WO2003069391A1

WO2003069391A1 - Device for confocal, optical microanalysis

Info

Publication number: WO2003069391A1
Application number: PCT/EP2003/000765
Authority: WO
Inventors: Norbert Czarnetzki
Original assignee: Carl Zeiss Microelectronic Systems Gmbh
Priority date: 2002-02-14
Filing date: 2003-01-25
Publication date: 2003-08-21
Also published as: DE10206004A1

Abstract

The invention relates to a device for confocal optical microanalysis comprising a microscope stage (1) that can be moved for optically scanning an object on an X-Y plane; a lighting device (2) for illuminating the object; an illumination optics arranged between the microscope stage (1) and the lighting device (2), said illumination optics having a first array (10) consisting of a plurality of optical microelements (7) for producing a grid consisting of a plurality of illumination points on the object to be analyzed, and a receiver device (12) having a plurality of receiver cells (13) for receiving the light emitted from the object after interaction in relation to the individual illumination points. A second array (14) consisting of optical microelements (15) arranged in the form of a grid is assigned to the receiver device (12), said microelements directing the light reflecting on the object onto the receiver cells (13). The inventive device makes it possible to detect large object fields by optimizing lateral and depth resolution. The device is particularly suitable for conducting analysis of fluorescent substances, wherein a plurality of samples can be simultaneously analyzed.

Description

Device for confocal optical microanalysis

The invention relates to a device for confocal optical microanalysis, comprising an object table for receiving an object to be examined, which can be moved for optical scanning of the object in an XY plane, an illumination device with one or more light sources for illuminating an object on the object table - Conceivable object, a lighting optics arranged between the lighting device and the object table with an array of a plurality of grid-shaped, optically - namely refractive, diffractive and / or reflective - effective micro-elements for generating a grid from a multiplicity of lighting points the object to be examined, and a receiving device with a plurality of receiver cells for receiving the light coming back from the object in an individual assignment to the individual lighting points.

Such a device is known from DE 40 35 799 C2. With the device described there, surface profiles of an object to be examined microscopically can be recorded in a relatively short time. For this purpose, the object is illuminated with light points arranged in a grid. The light point structure is predetermined by an aperture provided in the illumination beam path with a plurality of openings arranged in a grid. The illumination grid is imaged with a grid dimension on a receiving device, namely a CCD receiver, which corresponds to the grid dimension of the light-sensitive areas of the CCD receiver or is an integral multiple thereof. Furthermore, DE 40 35 799 C2 discloses the possibility of generating the illumination grid by means of an array from a multiplicity of lenses, which generates small light spots by means of sufficiently good imaging properties from an almost punctiform light source.

In order to achieve a high luminous efficacy in this device, however, a very precise adjustment of the lens array with respect to the positions of the receiver cells of the receiving device is necessary, which has undesirable restrictions on the freedom of design.

Starting from this prior art, the object of the invention is to further improve a device of the type mentioned at the outset. In particular, a device for fast analysis methods is to be made available, in which on the one hand a high efficiency of the lighting is to be ensured and on the other hand an evaluation with electronic methods is possible with a differentiated transformation of the image information from the object to be examined into an image plane.

According to the invention, in a device of the type described in the introduction, a second array is assigned from a plurality of optical micro-elements arranged in a grid, which direct the light coming back from the illumination points on the object onto the receiver cells of the receiving device.

By moving the object table and thus an object placed on the object table, this becomes Fixed lighting optics scanned in cells. The image information individually read out during a scanning process in synchronism with the movement of the object table at the receiver times of the receiving device can be electronically combined to form an overall image. It is not necessary to move the arrays. Rather, they remain stationary during a scanning process.

In this context, it is advantageous to arrange the optical microelements of the first array rhombically. The same applies to the micro elements of the second array. When the object to be examined is shifted, the confocal light bundles are continuously guided over the object and a complete image of the object to be examined is built up according to a certain displacement path.

