HK1184547B - Method and device for focusing substrates in an automated manner in fluorescence microscopy - Google Patents

Description

Method and apparatus for autofocusing a medium in fluorescence microscopy

Technical Field

The invention relates to a method and a device for the automatic inspection of biological material, comprising: a microscope on whose cross stage the biological material is arranged between a slide or analysis plate (Analystate) and at least one cover slip; at least one light source for illuminating the biological material and a camera unit which takes at least one picture of the biological material enlarged by means of an objective of the microscope and transmits it to the evaluation unit. The described solution provides a marking that can be recognized by a device for automatic focusing of biological material, wherein the focusing can be carried out by targeted movement of the cross table on the basis of the recognition of the marking.

Background

Fluorescence microscopy is a standard method used to examine patient samples in the field of medical diagnostics. In this case, further development efforts in this area for this technology have also been: the degree of automation is increased in order to thus minimize the error probability and improve the economy of microscopy.

In order to achieve an increase in the degree of automation in the laboratory, it is conceivable, on the one hand, to further automate the processing, in particular the dilution and incubation steps, of the sample to be examined, or, on the other hand, to more efficiently design the process for the processing, in particular the visual analysis, of the processed sample. The invention, which will be explained further below, relates to the field of sample analysis or diagnosis, wherein it is also basically known in fluorescence microscopy to generate digital image data of a sample to be examined and to transmit these image data to a data processing unit, on which special laboratory software is installed, and to carry out computer-aided analysis processing in order to carry out a diagnosis.

In such at least partially automated fluorescence microscopy, generating and selecting high quality pictures is of great importance, in particular in order to be able to ensure that the high quality remains unchanged when the assay diagnosis is completed. The important quality features of the pictures taken are ultimately their sharpness, so that the focusing of the biological material to be examined is of particular importance. In order to focus the biological material or the culture medium to be examined, a large number of pictures are usually taken in different planes, of which pictures that are not of sufficient quality are discarded again during the electronic analysis process.

Various solutions are known for the automatic focusing of microscopes. Among them, the active autofocus system commonly used in incident light microscopes is superior here in that: the shape, position or size of the light spot is evaluated by projecting a light spot or a symbol on the surface of the sample to be examined or on a cover glass covering the sample by means of an auxiliary light source and is focused on the sample surface or on the cover glass surface on the basis of the evaluation. An active autofocus system of this type is known, for example, from DE3446727C2 and should be able to achieve rapid autofocus.

Passive autofocus systems are typically used in transmitted light microscopes instead of active autofocus systems. As is known, for example, from DE3439304C2, the focal plane, i.e. the plane in which the sharpest picture is recorded, is determined on the basis of a comparison of the recorded image data. However, such systems are often slow because multiple searches must be performed to achieve satisfactory results.

Another special active autofocus system is known from DE 102010035104. In this document, a device for automatic focusing of a weakly luminescent culture medium is described, which device should ensure rapid image focusing. Faster focusing should on the one hand improve the efficiency of the automatic microscopy and on the other hand minimize the discoloration of the fluorescent dye during focusing. The technical scheme is characterized in that: first, a plurality of shots in the transmission light mode are taken and from these the clearest pictures are determined by means of known analytical processing methods. After the transmission light source is switched off, the cross stage of the microscope is moved to the focal plane determined in the transmission light mode. To generate the fluorescence image, the excitation light source is now activated. However, since the fluorescence can vary in different planes with respect to the concentration of the medium, it is also necessary to adjust the focus plane in the fluorescence with respect to the focus plane determined in the transmitted light mode in general. However, to determine an exact focus plane, only a small number of pictures have to be taken within a comparatively small search range, due to the pre-focusing previously done in the transmitted light mode. The fluorescence image is finally recorded by means of a digital camera in a precise focal plane and is transmitted to a data processing unit for further diagnosis.

The aim of further developing autofocus systems in fluorescence microscopy has always been: the time required for focusing or the number of pictures taken during focusing and not used for subsequent media testing is minimized. For this purpose, DE10100247a1 discloses another method for automatically focusing a culture medium in fluorescence microscopy, by which the focal point of the objective lens is highly accurate and nevertheless can be determined within a comparatively short time interval. In this case, an interference microscope is described in which the surface of the slide unit is provided with a coating which can be detected by an optical microscope. The reflected light is detected by a detector and the phase in the target region of the interference microscope is deduced from the detection signal. In this way, the interference microscope can finally be calibrated in a suitable manner.

The autofocus or at least partially autofocus microscopes described above are used several times in order to visualize specific proteins with the aid of antibodies. In this way, it is possible to determine in which tissue a specific protein is present and in which compartment of the cell the protein is localized. Fixed tissues, which in many cases consist of tissue sections, are used for this antibody staining. Generating said tissue sections by: first, a frozen section of healthy tissue is made and placed on the surface of a carrier glass, particularly a slide or cover glass. The tissue is then thawed and dried. Typically, the tissue sections are mounted on standard slides in known test systems, wherein a separate standard slide is used for each tissue section.

