Abstract:
The invention is based on a microscope, in particular a fluorescence analysis microscope, comprising an illumination carrier and illumination units arranged thereon for a reflected-light illumination of a sample region. In order to be able to illuminate a sample region uniformly in a simple manner by means of a compact device, it is proposed that at least three illumination units for the simultaneous reflected-light illumination of the sample region from different directions are arranged on the illumination carrier.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is a continuation of U.S. patent application Ser. No. 11/922,088, filed on Dec. 12, 2007 and entitled MICROSCOPE, which in turn is a PCT National Stage of PCT Application No. PCT/EP2006/005665, filed on Jun. 13, 2006, and which claims priority from German Patent Application No. 10 2005-027 312.2 filed on Jun. 13, 2005, the contents of each being incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    DE 29 44 214 discloses a fluorescence analysis microscope in which a sample is illuminated from different directions by means of a plurality of illumination sources. In this case, the illumination sources emit radiation in respectively different wavelength ranges. 
         [0003]    It is an object of the present invention, in particular, to specify a microscope in which a sample can be illuminated as uniformly as possible. This object is achieved according to the invention by means of the features of claim  1 . Further configurations emerge from the subclaims. 
       SUMMARY OF THE INVENTION 
       [0004]    The invention is based on a microscope, in particular a fluorescence analysis microscope, comprising an illumination carrier and illumination units arranged thereon for the reflected-light illumination of a sample region. It is proposed that at least three illumination units for the simultaneous reflected-light illumination of the sample region from different directions are arranged on the illumination carrier. As a result of this, the sample region can be illuminated uniformly, whereby—particularly during the fluorescence analysis—a high and uniform emission can be obtained without any shading regions of the sample region. It is possible to detect samples having a large area, such as preferably having an area with a diameter of greater than 3 mm, in an image, and it is additionally possible to achieve a high resolution, preferably a resolution of greater than 8 Mpixels. 
         [0005]    In order to increase the symmetry, the illumination units are expediently shaped identically. The illumination units comprise in each case at least one illumination source. These illumination sources are expediently embodied identically in terms of type and emission characteristic, whereby a uniform illumination of the sample region can be achieved in a simple manner. The illumination sources can advantageously be driven separately by a control unit, such that the uniform illumination can be achieved particularly simply and reliably, for example by means of a calibration. The illumination units expediently emit radiation onto the sample region in each case at an identical angle, whereby a particularly homogeneous illumination can be achieved. 
         [0006]    A further advantage is achieved if illumination units with illumination sources which emit radiation with different wavelength ranges are arranged on the illumination carrier, whereby a plurality of tests or analyses can be carried out simultaneously and/or sequentially in succession, for example with two or more dyes. Furthermore, advantageous overall evaluations can be made possible. 
         [0007]    Furthermore, the microscope preferably has a multi-bandpass emission filter having a plurality of passbands, whereby an advantageous detection of emissions of a plurality of dyes can be achieved and it is possible to avoid changing a filter when carrying out different analyses. Moveable parts and resultant error sources and costs can be avoided. It is possible to increase the throughput or the number per unit time, whereby together with the saving of reagents a considerable cost saving is made possible. 
         [0008]    In a further configuration of the invention it is proposed that the illumination units are shaped identically and are arranged on a plurality of receptacle regions of the illumination carrier which are provided for them. As a result of this, it is possible to achieve a modular construction of the microscope, in which case illumination units can be exchanged in a simple manner and the illumination of the sample region can be adapted to desired measurements in a particularly simple and cost-effective manner. The illumination units are advantageously attached to the illumination carrier and can additionally be screwed thereto. 
         [0009]    The uniformity of the illumination of the sample region can be increased further if at least four illumination units are arranged on the illumination carrier, the illumination beam paths of said illumination units being arranged in ring-shaped fashion with respect to the sample region. It is additionally possible to achieve a compact arrangement of the microscope as a result of this. Given a high number of illumination units on the illumination carrier, the illumination carrier can have a cooling element, such as a cooling fin for example. In this case, the cooling element has a shaping that is provided for cooling purposes and is especially suitable for cooling purposes. 