The rhombic arrangement of the optical micro-elements also favors a high luminous efficacy when lighting. The proportion of the optical microelements in the area of the respective array is preferably 80% or more.

In an advantageous embodiment of the device, the receiving device is a surface camera or line camera, the object table being movable synchronously with the reading of the receiving device. For larger observation objects, an area camera in the form of a structured TDI camera (TDI: Time Delayed Integration) is preferably used, the line scan rate of which is coordinated with the travel speed of the object table and which then provides a continuous, digitized image of the object to be examined. delivers the object from a large number of drawing files or single lines. The high sensitivity of TDI cameras makes it possible to increase the exposure time for the individual pixels by means of the number of integration levels, which means that faint (fluorescent) object points can also be detected.

The optical microelements of the first array can be designed, for example, as spherical or aspherical micromirrors. In this way, a color-correct image of the light source (s) (eg several lasers) can be ensured. In addition, the spherical aberration is smaller in the case of openings of the illuminating beam bundles that are not so large in comparison with refractive microelements of the same refractive power. A vertical incidence of the illuminating beams, which is advantageous for images with shaped micromirrors, can be achieved, for example, by suitable polarization-optical or dichroic splitter elements.

If unpolarized white light sources are used in the illumination device, a polarizer is preferably provided in order to utilize the effect of a polarization-optical divider element. However, it is also possible to use one or more lasers as the light source in the lighting device, in which an intrinsic linear polarization is possible without additional loss of light output. The laser light is preferably coupled into the illumination beam path via polarization-maintaining optical fibers. When using aspherical micromirrors, the illumination side is preferably dimensioned such that the illumination points on the object do not exceed the size of the object structures to be resolved thereon and the illumination cones illuminate the aperture of the optical microelements of the second array that can be recorded on the image side.

The free diameter of the micromirrors is primarily adapted to the needs of a high area coverage, the distance between the micromirrors, however, having to take into account the requirements for confocality. The ratio of the mirror diameter to the mean radius of curvature is very small for the practical applications of interest, so that the imaging via the mirror takes place in the para-axial space.

Instead of reflective microelements, refractive or diffractive microelements can of course also be used, these being preferably optimized for several wavelengths (fluorescence excitation). Holographic micro-elements are also to be counted for this.

For arrangements with refractive arrays made from individual lenses, a color correction for imaging light sources in the image plane for two wavelengths is achieved by taking into account dispersive properties of the lens material and diffractive effects of the lens edges.

Furthermore, it is possible to provide a pinhole array between the first array and the object table, which has a large number of pinholes arranged in a grid. The grid of the pinholes corresponds to the grid of the micro elements of the first array and is conjugated to it. Above all, false pin effects can be kept low by the pin holy array.

As an alternative or in addition, the neutral, optically ineffective zones between the micro-elements can be blackened or made of light-absorbing material in order to avoid reflections and to prevent an illumination background. With the basic rhombic shape of the micro-elements, an illuminance of almost 100% can even be achieved.

To separate the illuminating and imaging beams, for example polarization splitters or dichroic splitters in the form of preferably optical cubes are provided. In conjunction with polarization splitters, a λ / 4 plate allows a good separation of illuminating and imaging light in the direction of illumination. In this context, a steep-edged formation of the spectral separating edge of the polarization splitter layer or of the dichroic splitter layer is advantageous. This can be supplemented by suitable excitation edge filters on the part of the lighting device.

As already mentioned, the lighting device generates monochromatic light or else light of defined but different wavelengths. If a white light source is used, the excitation wavelengths are fixed by suitable band filters. However, it is also possible the use of laser light sources or arc lamps with extracted lines of different wavelengths. Longitudinal color errors that may occur for different excitation waves are eliminated by focusing the object to be examined, eg. B. compensated by focusing the object table. A correction by focusing the two arrays is also possible.

As an alternative or in addition to the aforementioned light sources, the ends of one or more illumination fibers can also be used as point light sources, which are achromatically collimated.