In addition to the previously described possibility of examining patient specimens with tissue sections arranged on standard slides, the so-called biochip technology of the company Euromeuna (EuroimmunaG) is also known. This technique, unlike the traditional indirect immunofluorescence technique, which always fixes tissue sections on standard slides prepared for examination, offers the possibility of standardizing the determination of autoantibodies and infectious antibodies due to the miniaturization of the specimens to be examined. In this way the work in the laboratory is simplified and made more efficient.

The biochip of the company omon relates to a relatively small carrier with biological material. In this case, the following effects are fully utilized: very small tissue sections can be produced and used for testing, provided that they are placed on a section-carrying medium and are fixed together with this medium on a slide or an analysis plate. Standard cover glass refers to thin, rectangular or circular pieces of glass of about 100 to 200 microns, typically 18 x 18 square millimeters in area, while biochips are pieces of cover glass coated with a suitable biomaterial, so these pieces of cover glass typically have a smaller surface. The biochip is therefore distinguished by a significantly smaller space requirement on the one hand and by a significantly reduced amount of tissue required on the other hand, compared with standard cover glasses.

In the manufacture of biochips, a frozen section of the desired tissue is first likewise placed on a standard cover glass, which is then partially thawed and dried. The coated cover glass is then broken into sections by creating lines of notches in the area of the tissue section with a diamond knife or laser, along which the coated cover glass is broken and thus divided into sections, so that the tissue-bearing cover glass is broken into pieces. In order to be able to carry out better, in particular more efficient, tests, the biochips are placed on suitable reaction zones (reactions-feld) of the slide. In this connection, it is conceivable to provide a plurality of reaction regions for corresponding biochips on one slide, wherein it is also possible to provide more than one biochip, preferably with different tissues, on one reaction region. In order to design the examination of patient samples particularly efficiently, a suitable biochip mosaic is formed by preparing a large number of reaction regions with corresponding biochips on a slide^TM(Biochip-Mosaike^TM)。

For the examination of patient samples, either tissue sections on standard cover glass or biochips or special biochip-mosaics are used^TMIn principle, the procedure for processing the sample, in particular for diluting the patient serum, and for culturing the tissue sections and for visual examination by means of a microscope, is the same.

During the sample treatment, the tissue is cultured in diluted patient serum, which should be checked for the presence of specific antibodies, and in a conjugate (Konjugat) comprising antibodies usually obtained from animals, which are marked with fluorescent material and are directed against the antibodies suspected in the patient serum. As long as the patient serum has an antibody against the antigen of the tissue section, not only this antibody binds to the tissue section but also a fluorescently labeled secondary antibody (Sekundaerantikoerper) binds to the human antibody adhering to the tissue section. The fluorescent dye bound to the corresponding tissue structure can finally be found with the aid of fluorescence microscopy.

Cultured tissue sections are coated with a mounting medium, such as PH buffered (PH-gepuffert) glycerol, and covered with cover glass prior to examination with a microscope. The cover glass is arranged in such a way that it remains at a safe distance from the surface of the tissue section, but can be observed without problems with a microscope.

Disclosure of Invention

As already mentioned, the aim of further developing an automatic device for testing patient samples consists essentially of: all method steps from the input of the sample to the completion of the experimental diagnosis are designed as efficiently as possible and at the same time possible sources of errors are eliminated as far as possible. Starting from the general desire to make the laboratory work more efficient and more reliable, a particular object of the present invention is to further improve the technical solutions known in the prior art for automated microscopy of biological material in such a way that the microscopy of slides with biochips, in particular biochip-mosaics, located thereon can be automated in an advantageous manner. First, it should be possible to achieve the process of focusing the biological material disposed on the biochip in a relatively simple way and at the same time should be done quickly and with high quality. The technical solution to be explained should be easily integrated into known systems for automated microscopy of biological material and assist the personnel working in the laboratory. It is another object of the present invention to minimize the number of pictures taken during focusing and the number of pictures not required for image analysis processing thereafter.

