, it is checked in step 46 whether the end position of the objective piezo actuator entered or calculated by the user has been reached. If it is determined in step 46 that the end position of the lens piezo actuator has not yet been reached, the image of this focus plane is recorded in step 36 with the aid of an analog or digital camera, preferably a CCD camera. After that In step 38, the lens piezo actuator 16 is controlled via the control device 18 in such a way that the lens 10 is moved in such a way that the next z plane in which the object is to be recorded is in the focus of the lens 10 in the object 12. This loop is repeated until it is determined in step 34 that the end position of the table has been reached. In this control loop, the objective is accordingly controlled by the objective piezo actuator in n steps with a step size of Δzi at different distances from the object. When the lens has reached its end position, it has covered a total distance of n * Δzi = N. If it is determined in step 46 that the end position of the lens has been reached, then in step 48 the lens is moved back into its starting position with the aid of the lens piezo actuator and then the lens table piezo actuator is controlled with the control unit 20 so that it is adjusted by the value of the previous lens total deflection namely Δz ₂ = N + i z. ^ is shifted. Now the control loop of the lens repeats and it is shifted again step by step over a total path of n * Δz-i = N. If its maximum deflection position is reached, the lens returns to its starting position and the stage is moved by the stage piezo actuator by the path of the new total lens movement, namely Δz ₂ = N + ΔZ _T (relative to the current table position).

By repeating this control sequence lens movement - table movement, it is possible to examine a large area of the sample despite the small maximum adjustment paths of the piezos.

As soon as it is recognized that the objective table has also reached its end position, a multifocus image and then a 3-dimensional surface profile are generated in step 40 from the series of individual images obtained in this way. This image is stored in step 42 and the method is ended in step 44.

The procedure described can be reproduced using the information given in the following table for a lens and table control sequence for n (lens) = 3 and (table) = 3.

As can be seen, a targeted alternating movement sequence of the lens 10 and the table 14 by means of a piezo-controlled adjusting device on the lens and on the table can cover a much larger scan area with high resolution.

Overall, there is a total distance that can be covered with two piezos (one on the lens and one on the object (table))

Dz (total) = Dz, - (m + 1) * (n + 1) * Dz, [μm] where m = number of steps of the table / object piezo control system n = number of steps of the lens piezo system Dz, = step distance of the lens piezo (e.g. 0.5 μm).

An improvement in the image quality can also be achieved by using an object stage piezo actuator and an objective piezo actuator for adjusting the distance of the objective from the sample. For this purpose, in accordance with the method described in FIG. 3, by inserting step 41, the multifocus image is unfolded with the apparatus function. This process is shown in FIG. 11.

In the end, FIG. 12 shows the further improvement by unfolding the individual images in step 39, which can also be used in accordance with the method described in FIG. 4 when using an object stage piezo actuator with the advantages already described.

In a further embodiment of the invention, a device for examining an object with a piezo-controlled objective in combination with an electromechanically controlled object stage is shown schematically in FIG. 13. A lens 10 is used to examine an object 12 that is attached to an object table 14. In order to be able to record the series of individual images, a lens piezo actuator 16 is provided on the lens 10 and can be controlled by a control unit 18. The stage 14 can be shifted in all three spatial directions in steps Δx, Δy, and Δz ₂ , this shift being controlled by a control module 21. The control module can preferably be connected to a computer. With this combination of movable components, it is now possible to examine larger objects. Because the option of moving the table in the x, y plane, partial images in different x, y positions can be recorded in a single z plane with a camera. These partial images can then be combined with an image processing software to form a single layer image, the so-called image mosaic. size that can exceed the otherwise possible image recording area by a multiple. In addition, the possible scanning area is significantly expanded via the table 14, which can also be moved electromechanically; because focus planes can now be set which result from a shift Δz _{2 of} the table in the z direction and from a shift Δz of the piezo-controlled lens.