The device explained above is particularly suitable for the optical analysis of substances arranged in an object plane which are only present in small quantities there. In the main beam path of the device, the light in interaction with these substances is converted into light with other properties, for example changed polarization state o ^'of the changed wavelength, if certain substance properties are present (eg fluorescence label). The multiple illumination and imaging beam paths enable, for example, individual examinations on a large number of sample containers which are arranged on a sample carrier at a relatively large distance. The sample carrier and the sample containers then together form the imaging object to be arranged on the object table.

The invention is explained in more detail below on the basis of preferred exemplary embodiments in conjunction with drawings. The drawings show in: Fig.l shows a first embodiment of the inventive apparatus ^as' using a first array of micro-mirrors, Figure 2 shows a second embodiment of the inventive device with a first array of re- fraktiven or diffractive optical micro-elements,

3 shows a third exemplary embodiment of a device according to the invention with a telecentric device

Microlens pairing of the arrays,

4 shows a fourth exemplary embodiment of a device according to the invention with a pinhole array,

5 shows an example of a rhombic grid of microelements in the illumination beam path, and in

6 shows an example of a rhombic grid of receiver elements in the imaging beam path.

In a first exemplary embodiment, FIG. 1 shows a device for optical microanalysis with confocal illumination and imaging. This comprises an object table 1 for holding an object to be examined, for example a carrier with a large number of substances to be analyzed. The object table 1 can be moved in the coordinates X, Y (meandering) of a coordinate system X, Y, Z, so that the object or the individual samples can be scanned point by point and can be optically scanned. With regard to the compensation of longitudinal color errors, there is also the possibility of focusing the stage 1 perpendicular to the XY plane, ie in the Z direction. Furthermore, the device comprises an illumination device 2 with which monochromatic light is generated. The lighting device 2 has a white light source 3, which is followed by a collimator 4, a polarizer 5 and a wavelength-selective filter β in the direction of illumination towards the object table 1.

In the illuminating beam path between the illuminating device 2 and the object table 1 there is an illuminating optic with which the light from the illuminating source 2 is confocally directed onto the object table 1 in the form of a plurality of illuminating light beams running side by side, so that there is a grid in an object plane from a variety of lighting points. In Fig.l only a single illumination beam path and the associated imaging beam path is shown as an example.

The illumination optics comprises a first array 10 made up of a multiplicity of optical microelements 7 in the form of micromirrors which are arranged in a grid-like manner on a carrier plate 8. In the exemplary embodiment shown, all the micromirrors 7 are of the same design and are arranged rhombically to one another. Examples of rhombic structures are shown in Fig.5 and Fig.6. With such arrangements, there is a high use of space and thus a good light yield in the lighting.

The carrier plate 8 with the micromirrors 7 is arranged opposite the lighting device 2. A polarization splitter 9 ensures the confocal illumination of the object field on the object table 1. The lighting device device 2 illuminates with a collimated beam through the preferably narrow-band polarization splitter 9 through the first array 10 of micromirrors \ 1, which image the light source 3 in parallel in the object plane. For this purpose, the light reflected by the ^" micromirrors" 7 is deflected via the polarization splitter 9 onto the object table 1. A λ / 4 plate 11 is arranged between the polarization splitter 9 and the array 10 in order to achieve the 180 °.

The individual micromirrors 7 have a light-concentrating effect and are dimensioned such that the images of the light source 3 lie within the desired analysis volumes on the object to be examined.

The light of the individual illumination points reflected by the object, again through the polarization splitter 9, reaches a receiving device 12 with a multiplicity of receiver cells 13 arranged in a raster pattern that individual or groups of receiver cells 13 are each assigned to an illumination point.