         [0010]    A precise orientation of illumination beam paths with respect to a detection beam path can be achieved in a simple manner if the illumination carrier forms a channel for a detection beam path from the sample region to a camera. A detection optic can be inserted, for example plugged, into said channel, in which case a complicated alignment of the illumination beam paths with respect to the detection beam path can be obviated. 
         [0011]    It is additionally proposed that the microscope comprises a detection beam path from the sample region to a camera, the illumination carrier being arranged in ring-shaped fashion around the detection beam path. As a result of this it is possible to achieve a compact arrangement of the microscope in conjunction with homogeneous illumination of the sample region. A stable, compact microscope that can be assembled in a simple manner can be achieved if the illumination carrier is embodied in one piece. The illumination carrier is expediently produced by the injection-molding method. Equally advantageously, the illumination carrier expediently carries all the illumination units of the microscope. 
         [0012]    The compactness of the microscope can be increased further if the illumination units are arranged in two cone arrangements on the illumination carrier. A very homogeneous illumination of the sample region can be obtained by means of the conical forms. A top side and underside of the illumination carrier can be utilized for the compact arrangement of illumination units if a cone vertex of one of the cone arrangements is located above, and one below, the illumination carrier. 
         [0013]    The compactness of the microscope can be increased further if at least two illumination units form a double unit, the illumination beam paths of which are combined by a coupling-mirror. 
         [0014]    A particularly precise decoupling of the illumination of the sample region from the emission from the sample region can be achieved if the microscope comprises a detection beam path extending from the sample region to a camera and an illumination beam path extending from an illumination unit to the sample region, the detection beam path and the illumination beam path being guided completely separately from one another. 
         [0015]    An exact and low-loss orientation of radiation from the illumination units, particularly when using illumination sources of different emission wavelengths, can be achieved if the reflected-light illumination is effected in each case along an illumination beam path from an illumination unit to the sample region and the illumination beam paths are guided completely separately from one another. 
         [0016]    Illumination units of small construction with emission characteristics that are particularly suitable for fluorescence analysis can be achieved if the illumination units in each case comprise an illumination source with at least one LED. 
         [0017]    Furthermore, a microscope comprising a camera and comprising a computing unit is proposed, wherein the computing unit is provided for monitoring a process by means of the camera, that is to say is provided in particular for detecting image data during a process proceeding within a sample, such as in particular before and/or during a marking process of a sample by means of dyes. In this case, a computing unit should be understood to mean in particular a control and/or regulating unit which has one or more processors and in particular one or more memory units with a specific operating software stored therein. Furthermore, “provided” should be understood to mean specifically equipped, designed and/or programmed. 
         [0018]    By means of a corresponding configuration, additional useful information items can be obtained and the measurement result can be improved, to be precise in particular if the computing unit is provided for at least partly eliminating disturbing effects identified during the process, such as in particular by means of a software-aided correction method in order to reduce signal noise. A corresponding correction algorithm is preferably stored in a memory unit of the computing unit. 
         [0019]    It is furthermore proposed that the microscope has a computing unit provided for carrying out a calibration on the basis of a reference object, whereby the measurement result can be improved in a simple manner. In this case, the reference object can be arranged at different regions that appear to be practical to the person skilled in the art, such as, in particular, in the region of a sample carrier and/or on a sample itself, and/or it can also be formed by a known sample itself. 
         [0020]    If the microscope has a computing unit provided for evaluating detected image data, quantities of data that are to be processed by an operator can be reduced, to be precise in particular if it is possible to output results in numerical values and/or characteristic quantities which can be used for unambiguously identifying whether a test is positive or negative. 
         [0021]    In a further configuration of the invention it is proposed that the microscope has an at least partly automated adjusting unit for adjusting a position of a sample relative to an optical sensor, such as, in particular, a mechanical and/or optical adjusting unit, such as, for example, an autofocus unit. By means of a mechanical adjusting unit, in this case the sample can be moved relative to the optical sensor, and/or the optical sensor can be moved relative to the sample manually and/or advantageously in an at least partly automated manner. Preferably, the at least partly automated adjustment takes place in real time, in which case “real time” should be understood to mean adjustment directly before and/or during detection of measurement data. 