The above object is achieved by an apparatus for automatic inspection of biological material, comprising: a microscope, the biological material being arranged on a cross-stage of the microscope between a slide or analysis plate and at least one cover slip; at least one light source for illuminating the biological material; and a camera unit which takes a picture of at least one piece of biological material magnified by means of the objective of the microscope and transmits the picture to the evaluation unit, wherein a mark is provided which can be recognized by the device for automatic focusing of the biological material and on the basis of the recognition of the mark focusing can be carried out by a targeted movement of the cross table, characterized in that: the biological material is arranged on a biochip made by breaking and dividing a cover glass, which is arranged on the slide, and the markers are located on the surface of the biochip, wherein a target interval within which focusing of the biological material on the biochip is accomplished can be determined on the basis of determining the position and/or orientation of the markers, wherein focusing of the biological material on the biochip is achieved by performing an analytical processing of pictures taken in at least two different planes within the target interval. In addition, the object is also achieved by a method for automated testing of biological material, comprising: microscope, the biological material is arranged on the microscope cross table between the slide or analysis plate and at least one cover plate, in the method, the biological material is illuminated, at least one picture of the biological material is taken by a camera unit, the picture is magnified by the microscope objective and transmitted to an analysis processing unit, and a device for automatic focusing of the biological material is used to detect a mark, the position and/or location of the mark is determined, and the biological material is focused by the targeted movement of the cross table on the basis of the detected position and/or location of the mark, characterized in that: providing the biological material on a biochip made by breaking and dividing a cover glass, the biological material being arranged on the biochip and the markers being detected on the surface of the biochip, wherein a target interval is determined on the basis of the position and/or orientation of the markers, within which target interval the focusing of the biological material on the biochip is done, wherein the focusing of the biological material on the biochip is done by analyzing pictures taken in at least two different planes within the target interval. Advantageous embodiments of the invention are further elucidated in part in the following with reference to the drawing.

The invention relates to a device for the automatic examination of biological material, comprising a microscope, on the cross table of which biological material is arranged between a slide or an analysis plate and at least one cover plate; the device also comprises at least one light source for illuminating the biological material and a camera unit which takes a picture of at least one biological material magnified by means of a microscope objective and transmits the picture to the evaluation unit, wherein a marking is provided which can be recognized by a device for automatic focusing of the biological material and can be focused by a targeted movement of the cross table on the basis of the recognition of the marking, and the improvement is such that the biological material is arranged on a biochip produced by breaking and dividing a cover glass, which is arranged on a slide and the marking is located on the side of the biochip facing the biological material.

With the solution according to the invention it is ensured that the focusing of the biological material arranged on the biochip is performed highly accurately but nevertheless relatively quickly. As will be explained in more detail below, a simple and reliable focusing of the biological material is possible by providing the biochip with markings which are preferably applied to the cover glass already prior to the separation of the actual cover glass during the production of the biochip. The integration of the application mark in the manufacturing process of the cover glass offers here the advantages of: applying the label on a cover glass that has not yet fragmented is significantly simpler than applying the label on a smaller biochip.

Important for an efficient focusing process are: the markers are located on the surface of the biochip, which is relatively flat compared to the surface of the biological material and which enables an accurate distance determination. In this case, it is in principle possible to arrange the marking on the side of the biochip facing the biological material or on the back of the biochip facing away from this side.

The first step before the start of the focusing process is to perform a detection of the mark, in particular to determine the distance between the objective lens and the mark. The target separation is determined on the basis of determining the position and/or orientation of the marks, in particular the distance between the marks and the objective. The target spacing here has a maximum distance and a minimum distance to the objective lens, between which the markers and the sample arranged on the surface of the biochip are located. In a particular embodiment of the invention, the focusing of the biological material within the target interval is continued after the target interval has been determined. Since the search for the focal point is limited to the target interval in this way, the focusing process can be significantly shortened.

The focusing of the marks is preferably performed in the transmissive light mode, which has the additional advantage that: biological materials labeled with fluorescent dyes are irradiated only for a short time in the range of the excitation wavelength and can therefore be resistant in a suitable manner to premature fading of the fluorescent dye.

However, in principle, it is also conceivable to carry out focusing in the fluorescence mode. The focusing method according to the invention can be realized mainly in such a way that the time required for the focusing process is shortened with respect to the known methods. Alternatively or additionally, an auxiliary radiation source, such as a laser, is used for detecting the marking. The radiation of such a used laser is preferably reflected by the marking and received by the receiving device. The distance between the mark and the objective can thus be determined from an evaluation of the optical path and/or the transit time. This is possible because the auxiliary radiation source, in particular the laser, is located in a defined position relative to the objective lens.

Either pattern can be used as a mark to be recognized by the means for focusing. Advantageously, grid lines having an equal distance at least in the direction of extension are suitable. Such grid lines are arranged on the cover glass or on the biochip before the biological material, preferably a frozen section of a tissue, is placed on the biochip. In this case, the markers are designed such that the influence between the markers and the biological material arranged on the biochip can be excluded as much as possible.

A special improvement of the invention is that: the marker as a biochip is provided with at least three contours whose center points are equally spaced. This contour is recognized by the means for focusing, so that the objective of the microscope can be precisely adjusted to the surface of the biochip or to the biological material located there after focusing has been completed. In any case, the marking provided on the biochip according to the invention ensures that the position and/or orientation of the biochip, in particular the distance of the biochip surface from the microscope objective, is reliably recognized. The contours provided on the biochip surface are preferably configured as circles, their centers being equally spaced from each other. In this connection it is conceivable to apply such a label in the desired form on the surface of the biochip by means of suitable devices before or after the cover glass has been broken. However, it is also conceivable to coat the surface at least partially to form a plane and to provide a corresponding template for producing the desired marking or to produce a corresponding marking contour after the plane coating, for example by etching.

