Microscopy techniques for the detection of biological target objects

Abstract

Microscopy method for detecting target objects (32) that have a predetermined optical property in material to be examined (6), in which - an overview field of view (36) of a first objective (10) of a microscope optic (14) is directed in a first step onto an overview area of ​​a sample carrier (4) with the material (6) to be examined, - the material to be examined (6) is illuminated from an illumination unit (16) which shines onto the sample carrier (4) from outside an image field tube (48) and is recorded by a camera (8), - the material (6) to be examined is optically examined for the optical property in such a way that even a single target object (32) with the predetermined optical property is recognized as such in the material (6) to be examined, - in a subsequent second step, a target field of view (52) of a second objective (12) of the microscope optics (14) is aligned to a target area around the target object (32) using the known position of the target object (32), - the detected target object (32) is examined in a differentiated manner according to various further optical properties.

Description

[0001] The invention relates to a microscopy method for detecting target objects, in particular biological target objects, which have a predetermined optical property, in material to be examined.

[0002] In medical diagnostics, the detection of individual cells exhibiting a specific characteristic within an environment of extremely large numbers of similar cells is becoming increasingly important. Cancer diagnostics is one example where single-cell detection plays a crucial role. In a blood test for cancer detection, several milliliters of blood are examined to determine whether one or more cancer cells are present in the sample. In the most extreme case, this means identifying a single cancer cell within an environment of approximately 10 8 to recognize "normal" blood cells.

[0003] To identify individual cells, they are typically labeled. For this purpose, one or more markers are attached to each cell, tailored to its specific properties and indicating those properties. Fluorescent markers are commonly used; when irradiated with light of a specific excitation wavelength, these markers emit light at a specific emission wavelength, allowing for unambiguous identification.

[0004] Several different methods exist for detecting individual labeled cells in a blood sample. In digital microscopy, the blood is spread out on a sample carrier and examined microscopically. The appropriately labeled cells are detected based on their specific radiation characteristics and then microscopically examined for further properties, such as multiple different markers. This method has the advantage that the cells in the blood sample can be reliably and individually identified and further analyzed. However, a disadvantage is the duration of such an examination. For the examination of a sample containing approximately 10⁸ cells, where a single labeled cell is to be reliably detected, several days of microscopic examination are typically required.

[0005] A significantly faster method is flow cytometry. In this method, the blood sample flows through a very thin capillary illuminated with laser light. Labeled cells flowing past are identified and counted. Analysis rates of up to 150,000 cells per second are possible, meaning that the examination of approximately 108 cells takes no longer than 20 minutes. However, a disadvantage of this method is that the analyzed cells cannot be examined in greater detail; they are essentially "lost" in a lower collection container.

[0006] From DE 203 21 352 U1, a microscopy system for visualizing indocyanine green is known, wherein a first beam path of a microscopy optic serves to obtain fluorescence images of an object region. A second beam path serves to obtain images of the object in the visible region of the spectrum. A display system presents the fluorescence images and the visible light images in superimposition. In addition, depth profile data of at least one identified contiguous analysis region of the object field can be obtained.

[0007] WO 2006 / 133 899 A2 relates to a fluorescence analysis microscope with which a sample area can be uniformly illuminated by at least three illumination units arranged on an illumination carrier.

[0008] It is an object of the present invention to provide a microscopy method for detecting cells, with which individual cells of a predetermined optical property can be found from a relatively large quantity of material to be examined and subsequently examined in more detail, wherein a rapidly executable method is sought.

[0009] This problem is solved by a microscopy method of the type mentioned above, wherein according to the invention - an overview field of view of a first objective of a microscope optic is directed in a first step onto an overview area of ​​a sample carrier with the material to be examined, - the material to be examined is illuminated from an illumination unit that shines onto the sample from outside an image field tube, and is recorded by a camera, - the material to be examined is optically examined for the optical property in such a way that even a single target object with the predetermined optical property is recognized as such in the material to be examined, - in a subsequent second step, a target field of view of a second objective of the microscope optics is aligned to a target area around the target object using the known position of the target object, - the detected target object is examined according to various further optical properties and in particular - the target object is classified into at least one of several target object classes.

[0010] The material to be investigated is expediently biological material. The target objects are expediently biological target objects. Accordingly, the following refers to biological material and biological target objects, without limiting the method according to the invention to this.

[0011] This method combines the advantages of classical flow cytometry with digital microscopy. Flow cytometry allows for high-speed analysis because the flowing sample is intensely illuminated per unit area by the laser radiation. This enables the method to operate very quickly. By transferring this principle of intense illumination to digital microscopy, the process can be accelerated even further.

[0012] However, a rapid examination combined with a possible subsequent in-depth examination is only possible if the digital microscopy procedure is divided into a two-step process, with strong illumination being used in the first step. The two-step process consists of quickly examining the entire sample area to be investigated in the first step using a relatively low microscopic magnification.

[0013] Since rapid detection of individual cells in a relatively large overview field of view is only possible with strong illumination, a powerful light source is expediently used in the first step. By using a light source that shines onto the sample from outside the image tube, a high illumination power per unit area can be focused onto the overview field of view, which, although relatively large from a microscopic perspective, is still small from a macroscopic perspective.

