Abstract:
A device and method for adjusting mounting of a microscope stage ( 14 ), to which an objective ( 38 ) is mounted, to a microscope stand ( 12 ), including at least one first connecting element ( 56 ) arranged on the microscope stand ( 12 ), at least one second connecting element ( 62 ) arranged on the microscope stage ( 14 ) for mounting the microscope stage ( 14 ) to the microscope stand ( 12 ), at least one first mating part ( 76 ) arranged on the microscope stand ( 12 ), at least one positioning base ( 66 ) supporting a second mating part ( 74 ), wherein the positioning base ( 66 ) adjusts the platform ( 16 ) on the microscope stand ( 12 ) and is mounted movably in a plane of adjustment that is parallel to the platform ( 16 ) with the mating parts ( 74, 76 ) being engaged, and at least one locking device ( 80 ) for locking the positioning base ( 66 ) to the platform ( 16 ).

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority of German patent application number 10 2010 061 166.2 filed Dec. 10, 2010, the entire disclosure of which is incorporated by reference herein. 
       FIELD OF THE INVENTION 
       [0002]    The present invention relates generally to a device and a method for the adjusted mounting of a microscope stage to a microscope stand. 
       BACKGROUND OF THE INVENTION 
       [0003]    In the recent past, light microscopic methods have been developed with which, based on a sequential, stochastic localization of individual point objects, in particular fluorescence molecules, image structures can be imaged that are smaller than the diffraction-dependent resolution limit of conventional light microscopes. Such methods are, for example, described in WO 2006/127692 A2; DE 10 2006 021 317 B3; WO 2007/128434 A1, US 2009/0134342 A1; DE 10 2008 024 568 A1; “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)”, Nature Methods 3, 793-796 (2006), M. J. Rust, M. Bates, X. Zhuang; “Resolution of Lambda/10 in fluorescence microscopy using fast single molecule photo-switching”, Geisler C. et al, Appl. Phys. A, 88, 223-226 (2007). This new branch of microscopy is also referred to as localization microscopy. The applied methods are known in the literature, for example, under the designations (F) PALM ((Fluorescence) Photoactivation Localization Microscopy), PALMIRA (PALM with Independently Running Acquisition), GSD (IM) (Ground State Depletion (Individual Molecule return) Microscopy) or (F) STORM ((Fluorescence) Stochastic Optical Reconstruction Microscopy). 
         [0004]    The new methods have in common that the structures to be imaged are prepared with markers that have two distinguishable states, namely a “bright” state and a “dark” state. When, for example, fluorescent dyes are used as markers, then the bright state is a state in which they are able to fluoresce and the dark state is a state in which they are not able to fluoresce. For imaging image structures with a resolution that is higher than the conventional resolution limit of the imaging optical system, a small subset of the markers is repeatedly brought into the bright state and thus it is so to speak activated. In this connection, the activated subset is to be chosen such that the average distance of adjacent markers in the bright state is greater than the resolution limit of the imaging optical system. The luminance signals of the activated subset are imaged onto a spatially resolving light detector, e.g. a CCD camera. Thus, of each marker a light spot is detected whose size is determined by the resolution limit of the imaging optical system. 
         [0005]    In this way, a plurality of raw data single frames is captured, in each of which a different activated subset is imaged. Using an image analysis process, then in each raw data single frame the centroids of the light spots are determined which represent those markers that are in the bright state. Thereafter, the centroids of the light spots determined from the raw data single frames are combined to a total representation. The high-resolution image created from this total representation reflects the distribution of the markers. For a representative reproduction of the structure to be imaged sufficient signals have to be detected. Since however the number of markers in the respective activated subset is limited by the minimum average distance which two markers may have in the bright state, a great many raw data single frames have to be captured to completely image the structure. Typically, the number of raw data single frames is in a range between 10,000 and 100,000. 
         [0006]    The time required for capturing one raw data single frame has a lower limit that is predetermined by the maximum image capturing rate of the imaging detector. This results in relatively long total capturing times for a series of raw data single frames required for the total representation. Thus, the total capturing time can take up to several hours. 
         [0007]    Over this long total capturing time, a movement of the specimen to be imaged relative to the imaging optical system may occur. Since for creating a high-resolution total image all raw data single frames are combined after the determination of the centroids, each relative movement between specimen and imaging optical system that occurs during the capturing of two successive raw data singles frames impairs the spatial resolution of the total image. In many cases, this relative movement results from a systematic mechanical movement of the system, also referred to as mechanical drift, which movement is caused, for example, by thermal expansion or shrinkage, by mechanical strains or by the change in the consistency of lubricants used in the mechanical components. 
         [0008]    In the above-described high-resolution methods, it is of particular importance to ensure a drift-free positioning of the objective forming the imaging system relative to the specimen arranged on the platform. This can be achieved in that the objective is not, as usual, mounted to an objective revolver but directly to the platform. With such a design, the objective is arranged on the underside of the platform facing away from the specimen in the area of a through hole formed in the platform and images the specimen which is arranged on a specimen holder resting on the upper side of the platform through the through hole. As a result of the direct mounting of the objective to the platform, the path over which the objective is mechanically coupled to the specimen holder is relatively short, whereby a mechanical drift occurring between the objective and the specimen holder can largely be prevented. 
         [0009]    When the objective is directly mounted to the microscope stage, then, however, the alignment of the objective to the optical axis of the microscope stand is relatively complex. For instance, the microscope stage has to be re-adjusted relative to the microscope stand whenever it has been removed from the microscope stand for example for repair purposes. This is laborious and error-prone. 