         [0022]    If the microscope has an at least substantially telecentric emission optic, on the object side and/or on the image side, it is possible to avoid undesirable changes, in particular during focusing, and it is possible to achieve a uniform angle of light incidence over an entire field of view of an image sensor, whereby an advantageous homogeneous sensitivity can be obtained. Furthermore, it is possible to use microlenses whose performance is dependent on the angle of incidence. In this case “substantially” should be understood to mean in particular that optics having tolerance-dictated deviations from 100% telecentricity are intended to fall within the scope of protection. 
         [0023]    It is furthermore proposed that the microscope has at least one passive optical means for homogenizing an illumination intensity. In this case, a “passive optical means”, in contrast to an illumination source, should be understood to mean in particular a lens, a mirror, etc., use preferably being made of optical means having specific surface contours which are provided for example for at least partly compensating for intensity differences caused by an oblique angle of incidence. Furthermore, optical means with holographically produced microstructures are particularly advantageously used, whereby it is possible to achieve an advantageous homogeneity with at the same time high transmission. 
         [0024]    It is possible to achieve a microscope at least largely without moveable parts, in particular relative to an illumination module, which can be embodied in small, lightweight and robust fashion, whereby it is preferably suitable for portable applications, or which can scan an area of arbitrary size or complexity by movement. 
         [0025]    A microscope comprising an optical scattering unit is furthermore proposed, which scattering unit has optical means, in particular optical scattering means, wherein the scattering unit has at least one optical means which is provided at least for reducing a beam expansion of the scattering unit, in particular relative to a scattering unit with exclusively spherical microlenses and/or holographically produced optical means, whereby an advantageous uniform illumination can be achieved and losses can be avoided. A “scattering unit” should be understood to mean in particular amounting unit, such as a screen, in particular, which is provided for scattering the light. 
         [0026]    Advantageous effects can be obtained if the optical means has an axis running through an optical axis of the scattering unit, and in particular if the optical means is formed in substantially cylindrical, cylinder-segment-shaped, conical and/or cone-segment-shaped fashion, whereby—relative to an axis extending radially outward proceeding from the optical axis—it is possible to obtain an advantageous orthogonal scattering tangential to a round scattering unit. With optical means formed in conical and/or cone-segment-shaped fashion, it is possible to obtain an advantageous arrangement of the optical means alongside one another at least largely without any interspace. 
         [0027]    If different optical means are arranged in a radial direction proceeding from an optical axis of the scattering unit, it is possible to obtain different advantageous scattering effects in a targeted manner in different regions. In particular, specific optical means which are provided at least for reducing a beam expansion are advantageously arranged in an outer edge region of the scattering unit. 
         [0028]    The invention additionally relates to a method for calibrating a microscope. The uniform and shadow-free illumination of a sample region in an as far as possible precisely predetermined intensity is of great importance particularly during fluorescence analysis. The calibration of the microscope is expedient for this purpose. The object of this part of the invention is to specify a method for calibrating a microscope by means of which it is possible to achieve a uniform illumination of a sample region with predetermined intensity in a simple manner. This object is achieved according to the invention by means of a method for calibrating a microscope, in particular a fluorescence analysis microscope, with a plurality of illumination units which illuminate a sample region from different directions, in which a known sample is illuminated, the image thereof is evaluated by an image processing, an undesirably illuminated partial region of the sample region is determined and at least one illumination parameter of one of the illumination units is altered by means of a stipulation obtained from the determination. It is possible to achieve a uniform illumination of a sample region with predetermined intensity in a simple manner. The illumination parameter can be emission intensity of an illumination source of one of the illumination units. 