[0014] The invention further proceeds from the consideration that cell diagnostics known from conventional digital microscopy should be distributed across two steps to enable a detailed examination. Thus, the first step focuses on recognizing the target object as such within the mass of biological material. Further diagnostic examination is not required in the first step. This is only carried out in the second step, allowing the identified target object to be differentiated and classified.

[0015] By dividing the detection and further diagnostics into two steps, the illumination in each step can be optimized for the specific tasks of those steps. For example, in the first step, illumination optimized for cell detection can be selected. This might involve monochromatic illumination at a frequency particularly suitable for cell detection, but which would make further analysis difficult or impossible. In the second step, illumination suitable for differentiation and classification can be used, although initial detection might be difficult or very time-consuming with this type of illumination.Depending on the application, it may also be possible to use the same lighting or the same light source - possibly with different radiant powers or radiant frequencies - in both steps.

[0016] The method according to the invention is particularly advantageous for the detection of individual and rare cells, in the so-called rare cell detection method. In this diagnostically common method, individual and rarely occurring cells are examined within a large population of "normal" cells. Examples include the quantification of tumor cells in the blood or bone marrow, prenatal diagnostics, tumor surveillance, and therapy monitoring. Another advantageous application is the detection of free tumor cells in the circulatory system, the so-called circulating tumor cell detection (CTC). Individual tumor cells that have detached from the primary tumor and found their way into the circulatory system, either directly or via lymphatic vessels, pose a significant risk of metastasis. To reliably detect this risk and, ideally, even potential sites for such metastases, these individual tumor cells are sought in the circulatory system.Another application example is the detection of settled tumor cells, the so-called Disseminated Tumor Cell (DTC) diagnostic procedure. Tissue is examined, for example, liver tissue, bone marrow, or lung tissue, in which an accumulation of previously free tumor cells is suspected that has settled at the corresponding tissue site and has begun or is promoting metastasis.

[0017] However, other fields, particularly in medicine, are also conceivable and advantageous, in which the inventive method can demonstrate significant benefits. In general, in clinical diagnostics, the field of blood tests can benefit from the inventive method, for example, the testing for infections or blood disorders. In pharmaceutical research, the method can be applied to drug screening, toxicology, or efficacy studies of active ingredients. Further fields of application include, for example, neurobiology, where the method can be used for cell differentiation, monitoring cell growth, or ion influx. The biological targets could be cells. Advantageously, the method can also be applied to other organisms. Thus, the targets can also be cell clusters, bacteria, colonies, or other organisms.Other biological targets from medical diagnostics, such as so-called nanocodes, are also conceivable and advantageous. These biological targets can also be predominantly non-biological. For example, nanocodes might only carry biological material on a small area, with the majority being the non-biological coding component. It is also possible that the biological targets initially contain no biological material and only acquire it in a second step. The same applies to the biological material surrounding the biological targets.

[0018] The microscopy procedure is suitably a medical microscopy diagnostic method for identifying individual diagnostic objects. Accordingly, the sample carrier is suitably a glass slide, as used in medical diagnostics. A so-called microplate is particularly advantageous, as it has a multitude of separate vessel sections that can be examined individually. A suitable example of such a microplate is the so-called microtiter plate in the format 128 mm × 86 mm. Plates in credit card format (85 mm × 54 mm) are also commonly used in medical diagnostics. The overview field of view can extend over the entire sample area, i.e., a microplate, although sub-areas can be scanned sequentially.

[0019] Advantageously, a microscope comprising microscope optics and an image tube is used to carry out the procedure. The microscope optics expediently include several objectives to enable different magnifications. The image tube is expediently a tube arranged between such an objective and the camera, through which the beam path imaging the sample is directed towards the camera. It serves to improve the optical properties of the microscope, for example, by enabling long focal lengths and suppressing scattered radiation. The image tube expediently has a length of at least 10 cm and at least one aperture arranged within the tube to suppress scattered radiation.

[0020] The overview field of view is projected onto an image area of ​​the camera by the microscope optics. The corresponding imaging beam path runs through the image tube, so that the optical axis of the beam path also passes through the image tube. Apart from the fact that the first light source is located outside the image tube, the path of the illumination beams also advantageously runs entirely outside the image tube, i.e., not even through the objective lens. The path of the illumination beams from the light source to the specimen support advantageously runs entirely outside the image tube and the microscope specimen. Incident light at an angle to the specimen surface, preferably as reflected light, is advantageous. This arrangement of the light source and the illumination beam path allows for very strong reflected light illumination, while keeping scattered radiation within the microscope to a very low level.This allows a fine emission of a marker from the biological target object to be clearly distinguished from the scattered radiation, despite strong illumination, which is advantageous for a rapid execution of the procedure.

[0021] In the second step, the target field of view of the microscope optics, preferably a microscope objective, is aligned with the target area around the target object. The target field of view can have a higher magnification than the overview field of view. When aligning the microscope object with the target field of view, positional information of the target object from the first step is processed. In the first step, the position of a detected target object was conveniently recorded and stored, preferably as two-dimensional spatial information. Using this known position, the target area is now precisely controlled by the microscope optics, advantageously automatically and with a motor drive.