In a further embodiment of the invention: the area surrounding the marking is determined in which no pictures are taken by the camera unit and/or the pictures taken are not used or discarded or deleted during processing in the evaluation unit. The arrangement of the respective regions around the markers arranged on the surface of the biochip ensures that tissue regions or cells which may react with the markers are not taken into account when examining biological material or when making a diagnosis of a patient sample.

According to a further development of the invention, it is provided that: the label is configured in the form of a planar coating of the biochip surface. In this case, in an advantageous manner, in particular three regions of the biochip are provided with planar markings. In a very specific embodiment, the entire surface of the biochip provided for labeling is provided with corresponding flat labels. By means of a completely planar or partially planar coating of the surface of the biochip, a corresponding signal is then generated in the distance measuring sensor, likewise on the basis of reflection or transmission on the coating.

The silicon containing mark is advantageously suitable for identification during operation with an automated microscope. Alternatively or additionally, the marking can be provided with a metal, preferably chromium. Dielectric layers whose spectral reflection properties are optimized for the distance measuring sensor and do not adversely affect the image analysis can likewise be considered. If a metal or a dielectric is used for the label on the surface of the biochip, it is contemplated that the metal or dielectric is vapor-deposited or sputtered onto the surface. This has the advantage in the manufacture of biochips labeled accordingly that: the metal vapor diffusion plating is reliably completed and the implementation cost is low. In a special embodiment, a special contour can also be engraved by etching in the area of the surface of the complete vapor deposition.

According to another embodiment of the invention, it is conceivable: the device for automatically focusing biological material has at least one laser light source or a polychromatic radiation source and a detector which receives at least partially light reflected by the marking and generates a signal on the basis of the received light, which signal is evaluated and the cross table is moved in a targeted manner in order to focus the biological material taking into account the evaluated signal. The use of a laser light source and a detector is advantageously suitable for: the surface of the biochip is at least partially coated with a metal such as chromium and the laser beam is reflected in this way. The laser light source of the described detector can be used to determine the position and/or orientation of the surface of the biochip on which the biological material is arranged in a preferred manner.

In addition, the present invention is superior in a method for automatically inspecting biological materials. The method according to the invention comprises a microscope on whose cross stage biological material is arranged between a slide or analysis plate and at least one cover slip, in the method, the biological material is illuminated, in which method at least one image of the biological material is taken by means of a camera unit, enlarged by means of an objective of a microscope, and transferred to an evaluation unit, and in which method the markers are detected, the orientation and/or position of the markers are determined by means of a device for autofocusing the biological material, and the focusing of the biological material is carried out by targeted movement of the cross table on the basis of the detected position and/or position of the markers, so that the method is improved, that is, the biomaterial is provided on a biochip made by breaking and dividing a cover glass, the biochip is placed on a slide, and a mark on the surface of the biochip is detected.

According to an advantageous further development of the method according to the invention, provision is made for: an object space within which the cross table moves during the focusing period is determined on the basis of the detected position and/or position of the markers. By setting the target separation in relation to the detected position and/or orientation of the biochip surface carrying the biological material, a separation comprising areas above and below the biochip surface can be determined in a preferred manner. During the focusing process, the cross table is moved in such a way that the focal plane moves between the interval limits. The displacement of the cross stage during focusing is thus defined in a preferred manner to a suitable value.

In this way, on the basis of the detected position and/or position of the marking, the cross table is moved from at least a first position in which the objective has a first distance to the biological material into a second position in which the objective has a second distance to the biological material, and furthermore at least one image of the biological material is taken in each case in two different planes between the first and second positions and is transmitted to the evaluation unit. In this case it can be easily understood that when at least two pictures in different planes are taken, the pictures have different degrees of sharpness. By evaluating the pictures taken in at least two different planes in the target interval by means of well-known image data evaluation methods, the sharpest picture can finally be determined in a preferred manner. And then determining the focus plane by selecting the clearest picture.

The method described thus allows the focal plane to be determined in an advantageous manner on the basis of a comparison of the images taken in the evaluation unit. The pictures taken and also located within the focus plane are finally transmitted to the device for performing the diagnosis. Preferably, this is a data processing unit, in particular a computer with laboratory software installed thereon. With the aid of the laboratory software, the pictures taken for a particular patient sample are included in the patient data record and stored accordingly. The physician responsible for the diagnosis can allow the entire data relating to the patient sample to be examined to be displayed quickly on the display screen in a comparatively simple manner.