FIG. 14 shows the basic procedure of the examination of an object 10 with an electromechanically controlled object table 14 and a piezo-controlled lens 12 using a flowchart. After the start of the method 30 and a rough presetting of the lens distance from the object, the further course of the method steps in the device can preferably be carried out fully automatically. For this purpose, the stage 14 and the lens are first moved into a starting position. This is advantageously carried out by means of the piezo and table control system (18, 21) via a computer.

In three control loops, the table 14 is then moved step by step over a defined x-y-z range. The first control loop is defined via query 50, in which it is checked whether the table 14 has reached the end position in the y direction. The second control loop is defined via query 54, in which it is checked whether the table 14 has reached the end position in the x direction. The third control loop is defined via query 56, in which it is checked whether the lens has reached its end position. If the table 14 is within manually or automatically taught-in x-y limits, the table is moved step by step by one image field through the program loop. As with a checkerboard pattern, a given scan area is scanned field by field.

In each newly approached x-y position of the table, an image stack is now recorded in the z direction.

This is achieved in the third control loop, in which the image is recorded in step 36 and then the new focal plane is changed by shifting the lens 10 in step 38 by the value Δz. The images are recorded in step 36 with an analog or digital camera and cached. The focal plane is guided through the object 12 in n steps and the desired image stack is recorded.

After a focus image stack has been recorded, the lens is returned to its z start position in step 48. In step 58, the stage 14 is steered further by an image field in the x-y plane and a further stack of focus images is recorded with the piezo lens according to the above method.

After all the images have been recorded and stored in this way, a mosaic image is created in step 52, preferably automatically, for each of the focus planes. This mosaic image is created by juxtaposing the images recorded in a focal plane, that is, the images that were recorded in a focal plane within a predefined x-y range. The mosaic images are created by known methods such as image-overlapping composition methods (autocorrelation) or touching composition methods. Since the mosaic images are generated for all focal planes, a mosaic image stack is created that represents all images of the focal planes.

Due to the possible small deflections of the lens piezo actuator, very small distances between the individual focal planes can be achieved. This property allows you to use high-resolution lenses, e.g. H. To work with lenses with a shallow depth of field and to make very fine topological details of a microscopic object visible.

In step 40, the composition of all the image mosaics, that is to say many individual images, creates a highly resolved image in the xy direction, both a completely sharp image in the z direction and an xy direction. In 3-dimensional space, the resolution can be defined as a so-called voxel resolution. A voxel is a volume element that has pixels in 3 spatial directions. With a voxel resolution of, for example, 1 μm edge length of an image pixel, a resolution of 25400 dpi arises in x, y, z, which in the context of these representations should be regarded as “extremely high resolution”. As shown in FIG. 15, after the multifocus image has been generated, the multifocus image can also be unfolded with the apparatus profile of the recording system in step 41 in order to further improve the image sharpness in the z direction. It is also possible, as shown in FIG. 16, to unfold all the xy-mosaic images with the apparatus profile of the recording system in step 39 even before the multifocus image is generated in step 40. As already mentioned, this method is time-consuming, but it leads to a multifocus image which is again improved with regard to the sharpness of the image. An unfolding of the individual mosaic images in accordance with the method described in FIG. 16 with subsequent multifocus image generation accordingly leads to a more precise multifocus mosaic image. However, the computational effort is considerable since each mosaic picture of the stack has to be unfolded.

In contrast, a deconvolution of the multifocus image already created, in accordance with the method shown in FIG. 15, can be calculated much more quickly from the original mosaic image data (undeveloped mosaic images). The fastest calculation is carried out if the original mosaic images are omitted as described in the method according to FIG. 14. By directly calculating a multifocus image from the previously created mosaic images, the highest speed of the design variants presented here is achieved. The user can freely choose which of these process variants is selected based on the desired result.

When using the device described in FIG. 13 with an electromechanically adjustable table 14, the resolution in the z direction can be increased even further. For this purpose, the distance between the objective 10 and the object 12 is adjusted from a suitable combination of the Δz, the objective 10 and the Δz _{2 of} the table 14. In FIGS. 17-19, this process sequence is in each case for a method without unfolding (FIG. 17 ), a method with unfolding of the multifocus image (FIG. 18) and a method with unfolding of the individual images (FIG. 19) are shown.