[0022] After aligning the optics with the target field of view, the target object can now be examined in greater detail, for example, at a higher magnification than was possible in the first step. In the second step, the target object is examined for further and different optical properties, which can be generated, for example, by differentiated marking. However, shape analyses of the target object are also possible, particularly when examining nanocodes, and morphological analyses are also feasible.

[0023] These investigations are also advantageously carried out automatically, with the categorization of different classes being useful. For example, the optical properties are advantageously machine-readable and machine-classifiable, so that the detected biological target can be automatically sorted into one of several target classes.

[0024] The optical properties can be inherent in the target object, for example, as morphological properties. However, the artificial generation of optical properties, for example, by labeling such as fluorescent labeling, is particularly advantageous. Artificial generation of an optical property, e.g., labeling of the biological target objects, can occur before, during, or after the first step. The simplest method is to generate artificial optical properties before the start of the inventive process. However, it is also possible to generate or modify an optical property, for example, between steps, to enable more detailed investigations in the subsequent step. Generating or modifying an optical property during an investigation, particularly during the second step, is also possible and may be advantageous.Thus, markings can be changed or added – particularly according to requirements, i.e., expediently depending on previously determined test results. Especially in morphological investigations, the optical properties can change or develop, for example, through the administration of an active ingredient.

[0025] The labels can be fluorescent labels, luminescent labels, silver labels or colorimetric labels, for example for genes, nucleic acids, proteins in cells, cell bodies or receptors or the like.

[0026] It is also possible to use a combination of different markings for the two steps.

[0027] The examination of a target object can be carried out in a second step using a different magnification and / or a different optical method. For example, the target field of view can be magnified by at least a factor of 3, and in particular by a factor of 5, compared to the overview field of view, thus facilitating a more precise examination of the previously located target object.

[0028] If the target object is detected in the first step using a first optical method and the target object is differentiated in the second step using a second optical method different from the first, then the two methods can be optimally adapted to the different tasks of detection and differentiation.

[0029] In a further advantageous embodiment of the invention, the material to be examined is illuminated in the second step by a second, different light source. This light source can be optimized for the examination in the second step, so that the two different light sources are adapted to different tasks in the two steps. It is advantageous if the second light source enables differentiation of the target objects according to various other optical properties by illumination with different frequencies. In this way, for example, different markers can be detected, and a multitude of different properties can be distinguished through possible marker permutations.

[0030] It is particularly advantageous if the material is illuminated monochromatically from the first light source in the first step.

[0031] Monochrome light refers to a spectrum with a wavelength bandwidth of less than 50 nm, particularly less than 30 nm. This allows for high radiant power per unit area and frequency range, enabling rapid detection of individual cells. Since differentiation and classification are not required in the first step, this approach is advantageous for locating cells using specialized light.

[0032] In contrast, it is advantageous if the biological material is illuminated polychromatically from a second light source in the second step. This allows for simple differentiation, for example by excitation at different frequencies, particularly with fluorescent labels, and makes the process easy to implement. The second light source can have a continuous spectrum or comprise several discrete frequency ranges separated by essentially radiation-free frequency gaps.

[0033] An LED light source is particularly suitable as the primary spotlight. This allows for the simple and bright generation of monochromatic light, especially with relatively low heat loss.

[0034] In the first step, it is advantageous to illuminate a large area of ​​the sample carrier or sample region so intensely that rapid detection of a target object is possible even at relatively low microscopic magnification. However, a high applied radiation power per unit area has the disadvantage of also increasing the power of scattered radiation, thus making detection more difficult. Since reliable target object detection depends on the ratio of object radiation to scattered radiation, for example, twice the radiation power, which is associated with twice the amount of scattered radiation reaching the camera, is not helpful.

[0035] The invention, however, is based on the consideration that, to accelerate the process, it is advantageous to select the highest possible illumination power, even if this results in poor collimation of the illumination radiation before focusing it onto the sample area. However, it is essential to prevent a significant portion of the resulting strong scatter radiation from entering the camera. For example, an arrangement of intermediate apertures, preferably several, within the image field tube can significantly reduce the proportion of scatter radiation. This can, at least to a considerable extent, compensate for the disadvantage of poor collimation.

[0036] High radiant power can be achieved using area emitters, e.g., LEDs. Accordingly, in a further advantageous embodiment, the invention proposes that the second light source comprises an area emitter. This allows for a particularly high excitation radiant power to be applied to the sample.

[0037] To further increase performance, it is advantageous to minimize the optical path between the light source and the sample. This is particularly important for area emitters, such as LEDs, which are difficult to collimate. Ideally, the distance between the light source, e.g., an LED, and the sample holder should be less than 100 mm, and preferably less than 50 mm.

[0038] In the second step, the primary focus is on examining the target object in detail, so the high radiation power from the first step is not strictly necessary. A low scatter radiation rate is advantageous for precise and differentiated diagnostics. Accordingly, the invention proposes that the radiation from the second light source be collimated by the image field tube and, in particular, guided coaxially to the optical axis. This allows a good ratio of sample radiation to scatter radiation to be achieved with sufficient radiation power.