Drawings

The invention will be further elucidated below by means of embodiments with reference to the drawings without restricting the general idea of the invention. In the drawings:

FIG. 1 is a slide with a biochip;

FIG. 2 is a cross-sectional view of the reaction zone of a slide with a biochip;

FIG. 3 is a top view of a biochip with labels;

FIG. 4 is a schematic view of a microscope with autofocus;

FIG. 5 is a front view of a microscope with autofocus;

FIG. 6 is a side view of a microscope with autofocus, an

Fig. 7 is an isometric view of a microscope with autofocus.

Detailed Description

First, FIG. 1 shows a slide 1 on which a biochip 2 is disposed. For this purpose, the carrier plate 1 has ten reaction zones, which are embodied as small recesses in relation to the remaining surface of the carrier plate 1. A biochip 2 is provided on the reaction region 3. In principle, it is conceivable to arrange one or also a plurality of biochips 2 on one reaction zone 3. In this connection, it is of course possible to match the size of the reaction zone 3 in a suitable manner.

The biochip 2 relates to a small carrier with biological material, which is produced by coating a tissue section on a standard cover glass and then fragmenting the cover glass. Standard cover glasses relate to thin, rectangular or round glass plates of about 100 to 200 micrometers, which typically have an area of 18 x 18 square millimeters, while biochips 2 are cover glass fragments coated with a suitable biomaterial, which thus have a much smaller surface. In connection with the respective test properties (Untersucchungsprofil) and the customer requirements, a large number of reaction regions 3 can be provided on one slide 1 for the respective biochip 2. In this case, it is also conceivable to provide more than one biochip with different tissues on a reaction region 3.

Tissue sections which are arranged on the reaction zone 3 of the slide 1 and which are also coated with tissue are cultivated with various liquids, in particular patient specimens, during the examination in the laboratory. After the end of the culture and before examination with a microscope, the cultured tissue sections are coated with glycerol as a sealant which has been subjected to a pH buffer treatment and are covered with a cover glass 4. The cover glass 4 is arranged in such a way that it can be observed with a microscope without any problems, although it has a safe distance to the surface of the tissue section.

Fig. 2 shows a greatly enlarged sectional view of the reaction zone 3 of the slide 1. On the reaction zone 3 of the slide 1 there is a biochip 2 coated with a portion of a tissue section 6. The tissue slices are coated with a sealant 5 and covered by a cover glass 4. The surface of the biochip 2 carrying the tissue section 6 has markings 7 in the form of lines on the south side facing the microscope.

In addition to this, a top view of the biochip 2 arranged on the reaction zone 3 of the slide is shown in FIG. 3. The lines used as the marks 7 are arranged on the surface in groups of several concentric circles. During the microscopic examination, the marking 7 is identified and focused by means of a device for autofocusing the biological material 6 located on the biochip. Optionally, the expansion of the marks 7 in the Z-direction is taken into account during the focusing process in order to achieve an optimization of the focus. The marks provided on the biochip 2 are thus used to probe the focal plane. It is important for the label 7 that it does not react with the biological material 6 located on the biochip 2.

As an alternative to the embodiment of the markers 7 shown in FIG. 3 in the shape of concentric circle groups, it is conceivable to arrange a parallel line or grid pattern on the surface of the biochip 2. The markings 7, which may also be planar, in particular comprise a metal or a dielectric.

In addition, the markings 7 provided according to the invention can be provided either on the upper side of the biochip 2, i.e. on the side facing the tissue 6 lying thereon, or on the back side, i.e. on the surface facing the slide 1. It is always important: the marks 7 can be reliably recognized by means for autofocusing the biological material 6 located on the biochip 2. Furthermore, the expansion of the markers 7 in the Z direction and/or the thickness of the tissue-carrying biochip 2 are taken into account in the evaluation of the focal plane, depending on the embodiment and design of the markers 7.

For the examination of patient specimens, a slide 1 according to fig. 1 is provided, which is equipped with a large number of biochips 2 covered by a cover glass 4. In this case, various tissues 6 or biomaterials are arranged on different biochips 2. The slide 1 with the biochip 2 is positioned and locked on a cross table 9 of a microscope 8. In this connection, it is conceivable to position the slide 1 on the cross table 9 manually or by means of the handling device 13. In particular in a microscope 8 which is operated at least in part in an automated laboratory, the incubated slide 1 with the biochip 2 is stored in a suitable slide magazine 12 and is automatically moved between this slide magazine and the cross table 9 by means of a handling device 13. Suitable actuating devices 13 preferably have gripping devices, which can be moved either relative to the cross table 9 or together with the cross table.

In any case, the slide 1 has a title-or code-shaped marking 14 which enables an accurate identification of the patient sample located on the biochip 2 and preferably also of the tissue type located on the biochip 2.

