EP2038690A2 - Method and device for producing an image of a thin layer of an object - Google Patents

Abstract

The invention relates to a method for producing an image of a layer of an object (4) by means of a wide field optical element (5) on a resolving detector (6). According to the invention, the object (4) is illuminated in a focused manner on at least one object plane (3) having at least two binary illuminating patterns (26, 27; 33, 34) and for each illuminating pattern (26, 27; 33, 34), the corresponding images are detected. The illuminating patterns (26, 27; 33, 34) respectively comprise dark areas (27; 34) and light areas (26; 33), the light and/or the dark areas completely covering the object (4) when the illuminating pattern (26, 27; 33, 34) is superimposed. A layer image is determined from the detected images, said layer image comprising a partial segment that respectively reproduces a partial area of the object (4) that is arranged inside the light area of one of the used illuminating patterns such that said edges are arranged at a distance from the edges of the light area about at least one predefined minimum distance, and which are respectively determined with at least partial artificial light correction using at least two images which are respectively detected for different illumination patterns in which the partial area corresponding to the respective partial segment is arranged completely inside the light area of a first different illuminating pattern or completely inside a dark area of a second of the different illuminating pattern.

Description

 Method and device for producing an image of a thin layer of an object

The invention relates to a method for producing an image of a thin layer of an object, in particular by means of a wide field optics, as well as a corresponding device.

Biological samples or materials are often examined microscopically. In particular, corresponding objects for the detection of structures with wide field optics can be examined, which image the object or a thin layer, ideally a plane, of the object onto a spatially resolving detector. Such imaging examination can be done for example with normal microscopy or fluorescence microscopy. The thin layer may be, for example, a fluorescent layer on a support such as a slide or the bottom of a titer plate containing immobilized cells, tissue sections or DNA fields, preferably arranged in "microarrays".

However, quantitative fluorescence microscopy is frequently used to examine biological objects in particular. The aim of quantitative fluorescence microscopy is usually to excite by irradiation of a sample, in particular a thin layer of given thickness, fluorescence radiation whose intensity depends on the concentration of fluorescent substances to be measured in the sample. By measuring the intensity of the fluorescence radiation, a conclusion on the concentration of the fluorescent substances is possible. Therefore, it is less of an issue of extreme image sharpness than of a very reliable detection of the rays emanating only from the thin layer.

ιT \ OC \ r ~ OA on. Frequently, instead of high-resolution microscopes, so-called spatially resolving fluorescence readers are used, which can be regarded as microscopes optimized for quantitative fluorescence microscopy. The objects or samples may in particular be biochips which have been produced by photolithographic means or by means of a spotter.

In order to obtain as accurate as possible measurements of the intensity of the fluorescence radiation generated in the thin layer, two conditions must be taken into account. On the one hand, the fluorescence radiation from the thin layer should be detected as extensively and quantitatively as possible. On the other hand, radiation, in particular fluorescence radiation which does not originate from the thin layer, should be suppressed as well as possible, i. It should be achieved a good depth selection, in which only possible radiation in a layer around the focal plane is detected. Although not necessarily to be in the visible range of the optical spectrum, this optical radiation to be suppressed is also referred to below as a false light.

At least the following sources can be considered as sources for the misdirection. False light is produced, for example, by reflections and by scattered light on surfaces, in glasses, eg due to air inclusions, by intrinsic fluorescence of the glasses used, in sockets or in fluorescence measurements by non-suppressed excitation light. Furthermore, false light can also come from areas of the object or the sample, which lie outside the focal plane, preferably in the thin layer, for example, from fluorescent contaminants on the back of a slide or from one of the thin layer adjacent adjacent layer with a fluorescent liquid.

However, false light can also adversely affect the image of the object since it reduces or falsifies the contrast of the detected intensity distribution.

One way to avoid stray light is to use confocal laser scanners. With a confocal laser scanner, only a small area of the sample is illuminated by a few μπi ² , and only this small area is considered during the detection. If this is consistently carried out with the help of a well-matched pinhole, then false light is suppressed from the outset. Laser scanners, however, have a number of disadvantages compared to microscopes with wide-field optics. For example, in fluorescence microscopy excitation saturation and a strong fading of fluorophores due to the high intensity of radiation in focus can occur. Furthermore, there are significant limitations in the choice of wavelength. Many moving components, a high adjustment effort and a low quantum efficiency of the detector, usually a photomultiplier, are further disadvantages.

In order to avoid these disadvantages, methods are proposed in which images are acquired under different illumination patterns and an image of the thin layer is calculated from the acquired images.

For example, EP 972220 Bl describes a method in which three images of the object with the thin layer are detected, which, with illumination focused on the thin layer, are spatially sinusoidal, in each case one third of a third. period against each other shifted intensity profiles are detected. From the captured images, a picture of the thin layer is calculated.

DE 199 30 816 A1 describes a method and a device for depth selection of microscope images in which a one-dimensional periodic grating, e.g. a strip grid, used for lighting. At least n (n> 2) CCD camera images are taken with the structure of the illumination shifted by l / n of the lattice constant. From the at least three images, a confocal section of the sample is then calculated. This method is prone to artifacts when the grid does not produce sinusoidal illumination intensity on the sample.

WO 98/45745 A1 (DE 698 02 514 T2) describes an imaging system and method for microscopes, in which a structured illumination is provided by means of superposition of two coherent light beams. The method follows as well as the method described above according to DE 199 30 816 A1 _. The main goal is to generate optical sections in different object planes similar to a laser scanning microscope.

Both methods pursue the goal of obtaining a depth resolution of thick samples. They serve to obtain confocal sections of a sample or object that is thick in comparison to the depth of field with a wide-field optic. In both cases, the computational effort is relatively large because trigonometric equations must be solved.

- A - In the unpublished German patent application P 103 30 716.8 a device for carrying out a method for eliminating stray light in the imaging of heterogeneous, luminous or illuminated planar objects is described. This comprises a radiation source with downstream, the radiation homogenizing illumination optics for the homogeneous illumination of a subsequent field blanket plane, in which a structured field stop is arranged to produce an illumination structure superimposed on the object or the sample. This illumination structure is imaged by first optical means on the sample, wherein said first optical means may comprise a lighting tube, optionally a color splitter, and a lens. Furthermore, second optical means are provided for imaging the sample together with the superimposed illumination structure onto a spatially resolving detector, in particular for optical radiation. The arrangement further comprises adjustment means with which the illumination structure in the object plane can be positioned in a defined manner on the object or the sample. The detector is connected to an evaluation device for detecting and eliminating the stray light. It uses a structured bright field illumination with at least two different illumination patterns, in which dark areas do not overlap. From corresponding images, a dark image and a bright image can be determined. By subtracting the dark image from the bright image, a resulting image can be obtained.

The structured bright field illumination provided in this device, in which the object illumination and the imaging of the object take place together with the exposed field diaphragm structure by a single objective, For example, the excitation light in the objective can cause the occurrence of stray light, in particular due to intrinsic fluorescence of the glasses used. Furthermore, the back of an object, for example a biochip, is irradiated with almost the same excitation intensity as the focal plane. Therefore, due to the contamination of the backside fluorescence intensity can be correspondingly high and give rise to measurement errors. It was therefore proposed to use a structured dark field illumination instead of the bright field illumination in a second method for avoiding these disadvantages.

In either method, it is necessary to perform interpolation between non-illuminated areas to obtain a complete dark-field image.