         [0029]    The microscope according to the invention can be used in various analyses which appear to be practical to the person skilled in the art, but particularly advantageously in cell-based analyses, in immunofluorescence analyses, in genetic analyses, such as, for example, in a so-called ELISA analysis (enzyme-linked immunosorbent assay), in a PCR analysis (prepolymerase chain reaction), in a real-time PCR analysis, etc., and/or in particular in order to determine the number of elements of a sample, such as preferably blood cells in an HIV test. Furthermore, the microscope is very well suited to at least partly automated biological and/or chemical methods, such as, in particular, methods with “biochips” or “lab-on-chip”, relatively complex biochemical processes being carried out automatically under the microscope. 
         [0030]    Furthermore, a method with a microscope is proposed in which before a measurement in the case of a sample an illumination unit is activated, the spectral properties of which lie at least substantially outside an absorbance range of at least one dye used in the case of the sample, whereby a phasing of dyes can be at least largely avoided. “Lying substantially outside an absorbance range” should be understood to mean, in particular, that the band ranges relative to the absorbance range of the dye overlap by less than 10%, preferably by less than 5%, and particularly advantageously do not overlap. In order to use the illumination for an autofocus process, an illumination unit whose spectrum overlaps one or more wavelength passbands of an emission filter is advantageously used. 
         [0031]    If at least one unit of the microscope is moved over a sample during a measurement, relatively large regions can be detected or scanned in a simple manner, and disturbing effects caused by a movement of the sample can be avoided. If a movement of the sample under the microscope can be avoided, connections such as preferably electrical contacts, connections for reagents and/or fluids, etc. can advantageously be provided on a sample carrier or object carrier, to be precise in particular in order to supply a sample with power, reagents and/or fluids. 
         [0000]    It is furthermore proposed that a part of the imaging and/or of the measurement field is used for detecting “sample information items”, preferably simultaneously with the detection of the samples. “Sample information items” should be understood to mean markings such as bar codes, for example, which contain items of information about the samples, and/or markings which serve for alignment or spatial determinations, and/or reference samples which serve for calibration, in particular intensity and/or spectral. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0032]    Further advantages will become apparent from the following description of the drawings. The drawings illustrate an exemplary embodiment of the invention. The drawings, the description and the claims contain numerous features in combination. The person skilled in the art will expediently also consider the features individually and combine them to form practical further combinations. In the figures: 
           [0033]      FIG. 1  shows a microscope for fluorescence analysis, 
           [0034]      FIG. 2  shows an illumination carrier of the microscope in a perspective view with two illumination units illustrated in an exploded view, 
           [0035]      FIG. 3  shows the illumination carrier from  FIG. 2  in a sectional illustration with inserted illumination units, 
           [0036]      FIG. 4  shows the illumination carrier from  FIGS. 2 and 3  in a side view, 
           [0037]      FIG. 5  shows a sample region covered by irradiation regions in a schematic illustration, 
           [0038]      FIG. 6  shows a schematically illustrated method sequence, 
           [0039]      FIG. 7  shows a scattering unit by itself, and 
           [0040]      FIG. 8  shows an alternative scattering unit by itself. 
       
    
    
     DETAILED DESCRIPTION 
       [0041]      FIG. 1  shows, in a schematic illustration, a microscope  2  for fluorescence analysis or a fluorescence analysis microscope comprising a stand  4 , a stand base  6 , for example for mounting onto a table, and an object carrier  8 , in the central position of which a sample region  10  is represented for illustration purposes. The object carrier  8  has connections  94  formed by electrical contacts and by fluid connections, in particular for supplying samples. Positioned above the sample region  10  is an illumination module  12  for illuminating the sample region  10 , and above that a camera  14  and a control unit  16 , which controls methods that can be carried out automatically by the microscope  2  and comprises an image processing for analyzing recorded images of the sample region  10 . The control unit  16  is provided for evaluating detected image data so as subsequently to be able to output final results by means of a schematically illustrated output unit  92 . The control unit  16 , which is formed by a computing unit and is integrated in the camera  14 , has a processor unit  66  and a memory unit  68  with an operating software stored therein. Arranged above the control unit  16  is an eyepiece  18  in order to permit an operator to look directly at the sample region  10 . 