Fig. 4 shows a schematic view of a fluorescence microscope 8, which comprises a light-transmitting device, a vertically movable cross table 9 and a digital camera 17 for taking pictures. An excitation light device with a dichroic beam splitter 18 and an excitation light source 16 is additionally provided. The spectroscope 18 reflects the excitation light emitted by the excitation light source 16 toward the biomaterial 6 disposed on the biochip 2. In contrast, the transmitted light emitted by the transmission light source 15 and redirected by the turning mirror 23 is transmitted out in the direction of the biochip 2 with the biomaterial 6. Dichroic beam splitter 18 is preferably configured as a reflective filter and reflects all wavelengths less than 510 nanometers. In short, the dichroic beam splitter 18 thus acts as a turning mirror for the excitation light, while the light with the fluorescence wavelength passes through the beam splitter 18 unimpeded. In addition to the dichroic filter or the reflective filter 18 which reflects the excitation light completely, a long-pass blocking filter (langpasssperfilter) 19 is preferably provided which filters out light having a wavelength below 510 nm.

In the embodiment described herein, the fluorescent dye used was fluorescein having an absorption peak of 485 nm and an emission peak of 514 nm. The basic idea of the described technology is that: the digital camera 17, which is arranged behind the long-pass blocking filter 19 in the direction of the light path, must take a picture not only in the fluorescent light but also in the transmitted light. For this reason, the transmission light source 15 is configured as an LED having a wavelength within 520 to 535 nanometers. Light of this wavelength passes not only through the beam splitter 18 but also through the blocking filter 19.

For the focusing process, the transmission light means generate light with a wavelength in the emission wavelength range of the fluorescent dye used by means of the transmission light source 15. In this case, the light emitted by the transmission light source 15 is focused by means of a suitable mirror group 22 and then turned vertically upwards by means of a turning mirror 23 in order to be emitted from the underside through the biochip 2 with the biological material 6 located thereon.

The sample 6 applied to the biochip 2 is, for example, cultured Human epithelial cells to which Anti-nuclear antibodies are bound, and which are stained with Anti-Human antibodies (Anti-Human-antibodies) labeled with fluorescein. Since the absorption peak, i.e., the excitation wavelength of fluorescein, is 485 nm, the dye is not excited to fluorescence by transmitted light. As can also be seen in fig. 4, the transmitted light emitted by the transmission light source 15 in the horizontal direction is first focused by the mirror arrangement 22 and then deflected into the vertical direction by the deflection mirror 23. The transmitted light passes through the biochip 2 with the biological material 6, which is arranged on the cross table 9, is focused by the objective 20 of the microscope 8 and passes unimpeded through the dichroic beam splitter 18 and the long-pass blocking filter 19 in order then to reach the sensor of the digital camera 17. The digital camera 17 produces pictures of the cell walls of the biological material 6 produced with transmitted light with a relatively short exposure time of about 10 milliseconds.

In order to minimize the number of pictures taken in different planes perpendicular to the z-axis, which are required for focusing in the transmitted light mode, the biochip 2 has markings 7 on its surface and the microscope 8 has means for identifying the markings. Before the start of the focusing process in the transmissive light mode, the distance of the mark 7 to the objective lens 20 and/or its position relative to the objective lens 20 is now first evaluated. For this purpose, a light source or radiation source is provided which emits light which is finally reflected by the marking 7 and detected by a suitable sensor. The distance of the marking 7 from the objective 20 and/or its position relative to the objective 20 is determined taking into account the transit time and/or the optical path. Optionally, the thickness of the mark 7, i.e. the extension of the mark in the z-direction, is also taken into account in the above-described distance and/or position estimation. Taking into account the measured distance of the marking 7 from the objective, the cross table 9 is moved by means of the motor 24 in the z direction in such a way that the focusing in the transmission light mode is carried out only in a defined target region. Within this target area with a distance interval of a few micrometers (abstinds-interval) in the z-direction, a small number, preferably three, of pictures in different planes are taken in the transmission light mode. The respective sharpness value of each individual picture is subsequently or at least partially simultaneously evaluated by a connected data processing device (not shown) by means of the known adjacent pixel gray-scale variance method (SMD). The picture with the largest value is identified as the sharpest picture and the appurtenant (in the z direction) vertical position of the cross table is determined as the focus plane.

When evaluating the focusing surface, it is preferable to disregard the region of the picture reflecting the vicinity of the label irrespective of the kind and embodiment of the label on the biochip. This is done to ensure that the parts of the biological material 6 located on the biochip 2 that may be affected by the label are not taken into account in the analysis process.

After the transmission light source 15 is turned off, the cross table 9 is moved to the focal plane measured in the transmission light. To generate the fluorescence image, the excitation light source 16, which is in the form of an LED, is then switched on. The emitted light is focused by means of a suitable lens group 21 and impinges on the already described dichroic beam splitter 18, which reflects the excitation light downwards and in this way leads through the objective lens 20 onto the biological material 6 on the biochip 2. Where the excitation light impinges on a fluorescent dye which, as a result of the excitation, emits diffuse light having a dominant wavelength of 514 nm. For the purpose of being photographed by the digital camera 17, a small portion of the fluorescent radiation emerges vertically upwards, passing through the objective 20 and through the dichroic beam splitter 18 and the long-pass blocking filter 19.