However, all of the above methods have the disadvantage for quantitative fluorescence microscopy that the accuracy of measuring the concentration of fluorescent material in the thin layer can still be improved, although depth selection can be achieved and false light can be at least partially suppressed from areas adjacent to the thin layer ,

The present invention is therefore based on the object to provide a method for producing an image of a, in particular thin, layer of an object which simultaneously allows good depth selection and high quantitative accuracy in fluorescence measurements, and to provide an apparatus for performing the method. The object is achieved by a method for producing an image of a layer of an object by means of a wide-field optics on a spatially resolving detector, in which the object is illuminated in at least one object plane focused with at least two binary illumination patterns and for each of the illumination patterns corresponding images are detected, the illumination patterns each have dark areas and bright areas, of which the light areas and / or the dark areas completely cover the object when the illumination patterns are superimposed, and from the acquired images a tomogram is determined which comprises subsegments which each reproduce a partial area of the object, then Within a bright region of at least one of the illumination patterns used is that its edges are spaced from the edges of the bright region by at least a predetermined minimum distance, and which in each case at least partially false light correct are determined using at least two images, which were detected at each different illumination patterns in which the respective sub-segment corresponding sub-area within a bright area of a first of the different Beleuchtungsmυster or completely within a dark area of a second of the different illumination patterns.

The object is further achieved by a device for generating an image of a layer of an object, having a lighting device for focused illumination of the object in an object plane, a device for generating at least two predetermined illumination patterns for illuminating the object in the object plane with one in the beam path after the Lighting device arranged as a structured shutter acting element, the Be each of the bright areas and / or the dark areas completely overlaps the object when the illumination patterns are superimposed, an imaging optics preferably designed as a wide-field optic for imaging the object plane onto an image plane, a spatially resolving detector arranged in the image plane for detection the optical radiation emanating from the object, and an evaluation device for evaluating detection signals of the detector, which is designed to capture images on the basis of the detection signals and to carry out the steps of the method according to the invention following the image acquisition, and in particular to obtain a slice image from the acquired images determining that comprises subsegments each representing a subregion of the object which lies within a bright region of at least one of the illumination patterns used, that the edges thereof from the edges of the Hellb at least a predetermined minimum distance, and which are respectively determined under at least partially False correcting using at least two images that were detected at each different illumination patterns in which the respective sub-segment corresponding partial area entirely within a bright area of a first of the different illumination patterns or entirely within a dark area of a second of the different illumination patterns.

The method, which can be carried out with the device, is used to produce an image of a layer of an object, preferably of a planar object, in particular of a heterogeneous, luminous or illuminated object. In particular, this method may be a microscopy method, preferably a quantitative fluorescence microscopy method or a method using a fluorescence reader.

In the process, the layer does not necessarily have to be thin; rather, the layer may in particular be a whole layer, and not just a plane in which the illumination radiation is focused.

In the method, the layer of the object, within the scope of the invention, depending on the size of the imageable region in the object plane, is understood to be an entire sample or an entire article or only a partial region of an entire sample or an entire article to be examined or imaged. illuminated with at least two structured, binary illumination patterns. In this case, a binary illumination pattern is understood as an illumination pattern in which, for example, in contrast to illumination patterns with an intensity profile in the form of a sine profile, the structuring essential for the method is achieved by substantially interposing into the dark areas, at least in the context of geometric optics no illumination radiation is obtained and the transition from dark areas to light areas is very narrow compared to the extent of these areas (PLEASE SPECIFY QUANTITATIVE LIMIT, IF POSSIBLE). The light and dark areas of the object are those areas of the object that lie in the light and dark areas of the illumination pattern when illuminated with a respective illumination pattern.

To generate the structured illumination, the illumination device for dispensing of illumination light, which in the context of the present invention in addition to visible infrared or ultraviolet radiation is understood, and the means for generating at least two predetermined illumination pattern for focused, in particular structured illumination of the object provided at least in the object layer to be imaged. For this purpose, the device for generating the binary illumination pattern has in particular an element acting as a diaphragm, which preferably acts as a field stop. The element has translucent and opaque areas which correspond in the illumination patterns respectively to bright and dark areas which are adjacent. Accordingly, illuminated and non-illuminated areas appear in or on the object or the examined area with illumination light.

In the method, the object is sequentially illuminated with a different one of the illumination patterns. The illumination patterns used in the method are chosen so that their dark areas and / or their bright areas completely cover the object in the case of an imagined or fictitious overlapping of the illumination patterns.

The object illuminated with the respective illumination pattern is then imaged by means of the imaging optics, which may in particular comprise an objective, onto the spatially resolving detector which serves to capture the images of the object. As a spatially resolving detector preferably a CCD or CMOS matrix can be used. The signals of the spatially resolving detector are provided by the evaluation device, for example a data processing device with a video interface, a memory in which at least one computer program for carrying out the evaluation and in particular the method steps after the illumination of the object are stored, and a processor for executing the computer program Processed images.

The invention is based inter alia on the following observation: When focusing structured illumination radiation, such as illumination radiation with a sinusoidal intensity profile transversely to the beam direction, into a plane of the object, only a distribution of the illumination intensity determined by the properties of the optics used for focusing results along the beam direction , which may have its maximum in the plane, but falls parallel to the beam direction from the point of the maximum. When the plane is imaged onto the detector by imaging optics, an analogous phenomenon occurs so that a depth response function defined for the particular illumination pattern, which indicates the intensity received by the detector as a function of the distance of the source from the plane in the beam direction, also Having maximum in the plane; but in the region of the maximum, the depth response function is also curved, so that the received intensity for a given illumination intensity decreases with increasing distance from the focal plane. This drop means reduced sensitivity near the focal plane, so that the radiation emanating from the illuminated layer is not completely detected. Moreover, in any illumination pattern in each detected image of the illuminated areas of the object, which are illuminated with light areas of the respective illumination pattern, if present, corresponding light portions are directed to the areas of the image of the object corresponding to the dark areas of the respective illumination pattern in the object, ie unlit areas of the object. In these areas of the image, the proportions are then detected as false light.

To increase the sensitivity in the layer and the reduction of the effects of stray light is proposed according to the invention, on the one hand to use binary illumination pattern and on the other to combine such areas in the captured images for the correction of stray light within the light or dark areas of the detection The images used are illumination patterns and whose edges also have a predetermined minimum distance from the transitions between the light and dark areas. Thus, influences of the transitions between HeIl- and dark area are greatly reduced and there is surprisingly a Tiefenresponse function with a plateau near the maximum, so that there is a substantially constant sensitivity across the width of the plateau.

Moreover, the inventive method is characterized by its simplicity, since only simple summation and selection of intensity values are to be performed. These steps are much faster with computers, but also simpler processors or even unprogrammed circuits than trigonometric operations. rations. Accordingly, the device according to the invention can also be constructed very simply.