All of the methods are now characterized in that an additional check is carried out in step 66 to determine whether the end position of the scanning area Δz _{2 of} the table 14 is enough. Accordingly, the additional step 69 is then also necessary, in which the object table 14 is moved into a new position. In addition, returning the table to its starting position is required in the remaining loops, which is carried out in steps 70 and 72. With this procedure, therefore, a depth-of-field image and a 3-dimensional surface profile of this image can be generated by simultaneously using a normal object table in the xyz direction and a piezo-controlled lens in the z direction. This enables a much larger z range to be recorded with the highest resolution in the z direction. 17 shows the basic procedure using a flow chart. After a rough presetting of the distance of the objective 10 from the object 12 has been carried out, the further method steps can be carried out automatically. The object 10 and the electromechanically controllable specimen stage 14 are preferably moved to a starting position by means of the piezo control device 18 and the table control system 21 via a computer. In three control loops, which are defined by the query steps 50, 54 and 66, the table 14 is now moved step by step over a defined xyz range. The first control loop is triggered by query 50 and checks the table position in the xy direction.

If the table is within manually or automatically taught-in xy limits, the table 14 is moved step by step by one image field through the program loop. As with a checkerboard pattern, a given scanning area is traversed field by field. An image stack in the z direction is now recorded in each newly approached xy position of the table. This is carried out by the control loop, which checks in step 56 whether the end position of the adjustment path of the objective piezo actuator has been reached. As long as this has not been reached, an image is recorded in step 36, stored and in step 38 the position of the objective is changed by Δzi via the objective piezo actuator. This procedure can be illustrated using the following example: After the start of the method, in the control loop defined by step 56, the objective is now controlled by the objective piezo actuator in Δzi steps at different distances from the object. If the lens has reached its end position, it will have covered a total distance of n * Δz, = N. Now in step 68 the lens is controlled via the lens piezo actuator back to its starting position. Furthermore, the table 14 is shifted by the value of the previous total lens deflection - Δz ₂ = N + Δz-i.

The control loop is now repeated and it is shifted again step by step over a total path of n * ΔZi = N. If its maximum deflection position has been reached, the lens 10 returns to its starting position and the table 14 is displaced relative to the current table position by the path of the new total lens movement by - Δz ₂ = N + Δz.

By repeating this lens movement and table movement, a much larger area of the object can be scanned with high resolution - since piezos can realize very small displacements. In each focal plane reached by the lens shift Δzi, an image is recorded with an analog or digital camera and initially saved (in RAM, on the hard disk, etc.).

Specifically, for the following lens and table control sequence for n (lens) = 3 and m (table) = 3, there is a procedure as shown in the following table.

As can be seen, a much larger scan area can be covered with a high resolution by means of a piezo-controlled adjusting device 16, 18 on the lens and the position control 21 of the table 14 by means of a targeted alternating movement sequence of the lens 10 and the table 14. This results in an overall distance that can be covered with this method to:

Δ z (total) = Δz, - (m + 1) * (n + 1) * Δz, [μm]

m = number of steps of the table / object control system (electromechanical) n = number of steps of the lens piezo system Δz1 = step distance of the lens piezo (e.g. 1 μm)

After a focus image stack has been recorded after said interaction between table and lens movement, the piezo lens 10 returns to its z-start position in the step. The table 14 becomes an image field controlled further in the xy plane and a further focus image stack is recorded with the piezo lens according to the above method.

As soon as all the images have been scanned, the mosaic image is generated for each scanned z-plane in step 52, this step preferably being carried out automatically by a special program first. In this way, a mosaic image is created for each focus level. A multifocus image is then generated again in step 40.

To improve the image quality, once again, as shown in FIG. 18, one can also use this method after generating the multifocus image

Unfold the multifocus image with the apparatus profile of the recording system in step 41 in order to further improve the image sharpness in the z direction.