Due to the long exposure time of about 500 milliseconds, the camera 17 generates a fluorescence image. Since the position of the fluorescence varies within the height of the biological material 6, the focal plane within the fluorescence has a deviation from the focal plane found in the transmitted light. To determine the exact focal plane, several fluorescence images are now also taken in a search area only a few microns large. As in the case of transmitted light, the cross table position is here moved in the vertical direction (z direction) in an electrically driven manner for each picture. The clearest fluorescence image is measured by means of the adjacent pixel grayscale variance method (SMD).

The area within which the focal plane must be evaluated in the fluorescence is comparatively small because of the focusing in the transmitted light which is carried out first. In this way the time during which the fluorescent dye is excited for emission of radiation and is therefore at least partially used can be minimized. Furthermore, the exposure time in transmitted light is significantly shorter than in fluorescence. In order to achieve a further reduction in the time required for autofocusing, it is ensured by means of the markings 7 provided on the biochip that only a few, preferably 3, pictures in the transmitted light have to be taken. In the case where no corresponding marker is provided, about 100 pictures in transmitted light are usually taken during focusing. The duration of the transmitted light focusing is shortened from 1 second, 100 × 10 ms, to 30 ms, 3 × 10 ms, taking into account the corresponding exposure time.

If the entire autofocus process is carried out in fluorescent light, the duration of the focusing even rises to about 200 × 500 ms to 100 s. Even in the case where the calculation of the image Sharpness (SMD) can be performed simultaneously with the image capturing because the exposure time in the fluorescence is long and thus the image capturing can be interrupted immediately once the clearest picture is obtained, an accumulated exposure time of about 50 seconds is required on average.

Fig. 5, 6 and 7 each show a fluorescence microscope 8 for the automatic examination of a biological sample 6. Like components are still provided with like reference numerals. The microscope 8 shown has a device for autofocusing the biological material 6, which is designed such that it recognizes the markings 7 provided on the biochips 2 which are provided on the slide 1 and on the basis of this recognition determines the target region in the z direction within which the focal plane is located. In order to determine the focus plane precisely, only a few more image shots are required, so that the autofocus can be accelerated considerably compared to the known systems.

The fluorescence microscope 8 has a receptacle, to which a slide magazine 12 for storing and supplying a plurality of slides 1 can be attached. The handling device 13, which is designed as a gripping device fixed to the cross table 9, can be used to remove the slides 1 required for the examination from the slide magazine 12 in a targeted manner and then place them again in this slide magazine. Both the slide magazine 12 and the individual slides 1 have a label-like or code-like marking in order to ensure unambiguous identification. The preparation, processing and testing of the samples is controlled by means of laboratory software, by which the test results are also stored and output.

In order to prepare the microscopically examining process, the so-called carrier 25, in which the desired slide 1 is fixed, is removed from the slide magazine 12 and is locked in the desired position on the cross table 9 by means of the handling device 13.

Fig. 8 exemplarily shows the configuration of the bracket 25. The illustrated carrier 25 is frame-shaped and has five receptacles for the slides 1. The slide 1 with the biochips 2 arranged thereon, which are arranged on ten reaction zones 3, respectively, is securely held by the holder 25 and can in this way be reliably transported and transported.

After the removal of the carrier 25 from the slide magazine 12 by the handling device 12 in the form of a gripper device, the positioning is carried out in such a way that the biochip 2 provided for inspection is finally located below the objective 10 of the microscope. A digital camera 17 is positioned above the objective lens 20, by means of which the desired picture is taken. The arrangement of the transmission light source and the excitation light sources 15, 16 and the remaining optical components corresponds to the arrangement described in connection with the description of fig. 4.

The already described autofocus process, which includes the detection of the markings 7 on the biochip and the taking of pictures in different planes, is first carried out in the central region of the biochip for each examination. After this, focusing and the image recording associated therewith are also continued in two further regions of the biochip 2, which are located to the left or right of the center. For this purpose, the cross table 9 is correspondingly moved in the horizontal direction.

It is always important: the marks 7 on the biochip 2 are first detected during focusing in order to thus define in a rational manner the horizontal area within which the focus plane is expected to lie and to optimize the autofocus process in terms of the time required.