[0039] Furthermore, it is advantageous if the power emitted onto the sample from the first light source per nm frequency range is at least three times higher, and in particular at least ten times higher, than the corresponding radiant power from the second light source. In this way, the entire overview field of view can be illuminated so intensely that the radiant power per area is sufficient to generate a sufficiently strong backscatter from a single target object to enable its detection even at low microscope resolution.

[0040] In a further advantageous embodiment of the invention, all biological target objects to be detected exhibit a general marker as a predetermined optical property, and the biological material is examined in the first step only for this general marker. In this way, the detection of individual cells within a large quantity of other biological material can be easily achieved. In particular, cell detection is possible at a ratio of 10 7 , in particular 10 8 , unmarked cells per marked cell quickly possible.

[0041] Furthermore, it is advantageous if the biological target objects also exhibit a classification mark as an additional optical property, and if the biological target objects are examined for this mark in a second step. This enables a simple, differentiated examination and classification.

[0042] Advantageously, the biological target is first examined for its coordinates on the sample carrier, and only in the second step, at higher optical magnification, are target-specific properties investigated. The coordinates can be two-dimensional, lying on a plane, particularly perpendicular to the optical axis of the imaging radiation emitted by the sample. However, it is also possible to incorporate the third dimension into the determined coordinates using special autofocus techniques. Typically, though, the third dimension is only captured in the second step, for example, through autofocus methods.

[0043] Advantageously, the target-specific properties are color and / or shape properties of the target object. Fluorescence and / or luminescence markings, color markings, nanocoding, or morphological properties are particularly suitable.

[0044] Especially with a large volume sample, for example 7.5 ml of blood with approximately 5 × 10 7 For blood cells, it is advantageous to first perform a raster scan of a sample area larger than the overview field of view of the microscope optics. Accordingly, it is proposed that, in the first step, a sample area of ​​the sample carrier be sequentially scanned face by face, the position of the detected target object within the overview area be determined, and in the second step, the detected target objects be individually approached and examined microscopically. The overview area on the sample surface can correspond in size to the overview field of view of the microscope optics.

[0045] The invention further proposes that both the first and second illumination units illuminate the material under investigation exclusively from one side. This preserves access to the sample from the other side.

[0046] Advantageously, both illumination units illuminate the sample or sample carrier from below, while the sample remains accessible from above. This allows for easy manipulation of the material under investigation. For example, a robot can add or remove material to be examined, or another substance such as an active ingredient, from the sample carrier during the microscopy process.

[0047] The invention also relates to a microscope device for classifying labeled cells in biological material, in particular for autonomous, automated classification. The microscope device can be connected to a sample carrier and have microscope optics aligned with the sample carrier. Furthermore, the microscope device includes a first illumination unit with a first light source comprising several illumination sources arranged outside an image field tube and in a ring around the image field tube, conically aligned with the sample carrier. In this way, the two-step procedure described above can be carried out particularly quickly and reliably.

[0048] An advantageous embodiment of the invention proposes that the microscope apparatus includes a second illumination unit for illuminating the sample carrier coaxially to the optical surface through the image field tube. This second illumination unit need not have its own light source, as the supply of radiation from an external light source can also suffice. In such an externally illuminated case, the illumination unit consists of corresponding light guiding elements for directing radiation from the external light source onto the sample carrier.

[0049] It is further proposed that the first light source comprises several illumination sources arranged in a ring around a microscope objective. This enables a conical radiation path from the illumination sources to the sample carrier or the biological material it supports, thereby allowing for a high radiant power per unit area.

[0050] To achieve the highest possible radiant power from the first illumination unit onto the sample carrier or the biological material on it, it is advantageous for each illumination source to have an output optic, with the output optics arranged adjacent to one another in a ring-like fashion around the image field tube. This allows for optimal radiant output from the illumination sources onto the sample carrier or the biological material.

[0051] It is further proposed that the microscope apparatus comprises a first filter unit with a first filter medium arranged between the illumination sources and the sample holder. Such a first filter medium can be an excitation filter that limits the radiation incident on the sample to a filter band that does not exceed a width of 50 nm, and more specifically, 25 nm. Furthermore, a second filter medium is advantageously arranged in the optical beam path between an objective of the microscope optics and the camera. Compared to the first filter medium, this second filter medium is advantageously designed to be transmissive only in a second spectral range, which lies outside the spectral range in which the first filter medium is transmissive. This measure also significantly reduces the scattered radiation incident on the camera.

[0052] To further reduce scattered radiation, it is advantageous to arrange several intermediate apertures in the image field tube, through which at least 90% of the scattered radiation from the illumination sources of the first illumination unit that passes through the lens is absorbed.

[0053] The preceding description of advantageous embodiments of the invention contains numerous features, some of which are summarized in the individual dependent claims. However, it is advantageous for those skilled in the art to consider these features individually and combine them into meaningful further combinations. In particular, these features can each be combined individually and in any suitable combination with the method and apparatus according to the independent claims.