It is also possible, as shown in FIG. 19, to unfold all x, y mosaic images with the apparatus profile of the recording system in step 39 before the multifocus image is generated in step 40. As already mentioned, this method is time-consuming, but it leads to a multifocus image which is again improved with regard to the sharpness of the image.

In a further embodiment of the invention, a piezo actuator 16 with an associated control 18 is arranged on the objective 10 and a piezo actuator 22 with an associated control 20 on the table 14. The table 14 is also coupled to an electromechanical controller to provide movement of the stage in the x, y, and z directions. This constellation is shown in a schematic representation in FIG.

Thus the following three adjustment elements are now available for setting the distance of the objective 10 from the object 12, that is to say for the total possible z displacement:

1.electromechanical table: Δz ₂

2.Table piezo actuator Δz ₃

3. Objective piezo actuator Δz, There are two adjustment elements for adjusting the stage in the x and y directions:

1. electromechanical table Δx ₂ ; Δy ₂

2. table piezo actuator Δx ₃ ; Δy ₃ The possibilities of the piezo combination enable a fine scan range to be achieved which is defined by the adjustment range covered by the two piezo actuators.

In accordance with the adjustment path Δz ₃ now available in addition to adjusting the distance of the objective 10 from the object 12, the methods described so far are expanded by a further control loop. This method is shown in FIG. 21 using a flow chart. For this purpose, it is checked in step 76 whether the end position of the object stage piezo actuator has already been reached. Depending on this result, either the current image or the lens 10 and the table piezo 22 are moved to their new position by step 78 and 80 in step 36. In addition, in the

The overall sequence also includes steps 82, 84 and 86, in each of which the table piezo 22 is moved back to its starting position

Basically, the scanning principle corresponds to that already described in connection with FIG. 11, in which a lens piezo actuator 16 and a table piezo actuator 22 are also used. A very precise, high-resolution and fast acquisition of the individual image planes for the reconstruction of a 3-dimensional surface image and a high-resolution image can take place here. Through the additional use of an electromechanical table 14 which can be adjusted in the three spatial directions x, y, z, the method according to FIG. 11 can be advantageously combined with the method according to FIG. 17. Both the coarse and the fine range can be coordinated in such a way that the fine scanning areas can be seamlessly joined together by a suitable degree of movement of the electromagnetic table in the z direction. The combination of the two exemplary embodiments thus allows a substantial increase in the fine scanning range available in the z direction. As shown schematically in FIG. 21, after each xy shift of the electromechanical microscope table 14, the exact desired position can be finely adjusted by means of the table piezo 22. This is achieved by an offset of the position of the object 12 by the table piezo 22 which is suitable in x and y, after the new xy position of the object has been preset by the electromechanical table 14.

This makes it possible to dispense with the calculation of the overlap area of adjacent images when creating the image mosaic, since the individual individual images contributing to the mosaic can be approached so precisely that adjacent images touch one another with pixel accuracy. Thus, the individual mosaic images can be put together using the touch method, the so-called touche method. This method of creating a mosaic is currently the fastest and most robust, since the quality factors determining the image mosaic depend exclusively on the exact positioning of the object12. This is achieved by an exact electromechanical positioning of the table 14 and the precise control of the table piezo 14 in the xy and z directions. This means that there is no need for an image analysis to calculate the overlap area of adjacent images. By eliminating the need for this time-consuming autocorrelation process, in addition to robustness and image content independence, a high speed is also achieved and rapidly changing processes on microscopic surfaces can be recorded with high resolution.