On the way to the receiving device 12, which is, for example, a CCD camera, the reflected light passes through a second array 14 made up of a plurality of optical micro-elements 15 arranged in a grid. The micro-elements 15 of the second array 14 are here as refractive or diffractive optical ones Elements formed and concentrate that from the lighting points' on the object emitting light onto the respectively assigned receiver cells 13 on the receiving device 12. For this purpose, the raster of the optical microelements 15 on the second array 14 is also conjugated to the raster of the picture elements or that of the first array 10. However, the rhombic basic arrangement is aligned in mirror image to the first array 10. Again, all the micro elements 15 are of the same design. They all have the same focal length and the same diameter and are formed uniformly on a carrier plate 16. As a result of the good use of space on the two arrays 10 and 14, a parallel confocal illumination and imaging is created, which uses up to 75% of the luminous energy of the illumination source 2.

When examining fluorescent substances, the actual image of the object to be analyzed or the samples to be analyzed arises primarily as a fluorescence image in reflection, i.e. the lighting points fluoresce back into the room. Each micro-element 15 of the second array 14 transforms the fluorescent light from the assigned illumination point into the image plane of the receiving device 12 in accordance with its receiving aperture.

Due to the imaging effect of the microelements 15, only the ^{'illumination} points of the object plane is transmitted into conjugated halftone dots of the image plane. The effect is a confocal transmission, which cuts out defined thin layers from the depth and width of the transformable object space and only allows them to contribute to the composition of the image. The overall picture of a certain object field is obtained by moving the object table 1 in the XY plane achieved. Using known methods of image data processing, the rhombically structured raster images are combined to form an overall image or can also be strengthened in the sum formation.

In the case of extended objects, the complete object field is detected by the object table 1 continuing to scan in a meandering manner. The step speed of the displacement of the object that can be achieved and the image formation that occurs synchronously with this by reading out the respective receiver cells depends, inter alia, on the sensitivity of the receiving device 12 or camera used, the brightness of the fluorescent examination object and the spacing of the illumination points. In a particularly advantageous variant of the receiving device 12, a TDI camera is used as the receiving device 12, the receiver surface of which is rhombically structured, the structure being oriented in mirror image to the first array 10. The diameters of the “light-sensitive islands” of the receiver surface preferably correspond to the Airy discs that can be focused by the microelements 15 of the array 14. By moving the object table 1, the image of the object runs transversely to the line direction of the TDI camera, the optoelectrically generated ones Charges of the object points are pushed across the lines at the same speed as the scanning object points, thus increasing the sensitivity of the line camera by increasing the exposure time of each image point, ie the length of time it stays on the receiving device 12. A correspondingly quick image build-up follows from the current one Composition of each because completely exposed end line of the TDI camera to a picture format of virtually any length.

In a further variant of this exemplary embodiment, a dichroic divider in the form of a divider cube can also be provided instead of the polarization divider 9. In any case, the broadband, longer-wave fluorescence radiation is transmitted by a suitable choice of the bandwidth of the polarization splitter 9 or the long-pass reflection edge of the dichroic splitter, so that a corresponding image of the longer-wave pixels is obtained at the receiving device 12.

A second exemplary embodiment shows in FIG. 2 a device for confocal optical microanalysis which differs from that of the first exemplary embodiment by the illumination optics. The object table 1, the lighting device 2 and the receiving device 12 can be designed as in the first exemplary embodiment, so that only the differences in the lighting optics need to be explained in more detail here. This in turn comprises a first array 17 for generating an illumination point grid in an object field. In contrast to the first exemplary embodiment, this first array 17 has a plurality of refractive or diffractive optical microelements 18 instead of micromirrors. In particular, holographic micro-elements 18 are also possible. The optical micro-elements 18 are configured in a rhombic arrangement with respect to one another on a carrier plate, the distance and aperture being selected such that the light from the illuminating device 2 is multiplied directly into the object plane. In the second exemplary embodiment, a divider element 19 is provided for this purpose, which directs the light coming from the lighting device 2 and passing through the first array 17 onto the object table 1. The light reflected by the object is reflected by the divider element 19 onto the receiving device 12 without being deflected. In order to concentrate the individual imaging beams of the illumination points on the receiver cells 13 of the receiving device 12, the divider element 19 is followed by a second array 20 made up of a large number of optical microelements 21 which act refractive or also diffractive. The optical microelements 21 in the imaging beam path are in turn formed on a carrier plate 22 and have a mirror-image rhombic basic arrangement to the first array 17. All micro-elements 21 are designed in the same way. In particular, they all have the same focal length and the same specific diameter.