[0054] The properties, features, and advantages of this invention described above, as well as the manner in which they are achieved, will become clearer and more readily understandable in connection with the following description of the exemplary embodiments, which are explained in more detail in conjunction with the drawings. The exemplary embodiments serve to illustrate the invention and do not limit it to the combination of features specified therein, including functional features. Furthermore, suitable features of each exemplary embodiment can also be considered explicitly in isolation, removed from one exemplary embodiment, incorporated into another exemplary embodiment to supplement it, and / or combined with an independent claim.

[0055] They show: Fig. 1 A schematic drawing of a fluorescence microscope with two objectives and two illumination units during a coarse scan, Fig. 2 a top view of a sample carrier with many sample containers, Fig. 3 a schematic representation of the second illumination unit arranged around a microscope objective, Fig. 4 a light source of the second lighting unit, Fig. 5 a beam path of scattered radiation emitted by a sample and largely absorbed in an image field tube, Fig. 6. The fluorescence microscope during a detailed examination, Fig. 7. A target field of view around one of the biological target objects under four different illuminations. Fig. 8 a target field of view directed at a bacterium at five different time points and Fig. 9 an overview field of view directed at a multitude of nanocodes.

[0056] Fig. Figure 1 shows a microscope apparatus 2 with a sample holder 4 on which biological material 6 is arranged. The microscope apparatus 2 comprises a camera 8 and two objectives 10, 12 with magnifications of 2.5x and 20x, which together form a microscope optic 14. The microscope apparatus 2 further comprises a first illumination unit 16, a second illumination unit 18 with a spectral filter 20, a beam splitter 22, and a spectral filter unit 24 with several spectral filters 26. The first illumination unit 16 comprises several light sources 28, each with a spectral filter 30.

[0057] A microscopy method for detecting biological target objects 32 in biological material 6 is based on the Fig. 1 to 7 are explained below.

[0058] First, the sample carrier 4 is placed in front of the first objective 10 of the microscope device 2 for examination. The sample carrier 4 is a microplate with, for example, 8 × 12 vessels 34, each containing a predetermined amount of body fluid. For the examination for tumor cells, for example, 1.5 ml of blood is distributed among the 96 vessels 26, so that 5 microplates provide a representative sample of 7.5 ml of blood.

[0059] The biological material 6 contains approximately 10 within the sample carrier 4. 7Cells, within which individual tumor cells may be present. The aim of the procedure is to determine whether tumor cells are present in the biological material and, if so, to classify them, differentiating between several categories or classes. The tumor cells thus constitute biological target objects 32 that can be identified. The biological material 6 is bound in a layer at the base of the vessels 34, whereby clusters and possibly smaller overlaps of cells are possible. The cells are thus held in a fixed position within the vessels 34 and do not change their position within the vessels 34 during the examination.

[0060] To locate and differentiate the tumor cells, the biological material 6 underwent a labeling procedure so that cells with specific receptors were labeled with corresponding markers, e.g., fluorescent markers. One of the markers is a general marker that adheres to all target objects 32, i.e., tumor cells in this embodiment. The other markers are differentiation markers assigned to target object classes, so that all target objects of a class carry one of the differentiation markers. The general marker has an excitation frequency of 488 nm and an emission frequency of 510 nm. The differentiation markers have excitation frequencies of 375 nm, 405 nm, 550 nm, and 649 nm, respectively.

[0061] In the first step of the example procedure, an overview field of view 36 of the first lens 10 is directed onto the biological material 6 on the sample carrier 4, so that an overview area of ​​the sample carrier 4 or of the biological material 6 is imaged in the camera 8. The in Fig. The indicated overview field of view 36 is chosen to be large enough that the contents of a vessel 34 lie completely within the overview field of view 36, so that the biological material 6 contained therein is completely imaged in the camera 8. On the other hand, the overview field of view 36 is chosen in its size and position such that the upper edge of the rim of the vessel 34 does not lie within it and is therefore not imaged.

[0062] The overview field of view 36 is now illuminated by the first illumination unit 16. For this purpose, the first illumination unit 16 contains a plurality of illumination sources 28, in this embodiment ten, which are arranged in a circle around the lens 10.

[0063] A top view of the lens 10 and the first illumination unit 16 is shown in Fig. 3 schematically represented. A representation of a light source 28 is shown in Fig. Figure 4 is shown. Each illumination source 28 comprises a light source 38, in this embodiment one or more LED emitters, a collimation optic 40, e.g. a collimation lens, an emission filter 42, which may be a spectral filter with a single transmission band, expediently having a width σ < 50 nm, and an output optic 44, which in this embodiment is a refocusing optic consisting of two lenses. The illumination sources 28 are positioned around the objective lens 10 such that their output optics 44 form a substantially uninterrupted ring around the objective lens, with the frames for the output optics 44 and a small amount of play between them being negligible.

[0064] The light sources 28 each shine obliquely onto the sample carrier 44 and together form a cone of light that converges on the sample carrier 4 into an illumination area onto which the objective lens 10 is directed. This illumination area is illuminated monochromatically by the light sources 28 together. The spectral filtering for this is achieved by the emission filters 42, which, in this embodiment, allow a spectral band with a width of σ < 20 nm to pass through from the radiation spectrum of the light sources 28 and are opaque to other radiation from the light sources 28. This excitation band lies, with its center of gravity and expediently entirely, in a higher frequency range than the excitation frequency of the general marker. Furthermore, the entire excitation band lies within the opaque frequency range of both the beam splitter 22 and the filter 26.