List of reference numerals

1 slide glass

2 biochip

3 reaction zone

4 cover glass

5 sealing agent

6 biological Material

7 labelling

8 microscope

9 Cross workstation

10 objective lens

11 device for automatic focusing

12 slide box

13 operating device

14 symbol

15 transmissive light source

16 excitation light source

17 digital camera

18 dichroic beam splitter

19 long pass blocking filter

20 objective lens

21 group of excitation light sources

22 lens group of transmission light source

23 steering mirror

24 motor

25 bracket

Claims (20)

1. Apparatus for the automatic inspection of biological material (6), comprising: a microscope (8), on the cross table (9) of which the biological material (6) is arranged between the slide (1) or the analysis plate and at least one cover slip (4); at least one light source (15, 16) for illuminating the biological material (6); and a camera unit (17) which takes a picture of at least one piece of biological material (6) magnified by means of an objective (10) of the microscope (8) and transmits the picture to an evaluation unit, wherein a marker (7) is provided which can be recognized by a device (11) for automatically focusing the biological material (6) and which can be focused by a targeted movement of the cross table (9) on the basis of the recognition of the marker (7), characterized in that: the biological material (6) is arranged on a biochip (2) produced by breaking and dividing a cover glass, which is arranged on the slide (1), and the markings (7) are located on the surface of the biochip (2), wherein a target interval can be determined on the basis of the position and/or orientation of the markings, within which target interval the focusing of the biological material on the biochip is carried out, wherein the focusing of the biological material on the biochip is carried out by evaluation of pictures taken in at least two different planes within the target interval.

2. The apparatus of claim 1, wherein: the markers (7) of the biochip (2) are configured as a grid line pattern.

3. The apparatus of claim 1, wherein: the markers (7) of the biochip (2) have at least three contours, the centre points of which are equally spaced.

4. The apparatus of claim 3, wherein: the contour is configured as a circle.

5. The apparatus of any of claims 1 to 4, wherein: a region surrounding the marking (7) is determined, in which no pictures are taken and/or in which the pictures taken are not used during processing in the evaluation unit.

6. The apparatus of claim 1, wherein: the marking (7) covers the biochip (2) at least partially in a ground plane.

7. The apparatus of claim 1 or 6, wherein: the marking (7) covers the side of the biochip (2) facing the biological material (6) in a planar manner.

8. The apparatus of any of claims 1 to 4, wherein: the marker (7) has silicon.

9. The apparatus of any of claims 1 to 4, wherein: the marker (7) has a metal.

10. The apparatus of any of claims 1 to 4, wherein: the label (7) has a dielectric.

11. The apparatus of any of claims 1 to 4, wherein: the marker (7) has chromium.

12. The apparatus of any of claims 1 to 4, wherein: the markers (7) are vapor-deposited and/or sputtered onto the biochip (2).

13. The apparatus of any of claims 1 to 4, wherein: the device (11) for automatically focusing the biological material (6) has at least one laser radiation source or polychromatic radiation source and a detector which at least partially receives the radiation reflected by the marking (7) and generates a signal on the basis of the received radiation, which signal is evaluated and a targeted movement of the cross table (9) is carried out for focusing the biological material (6) taking into account the evaluated signal.

14. Method for the automatic inspection of biological material (6), comprising: a microscope (8), on the cross-stage (9) of which the biological material (6) is arranged between the slide (1) or the analysis plate and at least one cover slip (4), in the method, the biological material (6) is illuminated, at least one image of the biological material (6) is taken by means of an imaging unit (17), said image being magnified by means of an objective (10) of the microscope (8) and transmitted to an evaluation unit, and detecting the markers (7) by means of a device (11) for automatically focusing the biological material (6), determining the orientation and/or position of the markers (7) and focusing the biological material (6) by means of a targeted movement of the cross table on the basis of the detected orientation and/or position of the markers (7), characterized in that: -providing the biological material (6) on a biochip (2) made by breaking and dividing a cover glass, on which the biological material (6) is arranged, and-detecting the markers (7) on the surface of the biochip (2), wherein-on the basis of determining the position and/or orientation of the markers-a target space is determined, within which focusing of the biological material on the biochip is done, wherein-the focusing of the biological material on the biochip is done by analyzing pictures taken in at least two different planes within the target space, respectively.

15. The method of claim 14, wherein: on the basis of the detected position and/or position of the marker (7), a target region is determined in which the cross table (9) is moved during focusing.

16. The method of claim 14 or 15, wherein: on the basis of the detected position and/or position of the marking (7), the cross table (9) is moved at least from a first position in which the objective (10) has a first distance to the biological material (6) into a second position in which the objective (10) has a second distance to the biological material (6), and at least one picture of the biological material (6) is taken in two different planes between the first and second positions and is transferred to the evaluation unit.

17. The method of claim 14 or 15, wherein: light of different wavelengths is used for detecting the marker (7) and for illuminating the biological material (6).

18. The method of claim 15, wherein: during focusing and during the period when the cross table (9) is within the target area, the biological material (6) is illuminated and at least one picture of the biological material (6) is taken in two different planes inside the target area and transferred to the analysis processing unit.

19. The method of claim 14 or 15, wherein: determining a focus plane on the basis of comparing the captured pictures in the analysis processing unit.

20. The method of claim 19, wherein: the picture in the focal plane is transferred to a means for performing a diagnosis.