This ensures both confocal lighting and confocal imaging. Exactly one microelement 18 of the first array 17 and exactly one microelement 21 of the second array 20 are assigned to each illumination point on the object. Interactions between the beams of the individual lighting points are largely eliminated. If necessary, the zones between the microelements on the arrays 17 and 20 that are not required for the optical effect are blackened or provided with a light-absorbing layer. In the second exemplary embodiment in particular, the divider element 19 can also be designed as a narrow-band polarization splitter, in particular for fluorescence applications, as a result of which the illumination beam path and the imaging beam path can be separated well. Alternatively, the use of a dichroic divider is also possible. In connection with a polarization divider, a λ / 4 plate 27 can again be provided between the divider element 19 and the object table 1.

The generation of an overall image from a multiplicity of raster images takes place analogously to the first exemplary embodiment.

The third exemplary embodiment shows in FIG. 3 a further device for confocal optical microanalysis in the form of a confocal microanalysis device with telecentric partial beam paths. The object table 1, the lighting device 2 and the receiving device 12 can be designed in the same way as in the first two exemplary embodiments. However, there are deviations for the lighting optics.

In this third exemplary embodiment, the collimated light coming from the lighting device 2 is directed onto the object table 1 via a divider element 23. To divide the illuminating light into a plurality of illumination points arranged in a grid, the divider element 23 is provided with a first array 24 ^″ in the direction of the object table 1 with a multiplicity of optical microelements 25, in particular refractive or diffractive micro- elements downstream. These micro-elements 25 are formed on a carrier plate 26 which is adjustable in the direction of the optical axis for the purpose of focusing. If necessary, depending on the purpose of the analysis, a λ / 4 plate 27 is again provided in the illumination beam path between the dividing element 23 and the object table 1.

Light reflected from an object reaches the receiving device 12 through the first array 24, the divider element 23 and a second array 28. The second array 28 in turn comprises a multiplicity of optical microelements 29 which are formed on a carrier plate 30. This carrier plate is arranged between the divider element 23 and the receiving device 12. Here, too, an unambiguous assignment of the micro elements 25 and 29 to an illumination point is ensured. The microelements 25 and 29 can in turn be arranged rhombically, with the receiving device 12 then likewise structuring the receiver cells 13 accordingly.

The telecentric arrangement enables simple adjustment of the two arrays 24 and 28, which can be moved in the direction of the optical axis for the purpose of focusing. In addition, the image scale can be easily changed.

If the illumination is carried out with a white light source, this can be designed as a modified Köhler illumination, so that a large number of images of a uniformly illuminated light field diaphragm through the optical micro elements 25 of the first array 24 in the object plane arise. An achromatic correction for two lines is achieved by a suitable choice of the dispersive properties of the material of the refractive optical microelements 25 in relation to their diffractive lens edges.

When using illuminating light with non-achromatic wavelengths, the object plane or the object table 1 is preferably focused in the direction of the optical axis, ie. H. here in the Z direction. The imaging beams which arise at the rear pass through the optical microelements 25 of the first array 24 and are approximately collimated in the process. As a result of the spectral changes in the illumination light occurring on the object, it is no longer deflected at the divider element 23, but rather passes through it and falls onto the second array 28. The telecentric imaging beam is imaged onto the receiving device 12 via this. If necessary, the second array 28 is finely focused in the direction of the optical axis by moving the second array 28 relative to the receiving device 12. A movement of the arrays 24 and 28 transversely to the optical axis for the purpose of scanning different pixels is not provided. Rather, the scanning is carried out solely by moving the object table 1 and the synchronous reading of the receiver cells assigned to the object points.