[0065] Due to the strong illumination of the biological material in the excitation band of the general marker, the marker emits light in a reflected beam, which in this embodiment lies at approximately 510 nm. Each biological target object coated with the general marker emits light so intensely that it is detected by the camera 8. The camera 8 can include a detection device, such as an image acquisition unit. For the sake of simplicity, a detection device and a differentiation device are described below as being associated with the camera 8.

[0066] To detect all target objects in all vessels 34 on the sample carrier 4, all possible areas of the sample carrier 4 where a target object could be located—in this embodiment, all vessels 34—are scanned sequentially. For this purpose, the overview field of view is directed successively at all vessels 34 and remains there for a predetermined duration, approximately 500 ms in this embodiment. Subsequently, the overview field of view 36 is positioned on the next vessel 36, and the radiation emitted by the biological material in the excitation band is detected again. Including the positioning times of the overview field of view 36, such a detection process takes approximately one minute in this embodiment and is performed automatically without requiring operator intervention.

[0067] Each object emitting radiation with an intensity above a threshold value within the radiation band is detected by the camera 8 as a target object 32. For each target object 32, its location or position within the overview field of view 36 is determined, and based on this, its position on the sample carrier is established, since the position of the overview field of view 36 on the sample carrier 4 is known by a corresponding positioning device of the camera 8. The position is expressed in two-dimensional coordinates x. i , y i The position of the first found target object 32 is in Fig. 2 accordingly indicated by x1, y1. On sample carrier 4 from Fig. There are 2 four target objects 32, all of which are found.

[0068] The very strong illumination of the biological material 6 causes it to emit a large amount of scattered radiation 46, which makes it difficult to detect the target objects 32. Several measures are taken to reduce this radiation 46 to a level that does not compromise the reliable detection of the target objects.

[0069] As a first step, the sample surface is illuminated by the first illumination unit 16 from outside the lens 10. This causes the illumination radiation to shine onto the sample carrier 4 at a different angle than the camera 8's field of view onto the sample carrier 4. In the exemplary embodiment from Fig. For example, the illumination angle is 45°, with the camera 8's field of view directed at the sample carrier 4 at an angle of 90°. The angle between the illumination angle and the viewing angle should be at least 30°. This ensures that radiation reflected from the sample carrier 4 does not, or only negligibly, enter the lens in the camera 8's line of sight.

[0070] This is in Fig. 5 indicated. Scattered radiation 46 reflected from the sample surface is reflected past the objective 10. Only such scattered radiation that originates from multiple scattering or from minor scattering at curved surfaces of the biological material enters the objective 10, as shown in Fig. 5 is indicated. However, the vast majority of this scattered radiation is not collimated and does not travel in the direction of view of camera 8.

[0071] To prevent this scattered radiation 46 from entering the camera, the microscope device 2 includes an image field tube 48, which has a length of at least 10 cm, between the objective 10 and the camera 8. Several apertures 50 are arranged within this image field tube 48, which absorb uncollimated radiation. In this way, at least 90% of the scattered radiation 46 entering the objective 10 is blocked.

[0072] By shading uncollimated scattered radiation 46, the further advantage is achieved that such scattered radiation 46, which can nevertheless pass through the collimating apertures 50, is largely collimated, so that it strikes the subsequent spectral filter 26 perpendicularly. With this radiation direction, the spectral filter 26 exhibits an optimal filtering effect, thus largely filtering out the scattered radiation 46.

[0073] In this way, despite very strong light exposure to the sample surface, it can be reliably achieved that the scattered radiation is so low that it does not significantly interfere with the reliable detection of even individual target objects 32 in the large overview field of view 36.

[0074] Once the entire sample area has been scanned and the position of all target objects 32 has been recorded, the second step of the procedure is carried out. For this, the microscope optics 14 are switched so that the second objective 12 is now used. This is in Fig. Figure 6 shows that the lens 12 has a 20x magnification and thus projects a smaller target field of view 52 onto the camera 8, which is shown in Fig. Figure 7 is shown. Based on the stored two-dimensional positions of the detected target objects 32, the target field of view 52 is now successively aligned with these target objects 32. Subsequently, the target objects are examined for further optical properties that expediently differ from the optical properties of the general marker.

[0075] For this purpose, the sample carrier 4 is illuminated in the area of ​​the target field of view 52 from the second illumination unit 18.

[0076] The second illumination unit 18, however, unlike the first illumination unit 16, emits a continuous spectrum in order to provide several excitation bands in different frequency ranges. Alternatively, an illumination unit would be possible that provides the desired excitation bands in the different frequency ranges monochromatically, so that, in contrast to continuous polychromatic radiation, discrete polychromatic radiation is available.