         [0042]    The illumination module  12  comprises an illumination carrier  20 —which is illustrated in a perspective view in FIG.  2 —for accommodating 16 illumination units  22   a ,  22   b , only two of which are shown in  FIG. 2 . The illumination units  22   a ,  22   b  are in each case arranged at accommodating regions  24  prepared for them and are held by two screws in screw holes  26  in each case at the accommodating region  24 . A channel  28  for a detection beam path  30  (see  FIG. 3 ) is led from the sample region  10  to the camera  14  centrally through the illumination carrier  20 . 
         [0043]    The illumination carrier  20  and the illumination units  22   a ,  22   b  arranged thereon are illustrated in sectional view in  FIG. 3 . A camera holder  32  with a telecentric emission optic  34  illustrated schematically is inserted into the channel  28 , the camera  14  (not illustrated in  FIG. 2 ) being plugged into said camera holder. In its interior, the camera holder  32  comprises a schematically illustrated adjusting unit formed by an autofocus unit  36  with a motor  38 , by means of which unit a lens  40  can be moved parallel to the detection beam path  30  upward or downward for focusing onto the sample region  10  or a sample arranged therein and it is possible to achieve an adjustment of a position of the sample relative to an optical sensor of the camera  14 . The autofocus unit  36  is controlled by the control unit  16 . The illumination carrier  20  is made of plastic and produced in one piece with the aid of an injection-molding method. It carries all the illumination units  22   a ,  22   b  arranged in ring-shaped or conical fashion around the detection beam path  30 . In addition, the microscope has a schematically illustrated automated mechanical adjusting unit  64  with guide rails and actuator units (not specifically illustrated) for adjusting a position of the sample relative to the optical sensor of the camera  14 . By means of the adjusting unit  64 , the camera  14  and the illumination module  12  can be moved relative to the sample in an automated manner, to be precise parallel and transversely with respect to the detection beam path  30 . The adjustment by means of the autofocus unit  36  and the mechanical adjusting unit  64  is effected during operation in real time, that is to say directly before and/or during detection of measurement data. By means of the mechanical adjusting unit  64 , the camera  14  and the illumination units  22   a ,  22   b  can be moved over the sample, under the control of the control unit  16 , for the purpose of scanning the sample. 
         [0044]    The in each case eight illumination units  22   a ,  22   b  are led—as is illustrated in FIG.  4 —around the detection beam path  30  in two cone arrangements, the detection beam path  30  lying in the cone axis of the cone arrangements. In this case, the imaginary cone vertex of one of the cone arrangements is located above, and the imaginary cone vertex of the other cone arrangement below, the illumination carrier  20 . The cone surfaces of the two cone arrangements are embodied at right angles to one another. Between the cone arrangements there are cooling fins  42  for cooling the illumination carrier  20 , which dissipate the heat generated by illumination sources  44   a ,  44   b , lenses  46  and spectral filters  48  to the surroundings of the illumination carrier  20 . The lenses  46  form passive optical means for homogenizing an illumination intensity, to be precise in that they have surface contours and/or holographically produced microstructures which are specifically adapted to an angle of incidence of the illumination beam path. 
         [0045]    An illumination beam path  50   a ,  50   b  respectively leads from the illumination sources  44   a ,  44   b  to the sample region  10 . The illumination beam paths  50   a ,  50   b  are combined in a coupling-in mirror  52  toward the sample region  10 . In this case, a respective illumination unit  22   a  with an illumination unit  22   b  arranged opposite in the direction of the detection beam path  30  forms a double unit, the beam paths  50   a  and  50   b , respectively, of which are combined by the coupling-in mirror  52 . In the coupling-in mirror  52 , the illumination beam paths  50   a ,  50   b  impinge on one another at right angles. 
         [0046]    The illumination beam paths  50   a ,  50   b -after they have been combined in the coupling-in mirror  52 —are arranged in ring-shaped fashion and at the same angle with respect to the sample region  10 . They are guided from the illumination sources  44   a ,  44   b  as far as the sample region  10  completely separately with respect to the detection beam path  30 . Moreover, the illumination beam paths  50   a  of all the illumination units  22   a  are guided from the illumination sources  44   a  as far as the sample region  10  completely separately from one another. 