In principle, in the method, narrow regions can occur between the subsegments, in which the brightness values can be determined by interpolation between the values of the adjacent subsegments. However, it is preferred in the method that the sub-segments join each other gapless or overlap. The device is for this purpose preferably designed so that the sub-segments connect to each other gapless or overlap. For this purpose, no interpolation between the sub-segments is necessary, which significantly speeds up the implementation of the method and increases the accuracy of the resulting layer image. In particular, since the dark areas of the illumination patterns completely cover the object when the illumination patterns are superimposed, a dark image of the entire object containing all of the false light portions to be eliminated can be obtained from the dark areas of the images without interpolation. In the event that subsegments or subareas corresponding thereto of the object overlap, it is possible to average over the redundant subareas of the different images. For example, when summing the images of the light regions, a renormalization in the overlapping regions may be performed to correct for double detection effects in those regions. If the subsegments of the illumination patterns overlap, they can preferably be made from different images via the redundant subregions of the images of the dark areas. In the method, it is then particularly preferred that the sub-segments overlap less than 10% of the minimum extent. For this purpose, the device is preferably designed so that the sub-segments overlap less than 10% of the minimum extent. In this way, only a small number of lighting patterns need to be used.

Basically, the minimum distances can be chosen arbitrarily. However, it is preferred in the method that the minimum distances are greater than 1/5 of the minimum spacing of adjacent boundaries of a light or dark region. The device is then preferably designed so that the minimum distances are greater than 1/5 of the minimum distance of adjacent boundaries of a light or dark area. This development has the advantage that in this way the intensity of radiation coming from a layer around the focal plane can be detected particularly completely.

The subsegments can be formed in different ways. In one variant of the method, a light image and a dark image can first be formed from the acquired images to determine the layer image, subregions of the acquired images which respectively reproduce regions of the object being used in a light or dark region during the detection of the image reflect the respective area of the object used in the respective image used and whose edge has the minimum distance from the transitions between the light and dark areas of the illumination pattern. By subtraction from the light and the dark image, an at least partially corrected layer image can then be generated. In the device then preferably the evaluation device to do so designed for determining the layer image first from the captured images to form a light and a dark image, wherein respective areas of the object reproducing sub-segments of the captured images are used, in a light or dark area of an illumination pattern used in the detection of the respective image reproduce lying region of the object and whose edge has the minimum distance from the transitions between the bright and dark areas of the illumination pattern, and to generate by subtraction of the light and the dark image, an at least partially corrected layer image. This variant has the advantage that a smoothing of the light and / or dark images can be easily performed.

In another variant of the method, an even number of illumination patterns may be used. First, at least partially false-corrected images can then be determined by forming a difference image from acquired images in which the respectively used illumination patterns are complementary to each other, and the slice image can be determined from the images corrected for at least partially false-light. The device is then preferably designed such that an even number of illumination patterns are used, and the evaluation device is designed to firstly determine at least partially false-corrected images by forming a difference image from acquired images in which the respectively used illumination patterns are complementary to one another, and from the at least partially false-corrected images to determine the layer image. This variant has the advantage that an assembly of sub-segments can be made easier. Under a joining is also understood that the segments partially overlap. The joining of the subsegments can also be understood as image montage. The sub-segments are arranged in the assembly according to the arrangement of the corresponding areas on the object relative to each other. The joining can be done for example by adding the pictures.

The illumination patterns themselves may have different structures, provided that the dark and bright areas and the sub-segments have the aforementioned properties. For example, illumination patterns with radially extending light and dark regions can be used, each of which is generated by rotating a corresponding basic pattern around a center by a predetermined angle.

In the method according to the invention, however, the illumination patterns are preferably given by a basic pattern which is offset differently in each case relative to the object. The device is preferably designed so that the illumination patterns are given by a basic pattern, which is offset in each case differently relative to the object. The illumination patterns thus have the same structure, but are offset from each other in the object plane. Such illumination patterns are easy to generate. In addition, the evaluation of the images generated with the illumination patterns is particularly simple.

Particularly preferably, in the method a basic pattern is used as a basic pattern, wherein the offset basic patterns are obtainable by shifting the basic pattern relative to the object. The device is to is preferably formed so that the basic pattern is a periodic basic pattern, the staggered basic patterns are available by shifting the basic pattern. In particular, respective offsets may depend on the period of the basic pattern. This type of illumination pattern allows not only a particularly simple generation, but also a simple evaluation of the captured images.

It is particularly preferred that the amount of displacement or shifts and the number of shifts and thus the illumination patterns are chosen so that a depth response function in the region of a focal plane in which the illumination patterns are focused, has a plateau. For this purpose, the device can be particularly preferably designed so that the amount of displacement or displacements and the number of shifts and thus the illumination patterns are selected so that a depth response function in the region of a focal plane in which the illumination patterns are focused, a Plateau. The depth response function is understood to mean the previously mentioned function. A plateau is understood to mean that this function assumes a constant value in a not only point-shaped region of the focal plane. Preferably, it falls from the plateau via steep flanks to a value close to zero, preferably to a value of zero.

The offset of the illumination patterns relative to the object can be achieved in various ways.

In a preferred embodiment of the method according to the invention, the object is displaced relative to the illumination pattern. In the device according to the invention is to a drive, with which the object or a slide is movable, controllable by the evaluation so that formed on the object offset by movement of an element of the structured aperture acting as a basic pattern of the illumination pattern, and that means of the evaluation images after each change in position of the object are automatically detectable. As drives preferably piezo actuators or eccentric drives can be used, which allow a very accurate positioning. If necessary, the drive can also be used for the positioning of the object, which is necessary in any case, relative to the optics. Preferably, an already existing motorized microscope stage can be used. The movement through the drive can be possible in one or two directions along the object plane, depending on the illumination patterns used. The evaluation device can be designed, in particular, to generate a sequence of illumination patterns on or in the object by driving the drive, wherein an image is detected after setting in each case one illumination pattern. Thus, the operation is much easier for the user of the device. In addition, by appropriate programming of the evaluation device, the dependence required by the method between the type of illumination pattern and necessary shifts can be automatically taken into account.

Alternatively, in order to avoid a movement of the object relative to the detector, the basic pattern can preferably be displaced by means of a mechanical device for the variant described above. In the device according to the invention, the element acting as a structured diaphragm is preferably a field stop. A drive with which the FeId Aperture or part of the field diaphragm is movable, can be controlled by the evaluation so that the illumination patterns are irradiated on the object. After each change of the illumination pattern images are automatically detected by the evaluation. A field stop is understood here to mean, in particular, a stop which has rigid, non-transparent or opaque elements for forming the structured illumination.

As an alternative to the movement of the illumination pattern, it may be provided that the element acting as a structured diaphragm is a field stop and a movable, light-deflecting element is arranged behind the field stop for generating the at least two illumination structures. The field diaphragm can be firmly positioned. In particular, a reflecting surface or a transparent plane-parallel plate which can be tilted about one axis or two orthogonal axes depending on the illumination patterns used can be used as the light-deflecting element. As drives preferably piezoelectric drives or eccentric drives can be used. In order to simplify the adjustment of the illumination patterns and the detection of the corresponding images, a drive, with which the light-deflecting element is movable, can be controlled by the evaluation device so that the illumination patterns are irradiated onto the object. After each change of the illumination pattern by means of the evaluation device images are automatically detected. This development has the advantages mentioned above in connection with the automatic control.

In both embodiments, the field stop has transparent and opaque areas, which are designed so that in the object plane, the desired illumination patterns are obtained.

If the use of mechanical means for shifting the basic pattern is to be avoided, an electrically controllable modulation unit for light can be provided, which is controlled so that the illumination patterns are generated. In the device according to the invention, for example, the element acting as a diaphragm can be designed as such an electronically controllable modulation unit. As electrically controllable modulation units for light, for example so-called DMDs ("digital mirror devices") or electronically controllable transmitted light or reflection liquid crystal screens or LCDs can be used. As before, the evaluation device is preferably controllable so that the illumination patterns are irradiated onto the area to be examined and images can be automatically detected after each change of the illumination pattern by means of the evaluation device. This embodiment of the invention not only allows to dispense with mechanical drives, but also allows easy switching between different types of illumination patterns.