[0077] The illumination radiation from the second illumination unit 18 is spectrally filtered in the excitation filter 20, whereby in this embodiment polychrome illumination of the sample carrier 4 is permitted. Alternatively, alternating monochrome illumination with alternating excitation filters 20 is possible. Regardless of these illumination variants, the radiation emitted by the sample is successively filtered at several emission frequencies in the filter unit 24, whereby several filters 26 with different transmission bands are used sequentially by inserting the filters successively into the imaging beam path. Alternatively, it is possible to allow several emission frequencies—corresponding to the markers used—to be simultaneously transmitted to and detected by the camera 8, in which case the camera must be a color camera with one color detector or with several monochrome detectors.

[0078] The illumination radiation from the second illumination unit 18 is guided through the image field tube 48 and the second objective 12, reaching the sample parallel to the optical axis of the objective 12 and to the viewing direction of the camera 8 on the sample. This also captures scattered radiation 46 directly reflected from the sample carrier 4 in the objective lens. This scattered radiation is absorbed less by the apertures 50 in the image field tube 48 than scattered radiation 46 from the first illumination unit 16. Although this scattered radiation 46 also strikes the filter 26 perpendicularly and is optimally filtered, a higher proportion of scattered radiation per captured sample area remains in the image than in the first step and with illumination by the first illumination unit 16. However, the higher magnification increases the emission from the target object 32, so that the stronger scattered radiation 46 does not significantly interfere with the differentiated examination of the target object 32 in the second step.

[0079] Fig. Figure 7 shows four target fields 52 in which the biological material 6 located therein is illuminated with four different frequencies, or rather, examined at four different frequencies. These images of the four target fields 52 are not taken simultaneously, but sequentially and stored.

[0080] At the in Fig. In the embodiment shown in Figure 7, a target object 32 is examined for several different optical properties, which, through appropriate staining or markers, allow conclusions to be drawn about the biological properties of the target object 32. In the first target field of view 52, ​​for example, the emission of FITC (fluorescein isothiocyanate) coupled to an immunoglobulin, in this case anti-cytokeratin, is detected. This cytoplasmic staining makes a large cell 32 with a specific antigenic surface property visible.

[0081] In the second target field 52, the emission of phycoerythrin is detected, which has an emission peak at 575 ± 10 nm and is bound to anti-CD45. This dye produces a mononuclear cell surface stain and, in this binding, is a marker indicating a normal cell nucleus.

[0082] It can be seen that in this target field 52 of camera 8, many blood cells are visible, but the target object 32 does not appear. The cytoplasmic staining in the first target field 52 and the absence of the target object in the mononuclear cell surface staining in the second target field 52 indicate that the target object 32 is a tumor cell migrating in the bloodstream. However, certainty about this will only be achieved with further markers.

[0083] Accordingly, the third target field 52 is examined for DAPI (4',6-diamidino-2-phenylindoles) with an emission at 461 nm. DAPI is a nuclear marker that visualizes DNA properties of the cell nucleus. The nuclear marking appearing in the third target field 52, together with the markings described above, allows the conclusion that a tumor cell is present in the target object 32.

[0084] In the fourth target field of view 52, ​​the filter unit 24 does not filter, as in the Fig. 1 and Fig. The rightmost filter field (number 6) indicates the result, creating a brightfield image. This can be used for further investigations, e.g., differentiation according to morphological properties.

[0085] To uniquely locate the target object 32 in all target visual fields 52 and to distinguish it from other depicted objects, the position of the target object 32 known from the first step is again used, which differs from surrounding objects, e.g., surrounding cells. In this way, the target object 32, detected in the first step, is automatically examined in the second step for a series of optical properties, based on which the target object 32 can be classified. For example, the target object 32 can be made from Fig. 7. A cell is classified as a tumor cell. If one of the tested optical properties is different, the classification will be different accordingly.

[0086] In the procedure described above, the same optical method, e.g., fluorescence analysis, can be used in both steps, i.e., both for the detection of the target objects 32 and for their differentiated examination for classification. However, it is also possible to choose a different optical method in the second step that facilitates the desired classification more easily than the first method. Bright-field microscopy has already been mentioned, but dark-field microscopy or other methods are equally possible.

[0087] An example of different procedures is shown below in the presentation in Fig. 8 explained. In the first step, biological material 6, which has been labeled with one or more color markers, is examined using fluorescence analysis. For example, specific bacteria are sought as target objects 32, which are labeled accordingly. The target objects and their position are recorded in the first step, as described above.

[0088] In the second step, the biological material 6 is again imaged onto the camera 8 using the target field of view 52. Fig. Figure 8 shows the target field 52, identical in position, at five different time points t1 - t5. For the sake of simplicity, only a bacterium is shown as the target object 32, although other objects may also be depicted without interfering with the investigation described below, since the target object 32 is captured with sufficient precision in its position to distinguish it from other depicted objects.

[0089] At the first time point t1, the target object 32 is examined for its morphological properties. This can be done directly through image processing or indirectly by measuring radiation levels. For example, in dark-field microscopy, the amount of radiation detected can depend on the size of the target object 32 in the target field of view. If the target object 32 grows, its radiation increases, and this growth can be detected indirectly via the received radiation power.

[0090] Before the second time point t2, an active ingredient is administered to the target object 32, which is located in Fig. 8 is indicated by hatching. The aim is to test how target object 32 develops under the active ingredient. For this purpose, target object 32 is examined in the second step as a function of time and classified according to this examination. In the Fig. In the example shown in Figure 8, the bacterium grows over time, suggesting a corresponding interaction with the active ingredient. The target object 32 is classified accordingly.