         [0047]    The illumination sources  44   a ,  44   b  are light-emitting diodes (LEDs), each illumination unit  22   a ,  22   b  having one LED in each case. In order to improve the below-described method for uniformly illuminating the sample region  10 , it is possible to arrange in each illumination unit  22   a ,  22   b  in each case a plurality of LEDs, in particular in a plane perpendicular to the illumination beam paths  50   a ,  50   b . As an alternative and/or in addition to conventional LEDs, it is possible to use laser diodes and/or solid-state lasers. 
         [0048]    The illumination units  22   a ,  22   b  embodied identically in terms of their geometry are plugged into the illumination carrier  20  and screwed there. The illumination sources  44   a  are embodied differently at least in part and emit radiation in different wavelength ranges during operation. Furthermore, the illumination sources  44   b  are embodied differently at least in part and emit radiation in different wavelength ranges during operation. In principle, the illumination sources  44   a  of the illumination units  22   a  could be embodied identically and emit radiation in the same wavelength range. The same applies to the illumination sources  44   b  of the illumination units  22   b . Furthermore, the illumination sources  44   b  are expediently embodied differently than the illumination sources  44   a  at least in part in terms of their spectral range, in order to minimize the coupling-in losses as a result of the coupling of the illumination beam paths  50   b  into the illumination beam paths  50   a  with the aid of the coupling-in mirror  52 . The illumination sources  44   a ,  44   b  can be divided into more than two spectral ranges in order to be able to carry out a plurality of fluorescence analyses simultaneously and/or else sequentially. In this case, in particular at least four colors or different spectral ranges are advantageous in order to be able to carry out a plurality of analyses simultaneously and in addition to have enough illumination sources  44   a ,  44   b  available per color so as to be able to uniformly illuminate the sample region  10  by the method described below. For this purpose, the illumination sources  44   a ,  44   b  can be driven separately by the control unit  16 . In terms of their spectral range, the spectral filters  48  are coordinated with the illumination sources  44   a ,  44   b . The coupling-in mirror  52  is embodied such that it is transmissive for radiation of the spectral range of the illumination sources  44   a  and reflective for radiation of the spectral range of the illumination source  44   b . In addition, a multi-bandpass emission filter  60  coordinated with the radiation sources is arranged in the channel  28 . 
         [0049]    Furthermore, the microscope  2  has scattering units  96  with optical means  100 ,  102  arranged downstream of the illumination sources  44   a ,  44   b  in the light beam direction. The optical means  100  are formed by spherical microlenses or by holographically created optical means, while the optical means  102  are formed by semicylindrical microlenses which have a cylindrical axis  108  running through an optical axis  106  of the scattering unit and are provided for avoiding beam expansion by the scattering unit  96  ( FIG. 7 ). Different optical means  100 ,  102  are arranged in the radial direction proceeding from the optical axis  106  of the scattering unit  96 , to be precise the optical means  100  are arranged within a radius R 1  and the optical means  102  are arranged outside the radius R 1  or in an outer edge region between the radius R 1  and a radius of the scattering unit  96 . 
         [0050]      FIG. 8  illustrates an alternative scattering unit  98  with conical optical means  104 , the cone or central axis  110  of which runs through an optical axis  106 ′ of the scattering unit  98  and extends as far as the edge of the scattering unit  98  proceeding from the optical axis  106 ′. The optical means  104  are provided, in a manner corresponding to the optical means  102 , for at least reducing or avoiding beam expansion caused by the scattering unit  98 . 
         [0051]    For the calibration of the microscope  2 , a known standard sample  54  (see  FIG. 5 ) is introduced into the sample region  10 . Said standard sample  54  contains structures which are identified by the image processing of the control unit  16 , whereby in an autofocus method the autofocus unit  36  is driven, the lens  40  is optimally positioned and the standard sample  54  is focused. Afterward, the standard sample  54  or the sample region  10  is illuminated by some or all of the illumination units  22   a ,  22   b.    