In a preferred variant of the method can be used as a basic pattern, a periodic stripe pattern with period p, the periodically alternating HeIl- and dark stripes are the same width and from which the other illumination patterns by shifting by the m / n times the period p transverse to the longitudinal direction the stripe being producible, where n is the number of illumination patterns and 0 <m <n. In the case of the device, the field stop preferably has a strip-shaped structure which differs from one another. has bleached transparent and opaque areas of equal width. The strips preferably extend over the entire examination area or the entire object. The more illumination patterns are used, the less excitation light is used in acquiring a corresponding image. As a result, the intensity noise in the background of the resulting image can be reduced almost arbitrarily. The dimensions of the transparent and opaque areas are chosen so that illumination patterns can be generated with the aforementioned parameters. When using an electrically controllable modulation unit for generating these illumination pattern sufficient training or programming of the evaluation.

Alternatively, illumination patterns each having a bi-directional array of light and dark regions may be used, the devices being offset from one another in at least one of the directions. When using a mechanically displaceable field stop, the field stop preferably has a two-directional periodic arrangement of transparent and opaque areas, the opaque areas adjoining each other. When using an electrically controllable modulation unit for generating these illumination pattern sufficient training or programming of the evaluation. The directions may be orthogonal to each other, but this may not necessarily be the case. In this embodiment, the marking of a direction is avoided by using stripes, so that the suppression of stray light becomes less directional. In addition, the modulation by the structured illumination vanishes faster outside of the focus area. ne or object level, so that light outside the depth of field of the imaging optics: better suppressed.

When periodic illumination patterns are used, it is possible that in the image cleared of stray light, there may still be conditional illumination inhomogeneities due to the periodicity of the illumination patterns. It is therefore preferred in the method according to the invention that the false light-corrected images are filtered low-frequency before formation layer image. In the case of the device, the evaluation device is preferably designed for this purpose such that the images corrected for false-correction are filtered at low frequency before the formation of the slice image.

If an object is to be examined with a dark field illumination, which may be desirable in particular in quantitative fluorescence microscopy, the illumination optics for imaging the element acting as a diaphragm on the object is designed for dark field illumination.

In the device according to the invention, the illumination optics should then be designed as an illumination objective with a small aperture, the optical axis of the illumination objective and the optical axis defined by the imaging optics enclosing an angle α. This training results in a large depth of field. The angle α should preferably be greater than 50 ° in order to minimize the radiation intensity on the underside of transparent objects or samples.

Preferably, in this case the illumination optics is a Scheimpflug optics. In this case, a larger numerical aperture for the dark field illumination can be provided, as the Focal plane of the lighting can be adapted to the top of the sample.

Furthermore, the imaging optics for imaging the object on the detector may include a Scheimpflug optics. In this case, the optical axis of the illumination lens is perpendicular to the surface of the sample, while the optical axis of the imaging lens is at an angle α to the optical axis of the illumination lens.

Both the method and the apparatus with which this method can be carried out can advantageously be used for reading out biochips, in quantitative fluorescence microscopy and in photometric measurements.

The invention will be explained in more detail by way of example with reference to the drawings. Show it:

Fig.l is a schematic representation of an optical structure of an apparatus for detecting an image of an object according to a first preferred embodiment of the invention,

2 is a schematic partial representation of a device for generating illumination patterns with a field stop with eccentric drive in the apparatus of Fig.l,

FIG. 3 shows four illumination patterns obtainable with the field stop of the device from FIG.

4 shows a representation of total depth response functions in the approximation of geometric optics for an illumination pattern with an intensity varying in one dimension according to a sine function and a binary illumination pattern with it the period of sine function repeating light and dark stripes,

5 shows a representation of total depth response functions in Fresnel's approximation for the illumination patterns in FIG. 4, FIG.

6 shows a schematic partial representation of a device for generating illumination patterns in a device for capturing an image of an object according to a sixth preferred embodiment of the invention,

7 is a bi-directional checkerboard illumination pattern in the apparatus and method according to the sixth preferred embodiment of the invention;

8 shows a representation of total depth response functions in the approximation of geometrical optics for an illumination pattern with an intensity varying in two dimensions according to a sine function and a binary illumination pattern with square light and dark stripes repeating the period of the sine function.

9 shows a schematic partial representation of a device for generating illumination patterns in a device for capturing an image of an object according to a seventh preferred embodiment of the invention,

10 is a schematic diagram of an optical arrangement of an apparatus for detecting an image of an object according to an eighth preferred embodiment of the invention; and FIG

11 is a schematic representation of an optical structure of a device for detecting a Image of an object according to a ninth preferred embodiment of the invention

A greatly simplified and schematically shown in Fig.l apparatus for forming an image of a layer of an object according to a first preferred embodiment of the invention comprises a lighting device 1, one of these downstream device 2 for generating illumination patterns in an object plane 3 of a on a support or Table arranged object 4, an imaging optics 5 for imaging the object 4 on an image plane B and arranged in the image plane spatially resolving detector 6. An evaluation 7 is connected via a detector connection to the detector 6 and via a control line to the device 2.

The illumination device 1 has a light or radiation source 8 which comprises a filter 9, a shutter 10 and, only optionally, the beam path homogenizing optical elements 11, such as e.g. a light guide rod or an internally mirrored glass hollow rod, and first illumination optics 12 and 13 for homogeneous illumination of a region of a field stop plane 14 are arranged downstream.

The device 2 has a arranged in the beam path of the illumination device 1, homogeneously illuminable by this, acting as a structured aperture, defined in the beam path in two mutually orthogonal directions in the field diaphragm plane 14 slidably arranged element, in the example a structured field stop 15, and a in FIG. l drive shown only schematically 16, with which the field diaphragm 15 is displaceable. The mechanical structure of the device 2 for generating illumination patterns is shown in more detail in FIG. Eccentric drives 17 and 18 are coupled to the field diaphragm 15 so that it can be laterally displaced in the field diaphragm plane 14 in two mutually orthogonal directions and positioned in this way defined.

The field diaphragm 15 has periodically arranged, strip-shaped, opaque areas, which are separated from one another by transparent areas, so that corresponding strip-shaped illumination patterns can be generated in the object plane 3, as will be explained in more detail below.

As further shown in FIG. 1, the field diaphragm 15 is imaged onto the object 4 to be examined or imaged, or the object plane 3, by means of a second illumination optical system 19, which comprises an illumination tube 20, a beam splitter 21 and an objective 22 , so that on the object 4 one of the position of the field stop 15 in the field stop plane 14 and its structure corresponding illumination pattern is irradiated.

The imaging optics 5, which represents a wide field optics, in the example comprises the objective 22, the beam splitter 21 and a imaging tube 23, and forms the object 4 illuminated with the respective illumination structure in a high-contrast manner onto the image plane B or the spatially resolving detector 6 for optical radiation.

The beam splitter 21 is formed in the example as a color splitter and has filters 24 and 25, with which unwanted or disturbing spectral radiation components can be filtered out. The beam splitter 21 and the filters 24 and 25 are components of a device for incident light fluorescence, wherein it is advantageous if the filters 25 and 26 are inclined by a few degrees, so as to remove disturbing reflections from the beam path.

