Abstract:
A microscope includes an interference contrast transmitted-light device having an analyzer disposed in the microscope imaging beam path, the analyzer causing a beam deflection. A fluorescence device is provided, the fluorescence device and the interference contrast transmitted-light device being selectably and alternatively insertable into the imaging beam path. A pair of glass wedge plates are arranged behind the analyzer in the imaging direction so as to compensate to zero for the beam deflection caused by the analyzer.

Description:
Priority is claimed to German patent application 103 21 091.1, the subject matter of which is hereby incorporated by reference herein. 
     The invention concerns a microscope having an interference contrast transmitted-light device that includes an analyzer in the imaging beam path. The invention further concerns a microscopy method for producing overlay images. 
     BACKGROUND 
     In microscopic examinations and documentation, it often happens that multiple exposures and assembled images that depict different aspects of the specimen are produced. The images thus produced are generated using various contrasting methods or various microscopy methods, and then combined into one image. The various image constituents are then displayed, for example, using so-called “false colors.” For example, the samples to be examined are often marked with various fluorescent dyes that can be excited and observed using different filter combinations. The images achieved in this fashion, each produced with a specific filter combination, are subsequently assembled into a single cumulative image. 
     The case considered here refers to the combination of images acquired by means of interference contrast transmitted-light (ICT) microscopy and fluorescence microscopy. The image generated with the ICT method then shows the unstained biological specimen as a whole. The image produced by means of fluorescence microscopy, on the other hand, shows only particular and specific markings at certain points. The images produced with the two methods are then assembled into one overlay image. 
     The overlay images produced in this fashion are each imaged onto a camera. It is desirable, in producing an overlay image of this kind, for the ICT image to be located on the CCD chip of the camera at exactly the same point as the fluorescence images or a bright-field transmitted-light image. The reason is that if the images are imaged onto the CCD chip with an offset from one another, evaluation of the image is negatively affected. 
     In the ICT method, a polarizing filter that serves as the analyzer is located in the imaging beam path of the microscope. This analyzer is usually arranged between the microscope objective and the tube lens. Since the analyzer has a transmissivity of only approx. 30% for unpolarized light, it is also usually introduced into the beam path only for the ICT method. For the fluorescence measurement or fluorescence observation, in which weak intensities generally occur, the analyzer is removed from the beam path. 
     Polarizing filters of planar configuration, such as those used in microscopy, usually comprise a stretched polarizing film that is cemented between two glass plates. Such polarizing filters have the disadvantage that incident light is slightly deflected. The reasons for this beam deflection include the stretched polarizing film itself, wedge-shaped cemented surfaces, and possibly wedge-shaped glass plates. Known polarizing filters of this type thus produce beam deflections of up to 3 minutes. If the microscope has, for example, a tube lens with a focal length of 200 millimeters, an analyzer of this kind with a beam deflection of 3 minutes produces approximately a 175-μm offset of the intermediate image of the sample, With a CCD camera having a pixel size of approx. 8 μm, the result is that the analyzer causes the image to be offset by approximately 20 pixels. 
     Until now, the offset produced on the CCD chip between the ICT image and the fluorescence image had to be tolerated, meaning that the associated loss in the quality of the overlay images necessarily had to be accepted. Alternatively, the image offset of the individual images in the overlay image was compensated for using software in the context of digital image acquisition or the processing of the overlay images, which required appropriate software and hardware and is time-consuming. 
     SUMMARY OF THE INVENTION 
     It is therefore an object of the present invention to provide a microscope in which the undesirable offset of the individual images in the overlay image is eliminated. 
     The present invention provides a microscope having an interference contrast transmitted-light device that encompasses an analyzer in the imaging beam path. The microscope includes a fluorescence device, the fluorescence device and the interference contrast transmitted-light device being selectably and alternatively insertable into the imaging beam path. Two glass wedge plates are additionally arranged behind the analyzer in the imaging direction, and are embodied and oriented in such a way that the total beam deflection of the two glass wedge plates compensates to zero for the beam deflection produced by the analyzer. 
     According to the present invention, two glass wedge plates are additionally arranged in the imaging beam path between the analyzer and the camera, i.e. immediately behind the analyzer in the imaging direction. The analyzer comprises an aligned polarizing filter, meaning that this polarizing filter is deliberately oriented (e.g. in the north/south direction) in terms of the vibration direction that is allowed to pass. The glass wedge plates can, in principle, be different. It has proven advantageous in terms of alignment, however, if two identical glass wedge plates are used. The glass wedge plates have a wedge angle α (=alpha) and impose on the passthrough beam a beam deflection δ (=delta), δ corresponding to approximately half the wedge angle. The wedge angle α and thus the beam deflection δ of the two glass wedge plates are selected so that δ is approximately half the magnitude of the maximum deflection resulting from the polarizing filter. This means that δ is equal to approximately 1.5′ (=minutes). 