If fixed excitation and analysis wavelengths are used, in a variant of the third exemplary embodiment it is possible to guarantee the conjugation of the confocal Formation, the two arrays 24 and 28 are firmly connected to the divider element 23, so that these components together form an advantageous technical unit.

The generation of an overall image from a multiplicity of raster images then takes place in accordance with the two exemplary embodiments explained above.

Another example of an illumination optics is shown in the fourth embodiment ^', the stage 1, illumination device 2 and receive device 12 may be formed as already explained above. In the case of the illumination optics according to the fourth exemplary embodiment, the parallel confocal illumination beam path is generated before the divider element 31. A collimated illuminated first array 32 with a large number of refractive or diffractive optical microelements 33 generates a rhombically screened confocal bundle, which is sharply confocally imaged onto a pinhole array 34 similarly screened to the first array 33. The pinhole array 34, a plate with a multiplicity of pinhole openings 35, minimizes false and stray light effects.

After the U deflection on the divider element 31, the illuminating light passes through a second array 36 with a large number of optical microelements 37 to the object table 1. The second array 36 has a dual task here, namely the task of mapping the pinhole array 34 into the object plane and further the task of the lighting points to be analyzed on the object in to map a receiver level, ie on the receiving device 12.

Axial color aberrations occurring for different illumination wavelengths are compensated for by focusing the receiving device 12 relative to the object table 1. An overall image is then generated from a multiplicity of raster images as already described above.

5 shows an example of a pinhole array 34 with a plurality of pinholes 35. Their rhombic arrangement is based on a right-sided displacement of the confocal pinhole rows by one pinhole radius each. In the illustrated example, one of the starting line, the a * n when moving to the object under examination corresponding to a path in the image space a scan period ^'of 10, after ten Pinholezeilen reached again completely corresponding pinhole, * r (r: Pinholeradius; n * r: From - stood between neighboring pinholes in integer multiples of the pinhole radius), namely ten times the line spacing, has illuminated each object point of a line-like object at least once. The displacement of the object table 1 takes place in adaptation to these distances, so that after such a length, ie a scanning period, the scanning of the line-shaped object has been carried out completely once in the example on which this is based. The 10 * 10 rhombic pinhole grid can be periodically multiplied in length and width in order to dimension the desired object scanning width or length and the number of scanning processes. In the example, n = 10 is chosen, which corresponds to a degree of coverage with pinhole areas of approx. 3%.

6 shows the associated second array 36 with the micro-elements 37. Because of the arrangement chosen in FIG. 4, the rhombic grid structure of the micro-elements 37 is a mirror image of that of the pinholes 35. In the example, after ten lines of optical microelements 37, an arrangement corresponding to the starting line is again created. When the object to be examined is moved by 10 * 2 * R (R: radius of a 'micro-element), namely ten times the diameter of the micro-elements 37, each object point is imaged at least once in an imaginary object line. In the example, at R * 10 = 2R, a degree of coverage with an imaging area of the microelements 37 of approximately 75% is achieved.

The advantage of all the devices described here is the possibility of detecting large object fields to be analyzed when using a parallel-confocal illumination and imaging method while optimizing the required lateral resolution and necessary depth discrimination. Instead of the lighting device with a white light source explained above, one or more laser light sources or discharge lamps can alternatively be used for confocal lighting.

The devices described are particularly suitable for examining fluorescence properties. By dividing the illuminating light into a large number of independent analysis beam paths, a large number of samples can be examined simultaneously. Besugsseichenliste

1 stage

2 lighting device

3 light source

4 collimator

5 polarizer

6 filters

7 optical micro elements

8 carrier plate

9 divider element

10 first array

11 λ / 4 plate

12 receiving device

13 receiver cells

14 second array

15 optical micro elements

16 carrier plate

17 first array

18 optical micro elements

19 divider element

20 second array

21 optical micro elements

22 support plate

23 divider element

24 first array

25 optical micro elements

26 carrier plate

27 λj. / 4-plate

28 second array

29 optical micro elements 30 carrier plate 31 divider elements 32 first array 33 optical micro-elements 34 pinhole array 35 pinhole 36 second array 37 optical micro-elements