The detector 6 comprises a matrix of CCD or CMOS elements and is in the example part of a CCD camera.

The detector 6 is connected to the evaluation device 7, which detects signals of the detector 6, performs the determination or elimination of the stray light in the imaging of the object 4 and generates a resulting slice image. For this purpose, the evaluation device 7 has a processor, a memory and corresponding interfaces. The resulting images may be stored and / or output via a display device or printer not shown in FIG.

The evaluation device 7 also serves as a control for the drive 16, with which the illumination structure generated by the structured field stop 15 is displaceable. The evaluation device 7 is designed such that it generates a sequence of illumination patterns by activating the drive 16 and the shutter 10 and detects a sequence of images of the examination area or of the object 4 respectively corresponding to the illumination patterns by detection of the signals of the detector 6. These images are processed after detection of the last image of the sequence in the evaluation device 7.

In carrying out a method according to a first preferred embodiment of the invention, four of Structure of the field stop 15 corresponding illumination patterns used, which are shown in more detail in Figure 3.

The first illumination pattern is a basic pattern. The other illumination patterns emerge from the basic pattern by shifting the basic pattern by the distance v. The basic pattern has a periodic structure of the period p with stripe-shaped dark regions 26 of width d and stripe-shaped bright regions 27 each arranged between the dark regions 26, which have the same width h as the dark regions 26.

The basic pattern is displaced three times by the distance v in a direction aligned orthogonal to the longitudinal direction of the striped dark regions 26 and light regions 27, respectively. The distance v, by which the successive illumination patterns resulting from displacement of the basic pattern are offset from one another, has in this example the length of one quarter of the period p. When using N illumination patterns instead of the four illumination patterns in this example, v may be chosen to be p / N in particular.

The period p is on the sample side preferably between lμm and lOOμm.

When the four illumination patterns in the object plane 3 are superimposed, the examination area or the object 4 is completely covered by the dark areas 26. When merging the dark areas 26, the object 4 is therefore completely covered by them, so that no interpolation between the dark areas 26 is necessary. The same applies to the bright areas 27.

In a method for producing an image of a layer of the object 4 by means of the wide field optics or the imaging optics 5 on the spatially resolving detector 6 according to a first preferred embodiment of the invention, the object 4 in the object plane 3 is successively with the four focused on the object plane 3 illumination patterns illuminated, for which the evaluation device 7 controls the drive 16 and the shutter 10 accordingly. For each of the illumination patterns, a corresponding image is automatically detected by means of the detector 6 and the evaluation device 7. The captured images optionally have in the areas in which the dark areas 26 are displayed, brightening by false light, which is emitted from illuminated with bright areas 27 sections of the object 4.

From the captured images, a slice image of the object 4 is then generated in the evaluation device 7.

For this purpose, a light and a dark image are first formed. To generate the light or dark image, only the subsegments are used which correspond to the subareas 37 and 38 of the light and dark regions 27 and 26, respectively, which form stripes in the center of the light and dark regions, respectively. These sub-segments correspond in this embodiment just the middle 50% of the images of the light and dark areas.

The light or dark image is then formed by combining these subregions 37 and 38 corresponding sub-segments. The arrangement of the areas of the captured images one another or the subsegments corresponds to the arrangement of the light or dark regions of the illumination patterns on the object 4 corresponding to the regions of the captured images.

The dark image is then smoothed by the evaluation device 7 for noise suppression using a corresponding low-pass filter.

Finally, in the evaluation device 7, the dark image is subtracted from the light image, resulting in a resulting layer image of the object 4, in which false light is suppressed, but the intensity in the layer is detected with high accuracy.

With this method it is achieved that an overall depth response function indicating, for points on the optical axis, the detected intensity of optical radiation emanating from a location on the optical axis at a distance z from the object plane, as a function of the irradiated intensity has such a form that a confocal suppression is achieved for planes far away from the object plane, thereby detecting fluorescence radiation arising in a certain depth range around the focal plane at full efficiency. This means that the total depth response function in the middle has a plateau with the value 1 or nearly 1.

The total depth response function for various structured illuminations can be calculated or estimated as follows. In the event that the structures that are thereby imaged on the object, clearly coarser than the resolution limit of the optical system, more precisely the Imaging optics 5, this calculation can be approximated with the models of geometric optics.

Geometric optics models can generally be applied when the optical system's transfer function, also referred to as PSF (Point Spread Function), can be approximated in focus by a delta function. In a first step, it should be assumed that this condition is met. In addition, it is assumed that the light wave field in the pupil plane corresponds to a homogeneously illuminated circle. From the ratio of the diameter of the illuminated field in the pupil and the focal length of the imaging optics 5, the numerical aperture of the imaging optics 5 can be calculated. The PSF as a function of the distance to the focal plane then results as a circle whose radius R _PSF depends on the distance to the focal plane z, the focal length f of the imaging optics 5 and the pupil radius Rp _up iii _e as follows:

R

R pupil

PSF = Z- f

The intensity distribution, which arises in a plane at a distance z from the object plane, then results as a convolution of the illumination structure IstrukturBiucht with the defocused PSF:

^ _obe (^ = PSF (z) <S> I _Slncture _ _Illumination _.

The detector 6 for the fluorescent light is in a plane conjugate to the focus or object plane. For the intensity _detector in the detector level, this yields: h _e ^ _or = PSF {z) ® [PSF (Z) ® I _Stnιktιιr . _Beleuchl ).

In this respect, PSF (z) can be understood as a deep response function. Since in this example the imaging optics 5 are used simultaneously for illumination, the overall depth response function is obtained by applying PSF (z) twice.

With the thus determined intensity distribution on the detector as a function of the defocusing z, the total depth response function can now be determined.

In the method described in the prior art, a sinusoidal intensity distribution is projected onto the surface and the modulation measured on the detector. That means that applies

Is _tr u _hur .B _ele uc _h ,. = 1 + S-II (O, Jf),

where ω _{x is} the spatial frequency in a plane orthogonal to the

Direction of the optical axis of the imaging optics 5 and the beam direction or the z-axis and x indicate a location in the plane.

This intensity distribution is folded twice with a circle, with a radius dependent on the depth z. The Fourier transform of the PSF (z) therefore corresponds to a modified Bessel function. The square of this function is known as Airyfunktion. The illumination function gives three peaks at zero, + ω _x and -ω _x . If one multiplies this function by the Airy function, then only at these three places do values differ from zero. By the Method of structured illumination, only the modulation of the intensity distribution is measured. Due to the two peaks at + a> _x and -ω _x , only two values are cut out of the Airy function. If one now varies the depth z, Airy functions with different scales result from which the Fourier transform of the illumination function cuts out only the values at + ω _x and -ω _x . In summary, therefore, one can say that in the model of geometric optics the total depth response function for illumination with a sinusoidal intensity distribution and the evaluation algorithms described above corresponds to an airy function.

If one considers a classical confocal point scanner with the models of geometric optics, this results in a total depth response function of (see the dashed curve in FIG.

^{Tresponse {z) = l ~} (2π Z ² ) ¹

An exact wave-optical description gives:

where a is a proportionality factor (see the dashed curve in Figure 5).