     During assembly, the two glass wedge plates are first mounted rotatably with respect to one another and with respect to the (already secured) polarizing filter. An alignment of the two glass wedge plates then takes place during manufacture of the microscope. This can be achieved, for example, using an autocollimation device with which very small beam deflections (on the order of one second) can be measured. In this operation, the glass wedge plates are aligned in such a way that the beam deflection of the two glass wedge plates compensates for the beam deflection of the polarizing filter. The two glass wedge plates are then immobilized in that position and thus prevented from rotating further. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The present invention will be explained in more detail below with reference to the schematic drawings. 
         FIG. 1  shows the arrangement of the two glass wedge plates directly behind the analyzer. 
         FIG. 2  is a vectorial depiction of the beam deflection in the arrangement shown in  FIG. 1 . 
         FIG. 3  shows a microscope having a fluorescence device in the working position. 
         FIG. 4  shows a microscope having in the working position an arrangement to compensate for the image offset produced by the analyzer. 
     
    
    
     DETAILED DESCRIPTION 
     In  FIG. 1 , a polarizing filter is arranged as analyzer  1 . The vibration direction that is allowed to pass is indicated schematically by a double arrow. Arranged directly behind analyzer  1  in the direction of the camera (not depicted), i.e. in the imaging direction, are a first glass wedge plate  2  and a second glass wedge plate  3 . They are located one above another within a receiving ring  4 , first glass wedge plate  2  and second glass wedge plate  3  being separated from one another by a spacer ring  5 . Spacer ring  5  permits a rotation of first glass wedge plate  2  with respect to second glass wedge plate  3  and vice versa. Glass wedge plates  2  and  3  can in this fashion be aligned with respect to one another in such a way that the total beam deflection cumulatively generated by the two glass wedge plates  2  and  3  together exactly compensates for the beam deflection of analyzer  1 . Analyzer  1  and glass wedge plates  2  and  3  are arranged on a common mount  40 . 
       FIG. 2  shows the beam deflections that occur in the arrangement of  FIG. 1 . Vectors are used for the illustration. Vector  6  reproduces the deflection as a result of analyzer  1 ; in this example, vector  6  is depicted as approx. ⅔ of the maximum possible deflection. Vector  7  shows the beam deflection resulting from first glass wedge plate  2 . Vector  8  shows the beam deflection resulting from second glass wedge plate  3 . Vectorial addition of vector  7  and vector  8  yields vector  9 , which indicates the resulting total beam deflection due to the combined action of first glass wedge plate  2  and second glass wedge plate  3 . As shown, the magnitude of vector  9  (i.e. the total beam deflection due to the two glass wedge plates  2  and  3 ) and the magnitude of vector  6  (i.e. the beam deflection resulting from analyzer  1 ) are identical. Since vector  6  and vector  9  are oppositely directed, the beam deflection of analyzer  1  is thus compensated for by means of the two glass wedge plates  2  and  3 . 
       FIG. 3  shows a microscope having a fluorescence device in the working position. A transmitted-light beam  12  proceeds from a transmitted-light source  11 . The light of transmitted-light source  11  passes successively through an illumination optical system  13 , a polarizer  14 , a first Wollaston prism  15 , and a condenser  16 . The beam emerging from condenser  16  illuminates a sample  17  that is placed on a microscope stage  18 . 
     The light passing through sample  17  is captured by an objective  19  and is imaged by it into an intermediate image plane  20 . Between objective  19  and intermediate image plane  20 , the beam passes through a second Wollaston prism  21  and a tube optical system  22  which, inter alia, determines the position of intermediate image plane  20 . 
     Arranged between second Wollaston prism  21  and tube optical system  22  is a device changer  23  that has at least two regions A and B for the reception of devices. In the embodiment depicted here, device changer  23  is mounted rotatably about a rotation axis  24 , such that by rotation about rotation axis  24 , one of the two regions A or B can be alternatively introduced into the beam path. In another embodiment (not depicted here), device changer  23  is constructed as a slider displaceable laterally in the beam path. Device changer  23  can furthermore comprise additional regions (not depicted here) having further devices, for example filter devices. 
     In the setting of device changer  23  shown here, its region A is located in the beam path of the microscope. A fluorescence device is arranged in this region. It comprises an excitation filter  25 , a semitransparent beam splitter  26 , and a barrier filter  27 . This fluorescence device is also referred to as a fluorescence cube. 
     To allow sample  17  to be examined using the fluorescence device, an incident-light beam  28  is conveyed to the fluorescence device. That beam proceeds from an incident-light source  29  and passes through an incident-light illumination optical system  30  having several lens elements  31  and apertures  32 . The light of incident-light beam  28  enters region A laterally and passes through excitation filter  25 , which permits only certain spectral fluorescence wavelength regions of the illuminating light to pass. 