[0091] Another example of an investigation will be presented using the illustration from Fig. 9 explained. The sample carrier 4 contains a multitude of so-called nanocodes, i.e., small carrier units 54, each of which has a machine-readable code, e.g., a barcode or other code. In the case of the Fig. In the embodiment shown in Figure 9, the code consists of points and lines whose arrangement relative to each other yields a predetermined content, e.g., a number. Each carrier unit 54 also contains a sample area 56 on which biological material is bound, optionally together with a marker. In this way, the biological material can be linked to the code, so that each of the carrier units 54 can carry different biological material, or differently marked biological material, which can be distinguished based on the code.

[0092] In the first step, many of the carrier units 45 are depicted together in the overview field of view, as in Fig.As indicated in Figure 9, depending on the presence of markers in the biological material, the sample surfaces 56 differ in their emission, so that the desired markers and thus the desired biological material can be found as target objects 32. The biological target objects 32 can be the carrier units 54, their sample surface 56, or the corresponding biological material.

[0093] Also in the first step, the encoding of at least those nanocodes that the detected target objects 32 bear or form is read. In this way, the target objects can already be assigned a code and thus further information in the first step. If necessary, classification into different classes is already possible in the first step.

[0094] In the second step, a higher magnification optical examination takes place, analogous to the procedure described above. Here, the target objects 32 can be examined in any desired manner, e.g., by means of fluorescence analysis, dark-field analysis, or other methods. Based on their further optical properties, the target objects 32 are automatically classified. Reference symbol list 2 Microscope device 4 sample carriers 6 biological material 8 Camera 10 lenses 12 Lens 14 Microscope optics 16 lighting units 18 lighting units 20 filters 22 beam splitters 24 filter units 26 filters 28 Light source 30 filters 32 biological targets 34 containers 36 Overview field of view 38 Light source 40 Collimation optics 42 emission filters 44 Output optics 46 Scattered radiation 48 Image field tube 50 aperture 52 Target field of view 54 carrier unit

Claims

[1] Microscopy method for detecting target objects (32) that have a predetermined optical property in material to be examined (6), wherein - an overview field of view (36) of a first objective (10) of a microscope optic (14) is directed in a first step onto an overview area of ​​a sample carrier (4) with the material (6) to be examined, - the material to be examined (6) is illuminated from an illumination unit (16) which shines onto the sample carrier (4) from outside an image field tube (48) and is recorded by a camera (8), - the material (6) to be examined is optically examined for the optical property in such a way that even a single target object (32) with the predetermined optical property is recognized as such in the material (6) to be examined, - in a subsequent second step, a target field of view (52) of a second objective (12) of the microscope optics (14) is aligned to a target area around the target object (32) using the known position of the target object (32), - the detected target object (32) is examined in a differentiated manner according to various further optical properties. [2] Microscopy method according to claim 1, characterized by , that the target field of view (52) has an increase of at least a factor of 3 compared to the overview field of view (36). [3] Microscopy method according to claim 1 or 2, characterized by , that the detection of the target object (32) in the first step is carried out using a first optical method and the differentiation of the target object (32) in the second step is carried out using a second optical method different from the first. [4] Microscopy method according to any one of the preceding claims, characterized by, that the material to be examined (6) is illuminated in the second step from a second and different illumination unit (18), which enables a differentiation of the target objects (32) according to various further optical properties by illumination with different frequencies. [5] Microscopy method according to any one of the preceding claims, characterized by , that the lighting from the first lighting unit (16) is LED light and the second lighting unit (18) has a spotlight with a continuous spectrum. [6] Microscopy method according to any one of the preceding claims, characterized by, that the material to be examined (6) is illuminated in the second step from a second and different illumination unit (18), which the sample carrier irradiates coaxially to the optical axis through the image field tube (48) and the first illumination unit (16) shines on the sample with a power per nanometer frequency range at least 5× higher than the second illumination unit (18). [7] Microscopy method according to any one of the preceding claims, characterized by , that all target objects (32) to be found have a general marking as a predetermined optical property and the material to be examined is only examined for the general marking in the first step, and the target objects (32) also have a classification marking as further optical properties and are examined for these in the second step. [8] Microscopy method according to any one of the preceding claims, characterized by, that the target object (32) is examined in the first step for its coordinates on the sample carrier (4) and only in the second step for target-specific properties at higher optical magnification. [9] Microscopy method according to claim 8, characterized by that the target-specific properties are color and / or shape properties of the target object. [10] Microscopy method according to any one of the preceding claims, characterized by , that in the second step the temporal sequence of at least one property of the target objects found (32) is examined. [11] Microscopy method according to any one of the preceding claims, characterized by , that in the first step a sample area of ​​the sample carrier (4) is sequentially scanned field of view, the position of the found target object (32) in the overview area is determined and in the second step the found target objects (32) are individually approached and microscopically examined. [12] Microscopy method according to any one of the preceding claims, characterized by , that both the first and the second lighting unit (16, 18) illuminate the material (6) to be examined exclusively from one side.

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