Here, too, the overall depth response function drops off in the immediate vicinity of the focal point. By contrast, the method according to the invention leads to a total depth response function which has a plateau with the value 1 in the immediate vicinity of the focus, ie in the immediate vicinity of the object plane.

Can sponsefunktion The basic principle for changing the Gesamttiefenre- follows p lausibiliert ^': one illuminates the sample with a rectangular strip grids and the method illustrated by the four images is used to process, it is ensured that all object points at least a quarter of a strip from the edge between bright and dark Strips are removed. Assuming that the transfer function PSF (z) corresponds to a circle with a radius dependent on the defocusing z, it follows that the information about an adjacent strip edge does not enter an object point until the defocusing becomes so great that the radius of the circle of confusion corresponds to the distance to the strip edge. This distance varies for the object points depending on their position between a quarter and a half of the strip width. Therefore, using the simplified models of geometric optics in the total depth response function, one has a plateau of value 1 having a width Z dependent on the location in the sample. The minimum width Z is calculated according to:

rectangle

8th

The maximum plateau width is just 2Z for the dots in the middle of the stripes. If more than four recordings are used, the minimum plateau width (the solid line in FIG. 5) can be further increased and achieved theoretically for infinite measurements the maximum plateau width 2Z (long dashed line in Figure 5).

This very simple explanation can be recalculated in Fresnelnäherung wave optics. The results to be expected can be distinguished in three cases:

1. The illumination structures are much larger than the optical resolution of the system. In this case, geometric and wave-optical descriptions will give the same results.

2. The illumination structures are not significantly larger than the resolution limit. In this case, a wave-optical calculation for the total depth response function must be applied. Depending on the size of the structures, the plateau of the total depth response function is "rounded off", which reduces the effective plateau width.

3. The size of the illumination structures is close to the resolution limit. In this case, only the first frequency component of the illumination grating is transmitted by the optical system. In this case, no plateau will occur in the overall depth response function. The total depth response function in this case will essentially correspond to the overall depth response function in geometric approximation for sinusoidal stripe illumination.

The second case can be simulated wave optically with the models of the paraxial Fresnellnäherung. The results are shown in FIG. Therein are the result for one Sinusoidal illumination structure shown by a dashed line and the result for the above-mentioned stripe structure of the same period. The transfer function PSF is calculated wave-optically in this case in the vicinity of the focal point and folded in accordance with the equation Iu _e Tektor with itself and with the binary stripe pattern. Fig.5 shows the obtained waveform. In this figure, total depth response functions for sinusoidal and binary illumination structures, represented by dashed and solid lines, respectively, are shown as a function of the position of points on the optical axis or z-axis, the zero point corresponding to the focal plane. It can be seen in comparison with FIG. 4 that the plateau of the overall depth response function is clearly rounded off.

In order to optimize the structured illumination in the form of a lighting grid for a specific measurement task, only the frequency of the illumination grid needs to be varied in otherwise unchanged evaluation. This variation has two effects. On the one hand, the confocal suppression of light from non-focal planes becomes the worse the larger the grating period of the illumination grating becomes. Essentially, the total depth response function and thus the degree of confocal suppression with the grid frequency scales. Secondly, the width of the plateau also scales in the total depth response function and thus the depth range of the sample, which is mapped onto the detector with intensity efficiency 1, at the frequency of the illumination grid. However, this simple linear dependence of the confocal suppression / plateau width with the grating frequency of the illumination grating is only valid for grating wavelengths which are significantly larger than the resolution limit of the optical system. If this condition is not met, the total depth response function must be calculated separately for each grid frequency. On this data, an optimization of the grid frequency for different measurement tasks can then take place.

A second exemplary embodiment differs from the first exemplary embodiment solely in that the generation of the slice image is modified and the evaluation device is modified accordingly. All explanations on the unchanged parts of the first embodiment therefore also apply here and the same reference numerals are used.

By subtracting the first image from the third image and the second image from the fourth image, two temporary images are thus generated in which an at least partially stray light correction has been carried out.

From the first temporary image, the partial segments 37 and from the second temporary image the partial segments corresponding to the partial regions 3.8 are then combined to form the tomographic image.

A third and a fourth exemplary embodiment differ from the first or second exemplary embodiment in that subregions 37 and 38 corresponding to sub-segments of the images of the light or dark regions do not now have the median strips of width p / 2, but those of width 3 / 4p are used, so that the sub-segments overlap. Since the subsegments overlap, the summation in the overlap areas results in intensity Elevations that are eliminated by referencing or renormalization.

The other steps are the same as in the first and second embodiments.

In a fifth preferred embodiment of the method and the device according to the invention, the field diaphragm 15 is not moved, but the object 4. For this purpose, a table can be used, on which the object is arranged and by means of a controlled by the evaluation device 7 drive in directions is movable parallel to the object plane 3. The evaluation of the captured images is analogous to the first embodiment. For positioning of the object 4 in the object plane 3 as actuators piezoelectric actuators, eccentric drives or other suitable adjustment mechanisms can be used, but preferably a motorized microscope stage. Accordingly, the second to fourth embodiments can be modified.

A device according to a sixth preferred embodiment of the invention differs from the device of the first embodiment in that a modified device 28, shown in principle in Figure 6, are used for generating illumination patterns and a correspondingly modified evaluation device. Since the other components are substantially unchanged, the same reference numerals are used for them and the explanations on the first embodiment apply accordingly. The device 28, which is partially shown in FIG. 6, now has, as an element acting as a structured diaphragm, a field diaphragm 29 with a checkerboard structure arranged in the field diaphragm plane 14, which has transparent and opaque square regions formed periodically in the same period with two orthogonal directions (see FIG .7). To produce different illumination structures, the device 28, as shown schematically in Figure 6, via a structured field stop 29 downstream in the light direction, plane-parallel glass plate 30 which is tiltable about two mutually orthogonal axes. For controlled, defined tilting of this glass plate 30 are provided as a drive 31 in an advantageous manner piezoelectric actuators 32 which are controlled accordingly by a control of the evaluation device, not shown. By tilting the glass plate 30 is known to be an optical beam offset of the beam path and thus a staggered image of the field stop 29 on the object 4. The arrows in Figure 7 marked the tilting directions of the glass plate 30th

To generate the beam offset, other suitable elements can be used. As drives for the glass plate 30 eccentric drives or other suitable drive mechanisms can be provided in another embodiment.

When the field diaphragm 29 is imaged in the object plane 3, a basic pattern shown in FIG. 7 is obtained with square light regions 33 and dark regions 34 arranged periodically in two mutually orthogonal directions, which form a checkerboard pattern. The basic pattern points in the first, in the following by the number 1 marked Direction a period pl and in the second, hereinafter indicated by the numeral 2 direction, the period p2, wherein in this embodiment, the periods are selected to be the same size.

To generate the sixteen illumination patterns in this example, the basic pattern, which itself constitutes a first illumination pattern, is first three times in succession by the distance vi in the direction 1, i. parallel to the in Fig.7 horizontal side of the bright areas 33, shifted, wherein a second, third and fourth illumination pattern arise. To generate a fifth illumination pattern, a displacement is then made by the distance v2 in a direction 2 orthogonal to the direction of the first displacement, i. parallel to the vertical in Fig.7 side of the bright areas. Thereafter, three further illumination patterns are generated by shifting by the distance vi in the direction of the first displacements. There are then further shifts by v2, then three times by vi, again by v2 and then three times by vi.