     The incident light is then deflected by means of beam splitter  26  toward objective  19  and is directed through objective  19  onto sample  17 . The incident light produces a fluorescence excitation in specific fluorochromes introduced into sample  17 . From sample  17 , the light travels through objective  19 , second Wollaston prism  21 , beam splitter  26 , and barrier filter  27 , into intermediate image plane  20 . The image of sample  17  generated there can be viewed by means of one or more eyepieces  33 . The image is also imaged onto CCD chip  34  of a camera  35 . 
       FIG. 4  shows the microscope of  FIG. 3  with an interference contrast transmitted-light device arranged entirely in the beam path, a compensation for the beam deflection of analyzer  1  being performed according to the present invention. 
     As already described, transmitted-light beam  12 , in which illuminating optical system  13 , one polarizer  14 , first Wollaston prism  15 , and condenser  16  are arranged, proceeds from transmitted-light source  11 . The light of transmitted-light beam  12  illuminates sample  17  and is imaged by objective  19  into intermediate image plane  20 . Behind objective  19 , the beam passes through second Wollaston prism  21  and enters device changer  23 . 
     With device changer  23  in the position depicted here, its region B is located in the beam path of the microscope. Device changer  23  is closed off at the side so that incident light from incident-light beam  28  cannot enter region B. An analyzer  1  is arranged in region B of device changer  23 . This analyzer  1 , together with polarizer  14 , first Wollaston prism  15  and second Wollaston prism  20 , constitutes an interference contrast transmitted-light device. 
     This analyzer  1  comprises a polarizing film and produces a beam offset of the beam passing through, as explained previously. This beam offset results in an image offset of the interference contrast transmitted-light image on CCD chip  34  of camera  35  with respect to the fluorescent image generated in the microscope setting shown in  FIG. 3 . 
     According to the present invention, this image offset is now compensated for optically. For that purpose, two glass wedge plates referred to as first glass wedge plate  2  and second glass wedge plate  3  are additionally arranged immediately behind analyzer  1  in the imaging direction. Glass wedge plates  2  and  3  are embodied and oriented in such a way that the total beam deflection generated by the two glass wedge plates  2  and  3  together compensates to zero for the beam deflection occurring behind analyzer  1 . 
     The image offset on CCD chip  34  of camera  35  of the interference contrast transmitted-light image with respect to the fluorescence image is thus compensated for. With precise alignment of the glass wedge plates  2  and  3  with respect to one another and with respect to analyzer  1 , the interference contrast transmitted-light image and the fluorescence image are imaged on CCD chip  34  of camera  35  with accurate superposition of each pixel. 
     An overlay image is generated in the following fashion: 
     A first microscope image is generated using the interference contrast transmitted-light device, and acquired with the camera. 
     A second microscope image is then generated using the fluorescence device, and likewise acquired with the camera. 
     Since compensation for the image offset of the first microscope image with respect to the second microscope image is accomplished with optical means, by the fact that according to the present invention, the two glass wedge plates  2 ,  3  are arranged in the interference contrast transmitted-light device directly behind the analyzer  1  in the imaging direction, an overlay image can be produced directly. The overlay image is produced by superimposition of the first microscope image and second microscope image, by transferring the first microscope image and the second microscope image respectively into an image memory and adding (stacking) them there to within pixel accuracy. The possibility also exists of adding up several (i.e. more than two) images of the aforesaid type, and performing further image processing steps. 
     The invention thus offers the advantage that a complex software-assisted compensation for the image offset is no longer necessary. Superimposition of the interference contrast transmitted-light image and the fluorescence image to within pixel accuracy results in an overlay image whose image quality is greatly improved, and which permits more accurate evaluation of the resulting overlay image. 
     PARTS LIST 
     
         
           1  Analyzer 
           2  First glass wedge plate 
           3  Second glass wedge plate 
           4  Receiving ring 
           5  Spacer ring 
           6  Vector of beam deflection by analyzer  1   
           7  Vector of beam deflection by first glass wedge plate 
           8  Vector of beam deflection by second glass wedge plate 
           9  Vector of total beam deflection by both glass wedge plates  2 ,  3   
           10  Vector of maximum beam deflection by analyzer  1   
           11  Transmitted-light source 
           12  Transmitted-light beam 
           13  Illumination optical system 
           14  Polarizer 
           15  First Wollaston prism 
           16  Condenser 
           17  Sample 
           18  Microscope stage 
           19  Objective 
           20  Intermediate image plane 
           21  Second wollaston prism 
           22  Tube optical system 
           23  Device changer 
           24  Rotation axis 
           25  Excitation filter 
           26  Semitransparent beam splitter 
           27  Barrier filter 
           28  Incident-light beam 
           29  Incident-light source 
           30  Incident-light illumination optical system 
           31  Lens elements 
           32  Aperture 
           33  Eyepieces 
           34  CCD chip 
           35  Camera