LIST OF REFERENCE NUMBERS

Objective Object Objective Surface of the object table facing the objective Objective piezo actuator Surface of the object table facing away from the objective Piezo control device Piezo control device Electromechanical displacement control Object table piezo actuator Start of the procedure Approaching the starting position Decision "End position reached?" Unfolding the individual images Generating the multi-focus image Unfolding the multi-focus image Saving the result End of the procedure Decision "End position objective piezo reached?" move the lens to the start position 50 Decision "End position of the table in the y direction reached?" 52 Generate picture mosaic

54 Decision "End position of the table in the x-direction reached?" 56 Decision "Objective piezo deposition reached?" 58 move the table to the next x position

60 move lens back to start position 62 move table back to x start position 64 move table to next y position 66 decision "end position table in z direction reached?" 68 move lens back to start position

69 move the table to the next z position

70 move table back to z-start position 72 move table back to z-start position 74 fine adjustment x, y-direction 76 decision "end position table in z-direction reached?"

78 Move lens back to start position

80 move table piezo to the next z position

82 move the table piezo back to the starting position

84 move table pie back to start position 86 move table pie back to start position d distance lens / object

Δx shift in the z direction

Δy shift in the z direction

Δz displacement in the z direction

Claims

claims

1. Device for the optical examination of an object (12) with a lens (10), an object table (14) for receiving the object (12) and an image recording device for recording a series of individual images of the object (12) in different planes, characterized that a piezo-controlled device (16, 22) for setting the distance of the objective (10) from the object (12) and also a device for generating a multifocus image from the series of individual images is provided.

2. Device according to claim 1, characterized in that the piezo-controlled device (16) is coupled to the lens (10).

3. Device according to claim 1, characterized in that the piezo-controlled device (22) is coupled to the object table (14).

4. The device according to claim 2, characterized in that a further piezo-controlled device (22) is coupled to the object table (14).

5. The device according to claim 3 or 4, characterized in that a piezo actuator (22) is applied to the object table (14) so that the object (12) on the piezo actuator (22) can be deposited.

Device according to one of claims 2-5, characterized in that the object table (14) can be moved in the x, y and z directions via an electromechanical adjusting device.

7. Device according to one of claims 1-6, characterized in that a device for unfolding the multifocus image is provided with an apparatus function,

8. Device according to one of claims 1-6, characterized in that a device for unfolding each of the individual images of the series is provided with an apparatus function.

9. Device according to one of claims 6-8, characterized in that a device for generating an image mosaic is provided.

10. A method for examining an object (12) using an objective (10) and an image recording device, the object (12) recording (36) a series of individual images with the image recording device, characterized in that the

The position of the focal plane of the objective (10) in the object (12) is set using a piezo-controlled device (16, 22) before taking a single image.

11. The method for examining an object (12) according to claim 10, characterized in that the positions of the focal planes of the objective (10) in the object (12) are set for each individual image so that they are equidistant from one another.

12. The method according to any one of claims 10 or 11, characterized in that after the recording of the individual images (36) or after the recording of the series of individual images, each of the individual images is unfolded with an apparatus function of the optical imaging device.

13. The method according to any one of claims 10 to 12, characterized in that a result image is determined from each of the individual images by extracting the subset of the image areas with high detail sharpness and in a further step (40) the plurality of result images are combined to form a multifocus image.

14. The method according to claim 13, characterized in that the multi-focus image unfolds in one step (41) with an apparatus function of the optical imaging device and thus a multi-focus image with increased detail sharpness is obtained.

15. The method according to any one of claims 10-14, characterized in that for adjusting the distance of the object (12) from the lens (10) a piezo actuator (16) attached to the lens and a piezo actuator (22) attached to the table of theirs respective control devices (18, 20) are controlled so that their extension changes in the z direction.

16. The method according to claim 15, characterized in that for the positioning of the object (12) the object table (14) is moved in the x, -y, - or z-direction with an electromechanical displacement device.

17. The method according to claim 16, characterized in that in each focal plane, in a first step 52, an image mosaic is first generated from the individual images for this level and each image mosaic is used in a second step (40) to generate a multifocus image.

18. The method according to claim 17, characterized in that each image mosaic is unfolded with the apparatus function of the optical imaging device before the generation of the multifocus image.

19. The method according to claim 17, characterized in that the multifocus image created from each image mosaic unfolds with the apparatus profile of the optical imaging device and thus a multifocus image with increased detail sharpness is obtained.