The periods pl and p2 are in the object plane preferably in the range between lμm and lOOμm. You can be chosen different sizes in other embodiments.

When the sixteen illumination patterns are superimposed, their bright areas 33 completely cover the object 4 in the object plane 3. The same applies to the dark areas 34.

The method for imaging the object 4 according to the sixth preferred embodiment of the invention is analogous to the first embodiment. The sub-segments correspond Now each square areas 37 and 38 in a square of the checkerboard pattern, the edge of the respective edge of the light or dark area has a distance of one quarter of the side of the bright and dark area.

The method has over the method of the first embodiment, i. the use of illumination patterns with stripes, the advantage that the modulation by the structured illumination disappears faster outside the focus plane or object plane, so that light outside the depth of field of the imaging optics is better suppressed.

FIG. 8 shows by way of example the total depth response function in the approximation of geometrical optics for the binary illumination pattern with checkerboard pattern-shaped intensity distribution (solid line) and for an illumination pattern with sinusoidal intensity distribution (dashed line) in two dimensions. As in FIGS. 4 and 5, the abscissa describes the distance from the focal plane in scaled, arbitrary units and the ordinate also describes the respective value of the total depth response function in arbitrarily scaled units. Thereafter, a closer approach to a box-shaped profile is achieved.

An apparatus for detecting an image of an object according to a seventh preferred embodiment of the invention differs from the apparatus of the first embodiment by another drive for the field diaphragm 15. All other components are unchanged, so that the same reference numerals are used for them and the explanations in connection with the first embodiment according to Fig.l apply accordingly.

9 shows a schematic partial representation of the modified device for generating illumination patterns. The drive controlled by the controller in the evaluation device comprises piezo actuators 35 and 36, which are coupled to the field diaphragm 15, so that the field diaphragm 15 can be adjusted by means of the piezo actuators 35 and 36 by lateral displacement along two mutually orthogonal directions in the field stop plane 14 , By mapping the field diaphragm 15 set in different positions onto the object 4, different illumination structures in the object plane 3 are generated as before, which are then imaged onto the detector 6 together with the object 3.

An eighth preferred embodiment of the invention differs from the previously described embodiments in that the means for generating illumination patterns now neither a field stop nor a drive, but instead acting as a structured aperture element arranged in the field stop plane 14 electrically controllable modulation unit 45 in Shape of a transmitted light LCD has. As shown schematically in FIG. 10, the modulation unit 45 is connected via a control line to an evaluation device 46, which is modified relative to the evaluation device 7 of the first embodiment such that light-dark patterns stored in the evaluation device are displayed on the transmitted-light LCD, so that corresponding to the object plane 3 corresponding illumination patterns are blasted. A device according to a ninth preferred embodiment of the invention, whose overall optical structure is shown schematically simplified in Figure 11, differs from the device of the first embodiment in that the element acting as a diaphragm for dark field illumination is sharply imaged on the object. For this purpose, the second illumination optical system 19 is replaced by a modified second illumination optical system 40, the beam splitter 21 is no longer necessary. For components that are substantially unchanged from the first embodiment, the embodiments of the first embodiment apply mutatis mutandis and the same reference numerals are used.

The device comprises the illumination device 1 with the light or radiation source 8, optionally the shutter 10 and, advantageously, the beam path homogenizing optical elements 11, such as. a Lichtleitstab or internally mirrored glass hollow rod, and illumination optics 12 and 13 for homogeneous illumination of arranged in the field stop plane 14 in the beam path means 2 for generating illumination patterns, which in the example comprises the structured field stop 15, are arranged downstream. This field diaphragm 15 is arranged positionable defined in the beam path in two mutually orthogonal directions of the field stop plane 14. It can therefore be moved by means of coupled to the field diaphragm 15 drive 16 in this plane 14.

Subsequent to the field diaphragm 15 downstream modified second illumination optical system 40, which in the example a lighting tube 41, a deflection mirror 42, an excitation filter 43, and an objective 44, which is structured as Aperture acting element, ie here the structured field stop 15, in dark field illumination on the object to be examined or measured 4 or the object plane 3 shown. The second illumination optics 40 forms a so-called Scheimpflug optics whose optical axis is arranged at an angle α to the perpendicular to the surface of the object 4 and the object plane 3 extending optical axis of the imaging optics 5. Advantageously, the angle α> 50 °. By means of this imaging optics 5, which comprises, for example, the objective 22, the filter 25 and the imaging tube 23, the object 4 is imaged in high contrast together with the illumination structure superimposed on it onto the spatially resolving detector 6 for optical radiation.

In an analogous manner, however, the imaging optics can also be designed as a Scheimpflug optics in a modified embodiment. In this case, the optical axis of the second illumination optical system 40 is perpendicular to the surface of the object 4 or the object plane 3. With this optical axis, the optical axis of the imaging optics then forms the angle α.

Reference sign list

1 lighting device

2 device for the production of

illumination patterns

3 object level

_C O

4 object

5 imaging optics

6 detector

7 evaluation device

radiation source

9 filters

10 closure

11 homogenizing optical

element

12, 13 first illumination optics

14 field stop level

15 field stop

16 drive

17, 18 eccentric drive

19 second illumination optics

20 lighting tube

21 beam splitter

22 lens

23 Figure tube

24, 25 filters

26 dark areas

27 bright areas

28 device for generating

illumination patterns

29 field stop

30 glass plate

31 drive 32 piezo actuators

33 bright areas

34 dark areas

35 piezoelectric actuator

36 piezo actuator

37 subareas

38 subareas

39 cells

40 second illumination optics

41 lighting tube

42 deflecting mirror

43 excitation filter

44 lens

45 modulation unit

46 evaluation device

Claims

claims

1. A method for producing an image of a layer of an object (4) by means of a wide field optics. (5) on a spatially resolving detector (6), in which the object (4) is focused in at least one object plane (3) with at least two binary illumination patterns (26, 27, 33, 34) and for each of the illumination patterns (26, 27, 33, 34), the illumination patterns (26, 27, 33, 34) each having dark areas (27, 34) and bright areas (26, 33), of which the bright areas and / or the dark areas are superposed the illumination pattern (26, 27; 33, 34) completely covers the object (4), and from the acquired images a slice image is determined which comprises sub-segments which each represent a subregion of the object (4) which is at least within a bright region one of the illumination patterns used (26, 27, 33, 34) is that its edges are spaced from the edges of the bright region by at least a predetermined minimum distance, and in each case at least partially Faltsichtkorrektu r are determined using at least two images, which in each case different illumination patterns (26, 27; 33, 34) in which the partial area corresponding to the respective partial segment is located entirely within a bright area of a first of the different illumination patterns (26, 27, 33, 34) or quite within a dark area of a second of the different illumination patterns (26, 27, 33 , 34).

2. The method of claim 1, wherein the subsegments join each other gapless or overlap.

3. The method of claim 2, wherein the sub-segments overlap less than 10% of the minimum extent.

A method as claimed in any one of the preceding claims, wherein the minimum distance is greater than 1/5 of the closest spacing of adjacent boundaries.

5. The method according to any one of the preceding claims, wherein, for determining the layer image first from the captured images, a light and a dark image are formed, each area of the object (4) reproducing sub-segments of the captured images are used, in a bright - or dark area of a used in the detection of the respective image illumination pattern (26, 27, 33, 34) lying area (37, 38) of the object (4) and the edge of the minimum distance from the transitions between the light and dark areas of Illumination pattern (26, 27, 33, 34), and by subtraction of the light and the dark image, an at least partially corrected layer image is generated.