         [0052]    By way of example, all the illumination sources  44   a  emit radiation in one spectral range or with one color, and all the illumination sources  44   b  emit radiation in another spectral range. In this case, for example firstly the standard sample  54  is illuminated by means of the illumination sources  44   a . Each double unit comprising a respective illumination unit  22   a ,  22   b  then illuminates the sample region  10  with an illumination field  56 , such that the sample region  10  is illuminated with eight overlapping illumination fields  56 . The standard sample  54  is examined by the image processing of the control unit  16  with regard to its brightness, it being ascertained, for example, that a partial region  58  of the sample region  10  is illuminated only inadequately. Those illumination fields  56  which at least partly cover the partial region  58  are then illuminated more brightly relative to the other illumination fields  56  in a manner such that the partial region  58  is illuminated in a desired manner in relation to the entire sample region  10 , such that the entire sample region  10  is now illuminated uniformly as desired. Afterward, the standard sample  54  is illuminated by means of all the illumination sources  44   b , which generate the same illumination fields  56  as the illumination sources  44   a , and the method is carried out analogously for said illumination sources  44   b . However, the calibration method can also be carried out simultaneously for a plurality of spectral ranges used. 
         [0053]    With the use of a plurality of LEDs per illumination unit  22   a ,  22   b , each illumination field  56  can be varied in terms of its brightness not only altogether but also regionally, whereby the entire sample region  10  can be illuminated particularly uniformly with the aid of the calibration method. 
         [0054]    With the calibration of the illumination of the sample region  10  thus achieved, quantitative evaluations can be carried out in a particularly reliably reproducible and simple manner with the microscope  2 . 
         [0055]    The control unit  16  is provided for monitoring a process by means of the camera, to be precise for detecting image data before and during a marking process by means of dyes and for eliminating disturbing effects identified during and/or after the monitoring at least partly by means of an algorithm. Furthermore, the control unit is provided for carrying out a calibration on the basis of a reference object  62  arranged on a sample—as is indicated in  FIG. 5  and/or on the basis of a reference object  62 ′ separated from a sample on the object carrier  8 . 
         [0056]    A schematically illustrated method sequence is illustrated by way of example in  FIG. 6 . In a method step  70 , a sample is inserted into the sample region  10 . In a subsequent method step  72 , a calibration is carried out on the basis of a reference object  62  arranged on the sample. Afterward, in a method step  74 , before a marking of the sample with dyes, image data of the sample are detected in order that subsequently, in a method step  76 , disturbing effects identified are eliminated by software technology. Sample information items provided laterally alongside the sample are additionally detected in method step  74 . 
         [0057]    In a method step  78 , the sample is marked with dyes. Afterward, in a method step  80 , the illumination units  22   a ,  22   b  are activated, the spectral properties of the latter lying outside the absorbance ranges of the dyes used in the case of the sample, and measurement-optimizing settings are performed. In a method step  82 , the sample is irradiated by means of illumination sources  44   a ,  44   b —which are coordinated with a first dye—with a first wavelength range, image data are detected and the number of cells of the sample that are marked by the first dye is determined. 
         [0058]    In a method step  84 , the sample is irradiated by means of illumination sources  44   a ,  44   b -which are coordinated with a second dye—with a second wavelength range, which differs from the first wavelength range, image data are detected and the number of cells of the sample that are marked by the second dye is determined. 
         [0059]    Afterward, in a further method step  86 , the sample is simultaneously irradiated by means of illumination sources  44   a ,  44   b  with the differing wavelength ranges, in order to detect the total number of marked cells. Afterward, the detected data are evaluated within the control unit  16  in a method step  88  and, in a method step  90 , a final result of the analysis carried out is output by means of the output unit  92  indicated schematically. Instead of a detection of image data in method steps  82  to  86  with a stationary camera  14 , the latter can be moved over the sample by means of the mechanical adjusting unit  64  during the detection of image data manually and/or else advantageously in automated fashion. In principle, however, other sequences which appear to be practical to the person skilled in the art are also conceivable.