10

Claims

claims

Claims 1. Device for confocal optical microanalysis, comprising - an object table (1) which can be adjusted in the direction of the coordinates X, Y, an illumination device (2) with one or more light sources (3) for illuminating one placed on the object table (1) Object, - a lighting optics arranged between the object table (1) and the lighting device (2) with a first grid-shaped array (10; 17; 24; 32) made of a plurality of optically refractive, diffractive and / or reflective micro elements (7; 18; 25; 33) for Erzeu- a grid of illumination points at the object and a receiving device (12) having a ^'plurality of receiver cells (13) supply in for receiving the returning light from the object in association with the individual illumination points, characterized that the receiving device (12) is assigned a second grid-shaped array (14; 20; 28; 36) made up of a large number of optical microelements (15; 21; 29; 37), through which the light reflected from the illumination points on the object is directed onto the receiver cells (13) of the receiving device (12).

2. Device according to claim 1, characterized in that the micro-elements (7; 18; 25; 33) of the first

Arrays (10; 17; 24; 32) are arranged rhombically and the micro-elements (15; 21; 29; 37) of the second Arrays (14; 20; 28; 36) are arranged in a rhombic mirror image of the first array (10; 17; 24; 32).

3. Device according to claim 1 or 2, characterized in that the proportion of the optical micro-elements on the surface of the first array (10; 17; 24; 32) is 75% or greater.

4. Device according to one of claims 1 to 3 ', characterized in that the receiving device (12) as

Surface camera or line camera and the object table (1) can be moved synchronously with the evaluation of the receiver cells (13) of the receiving device (12).

5. Device according to one of claims 1 to 4, characterized in that the optical micro-elements (7) of the first array (10) are designed as spherical or aspherical micromirrors.

6. Device according to one of claims 1 to 4, characterized in that the optical micro-elements (18; 25; 33) of the first array (17; 24; 32) are designed as refractive or diffractive optical elements optimized for several wavelengths.

7. The device according to claim 6, characterized in that a spatially parallel and simultaneous imaging of the light source or light sources (3) in a plane on the object via a rhombically arranged array of refractive microlenses with achromatized effect taking into account diffractive effects by the lens edge ,

8. Device according to one of claims 1 to 7, characterized in that between the first array (32) and the object table (1) a pinhole array (34) is provided which has a plurality of grid-shaped pinholes (35), wherein the The grid of the pinholes (.35) corresponds to the grid of the microelements (33) of the first array (32).

.

9. Device according to one of claims 1 to 8, characterized in that the ^' neutral zones between the optical micro-elements (7, 15; 18, 21; 25, 29; 32, 37) are blackened or consist of light-absorbing material.

10. Device according to one of claims 1 to 9, characterized in that a polarization splitter (9; 19; 23; 31) is provided for the separation of illuminating and imaging beams.

11. Device according to one of claims 1 to 9, characterized in that a dichroic splitter in the form of an optical cube is provided for the separation of illuminating and imaging beams.

12. Device according to one of the preceding claims, characterized in that a λi / 4 plate (11; 27) for the illumination wavelengths is provided in the illumination beam path between the illumination device (2) and the object table (1).

13. Device according to one of the preceding claims, characterized in that the lighting device (2) emits monochromatic light or light of several, defined wavelengths λi.

14. Device according to one of the preceding claims, characterized in that as light sources (3) in the lighting device (2) a plurality of lasers or multiline lasers with a fixed, but different wavelength λi are available.

15. Device according to one of the preceding claims, characterized in that the lighting device (2) comprises arc lamps with extracted lines of different wavelengths.

16. Device according to one of the preceding claims, characterized in that light sources (3) are formed by the ends of one or more illumination fibers, which are achromatically limited as point light sources.