6. Method according to one of claims 1 to 5, in which an even number of illumination patterns (26, 27, 33, 34) is used, and first at least partially false-light-corrected images are determined by forming a difference image from acquired images in which the respective used illumination patterns (26, 27; 33, 34) are complementary to each other, and from the at least partially false-corrected images, the layer image is determined.

A method according to any one of the preceding claims, wherein the illumination patterns (26, 27, 33, 34) are given by a basic pattern which is offset differently relative to the object (4).

8. The method according to claim 7, wherein the basic pattern used is a periodic basic pattern, wherein the offset basic patterns can be obtained by shifting the basic pattern relative to the object (4).

9. The method of claim 8, wherein the amount of displacement or shifts and the number of shifts and thus the illumination patterns (26, 27, 33, 34) are selected so that a Tiefenresponse- function in the region of a focal plane, in which the Illuminated pattern (26, 27, 33, 34) are focused, has a plateau.

10. The method according to any one of claims 7 to 9, wherein the object (4) relative to the basic pattern (26, 27) is moved.

11. The method according to any one of claims 7 to 10, wherein the basic pattern (26, 27, 33, 34) by means of a mechanical device (15, 9, 15, 16, 15, 31) is moved.

12. The method according to any one of claims 1 to 11, wherein an electrically controllable modulation unit (45) for Light is controlled so that the illumination patterns (26, 27, 33, 34) are generated.

13. The method according to any one of claims 7 to 11 or any one of claims 7 to 11 and claim 12, wherein the basic pattern is a periodic stripe pattern with period p is used, the periodically alternating light and dark stripes are the same width and from the other illumination patterns (26, 27, 33, 34) can be generated by shifting m / n times the period p transverse to the longitudinal direction of the strips, where 0 <m <n.

14. The method according to any one of claims 7 to 11 or any one of claims 7 to 11 and claim 12, wherein the illumination pattern (33, 34) each having a two-directional periodic arrangement of light and dark areas (33, 34) are used wherein the assemblies are offset from each other in at least one of the directions.

15. The method according to any one of the preceding claims, in which light and / or dark images are filtered low frequency.

16. A device for producing an image of a layer of an object (4), comprising an illumination device (1) for focused illumination of the object (4) in an object plane (3), a device (2; 28; 45) for generating at least two predetermined ones Illumination pattern (26, 27, 33, 34) for illuminating the object (4) in the object plane (3) with an im. Beam path after the illumination Device (1) arranged as a structured aperture acting element, wherein the illumination patterns (26, 27, 33, 34) respectively dark areas (27; 34) and light areas (26; 33), of which the light areas and / or the dark areas when superimposed the illumination pattern (26, 27, 33, 34) completely covers the object (4), an imaging optics (5), preferably designed as a wide field optic, for imaging the object plane (3) on an image plane (B), one in the image plane (B) arranged spatially resolving detector (6) for detecting the optical radiation emitted by the object (4), and an evaluation device (7, 46) for evaluating detection signals of the detector (6), which is adapted to capture images on the basis of the detection signals and to carry out the steps of a method according to one of the preceding claims following the image acquisition, and in particular to determine from the acquired images a slice image comprising sub-segments t, each of which reproduces a partial region of the object (4) which is thus located within a bright region of one of the illumination patterns (26, 27; 33, 34), the edges of which are spaced from the edges of the bright region by at least a predetermined minimum distance and which are each determined by at least partially straightening correction using at least two images which are respectively different for each illumination pattern (26, 27; 34), in which the partial area corresponding to the respective sub-segment is completely within a bright area of a first of the different illumination patterns (26, 27, 33, 34) or entirely within a dark area of a second of the different illumination patterns (26, 27, 33, 34).

17. The apparatus of claim 16, which is formed so that the subsegments join each other gapless or overlap.

18. The apparatus of claim 17, which is formed so that the sub-segments overlap less than 10% of the minimum extent.

A device according to claim 16 or 17, arranged such that the minimum distances are greater than 1/5 of the minimum distance of adjacent boundaries of a Hello dark area.

20. Device according to one of claims 16 to 19, wherein the evaluation device is adapted to first form from the captured images a light and a dark image to determine the layer image, wherein each region of the object (4) reproducing sub-segments of the captured images are used, which in a light and dark area of an illumination pattern used in the detection of the respective image (26, 27, 33, 34) lying area of the object (4) and whose edge the minimum distance from the transitions between the light and Dark areas of the illumination pattern (26, 27, 33, 34), and by subtracting the light and the dark image to produce an at least partially corrected layer image.

21. Device according to one of claims 16 to 19, the like is formed so that an even number of illumination patterns (26, 27, 33, 34) is used, and in which the evaluation device is adapted to first at least partially false-corrected images by forming a difference image of detected images to determine in which each used Illumination patterns (26, 27, 33, 34) are complementary to each other, and to determine from the at least partially false-corrected images, the slice image.

22. Device according to one of claims 16 to 21, which is formed so that the illumination patterns (26, 27, 33, 34) are given by a basic pattern which is offset differently relative to the object (4).

23. The apparatus of claim 22, which is formed so that the basic pattern is a periodic basic pattern, the staggered basic patterns are available by shifting the basic pattern.

24. Device according to claim 23, which is designed so that the amount of the displacement or displacements and the number of displacements and thus the illumination patterns (26, 27, 33, 34) are selected such that a depth response function in the region of a focal plane into which the illumination patterns (26, 27, 33, 34) are focused has a plateau.

25. Device according to one of claims 16 to 24, wherein a drive (16; 31), with which the object (4) or a support for the object (4) is movable, of the Evaluation device (7) is controllable such that on the object (4) moved with the carrier, a basic pattern (26, 27, 33, 34) generated by the element acting as a structured diaphragm images an illumination pattern (26, 27; 33, 34) , and by means of the evaluation device (7, 46) images of the detector (6) are automatically detected after each change in the position of the object (4).

26. Device according to one of claims 16 to 25, wherein ~ a drive (16; 31), with which the object (4) or a support for the object (4) is movable, from the evaluation device (7) is controllable in that a basic pattern (26, 27, 33, 34) generated by the element acting as a structured diaphragm is imaged on the object (4) moving with the support, and forms an illumination pattern (26, 27, 33, 34), and by means of the evaluation device (7 , 46) images of the detector (6) are automatically detectable after each change in the position of the object (4).

27. Device according to one of claims 16 to 26, wherein the element acting as a structured aperture is a field stop (15) and for generating the at least two illumination structures behind the field stop (15), a movable, light-deflecting element (30) is arranged.

28. The apparatus of claim 27, wherein the field stop (15) has a strip-shaped structure of alternating transparent and opaque areas.

29. The apparatus of claim 27, wherein the field stop

(15) has a bi-directional array of transparent and opaque areas, the opaque areas being adjacent to each other.

30. The apparatus of claim 27, wherein the element acting as a diaphragm is an electronically controllable modulation unit (45), in particular an LCD or a DMD.

31. Device according to one of claims 16 to 30, wherein an illumination optical system (40) for imaging the element acting as a diaphragm on the object (4) is designed as an illumination optical system for a dark field illumination.

32. Device according to one of claims 16 to 31, wherein the evaluation device is designed so that light and / or dark images are filtered low frequency.