         [0010]    From U.S. Pat. No. 3,572,889 A, a mechanism is known which serves to quickly mount different microscope stages to a microscope stand in an adjusted manner. This mechanism comprises an adjusting unit formed of a mounting bracket and a mounting plate arranged at the rear side of the mounting bracket. The mounting plate is fixed to a sliding element which is arranged vertically movably on the microscope stand. The microscope stage is coupled to the mounting bracket. The mounting plate and the mounting bracket have contact surfaces which allow a displacement of the mounting plate relative to the mounting bracket. 
         [0011]    In US 2005/0083569 A1, a microscope is described having a focusing column, a stage and a mechanism which enables the removal of the focusing column from the stage. This mechanism comprises a threaded shaft mounted to the focusing column and a knurled wheel which is provided with a matching thread and can be rotated within the stage to couple and decouple the focusing column to and from the stage. 
       SUMMARY OF THE INVENTION 
       [0012]    The invention is based on the object to specify a device and a method for the high-resolution light microscopy which enable an adjusted mounting of a microscope stage, to which an objective is mounted, to a microscope stand in an easy and reproducible manner. 
         [0013]    The invention solves this object by a device described herein having at least one first connecting element arranged on the microscope stand, at least one second connecting element which is arranged on the microscope stage and is connectable to the first connecting element for mounting the microscope stage to the microscope stand, at least one first mating part arranged on the microscope stand, at least one positioning base which is arranged on the underside of a platform of the microscope stage facing the microscope stand and supports a second mating part which is engageable with the first mating part, wherein the positioning base for adjusting the platform on the microscope stand is mounted movably in a plane of adjustment that is parallel to the platform with the mating parts being engaged, and at least one locking device for locking the positioning base to the adjusted platform. 
         [0014]    Thus, the invention provides to displace the platform in the plane of adjustment on the microscope stand, while a positioning base arranged on the underside of the platform is in an accurately fitting connection relative to the microscope stand via the engaging mating parts. For this, the positioning base is mounted movably in the plane of adjustment. This type of mounting can also be referred to as “floating”. 
         [0015]    When the desired positioning of the platform on the microscope stand has been attained, in which the objective mounted to the platform is precisely aligned to the optical axis of the microscope stand, the positioning base and thus the mating part held thereon is fixed to the platform by means of the locking device. Thus, the adjusted positioning of the platform on the microscope stand is defined in a reproducible manner. When the microscope stage is removed from the microscope stand for example for repair purposes, then it is subsequently simply re-placed on the microscope stand such that the mating parts engage with one another. A repeated adjustment is not necessary. 
         [0016]    In an advantageous development, a base receptacle is formed on the underside of the platform, in which the positioning base is arranged with clearance in the plane of adjustment. On the upper side of the platform facing away from the microscope stand, at least one insertion hole is formed which leads into the base receptacle. In the positioning base at least one recess is formed which is in communication with the insertion hole over the entire area in which the positioning base is movable in the plane of adjustment as a result of its clearance. The locking device comprises a clamp having a first clamping element seated in the base receptacle and a second clamping element which is seated in the insertion hole and can be tightened on the first clamping element to lock the positioning base arranged in the base receptacle between the two clamping elements. 
         [0017]    The part of the clamp arranged in the insertion hole or in the recess of the positioning base has a clearance in the plane of adjustment. In this embodiment, the positioning base is mounted in a floating manner in the recess formed in the platform. As a result thereof, the platform can be displaced on the microscope stand, while the positioning base is firmly seated on the microscope stand. As soon as the platform is adjusted, the positioning base can be locked in the recess by means of the clamp. For this, it is clamped between the two clamping elements. As the first clamping element is seated in the insertion hole formed on the upper side of the platform, it can be accessed from above. As a result thereof, the positioning base can easily be locked from above after the desired adjustment has been achieved. 
         [0018]    In a preferred development, at least one positioning bar is mounted to the microscope stand, at which positioning bar the first connecting element and the first mating part are arranged. The positioning bar can, for example, be mounted via standard mounting holes which are present on the microscope stand anyway for mounting the microscope stage. The positioning bar thus defines an interface for the mating parts to be engaged. It goes without saying that the inventive adjusting device does not necessarily require such an interface. In this case, the first mating part is to be provided directly on the microscope stand. 
         [0019]    According to a further aspect of the invention, a method for the adjusted mounting of a microscope stage to a microscope stand is described herein. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]    Exemplary embodiments of the present invention are described in more detail below with reference to the schematic drawings, in which: 
           [0021]      FIG. 1  shows a perspective view of a high-resolution light microscope as an embodiment; 
           [0022]      FIG. 2  shows a perspective view of the light microscope with lifted microscope stage; 
           [0023]      FIG. 3  shows a perspective bottom view of the microscope stage; 
           [0024]      FIG. 4  shows a view of the microscope stage, in which for illustrating the components forming the inventive adjusting device a part of the microscope stage is omitted; 
           [0025]      FIG. 5  shows a further view of the microscope stage, in which the adjusting device is partially sectionally illustrated; 
           [0026]      FIG. 6  shows a view in which a part of  FIG. 5  is illustrated to a larger scale; 
           [0027]      FIG. 7  shows further views corresponding to the view of  FIG. 6  and showing various adjusting states; and 
           [0028]      FIG. 8  shows a view of the microscope stage, in which the positioning bases and the locking clamps are shown from below. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0029]    In the following, at first the total structure of a high-resolution light microscope  10  is explained with reference to  FIGS. 1 to 3 . Here, only those components of the light microscope  10  are described which are essential for understanding the present invention. 
         [0030]    The light microscope  10  has a microscope stand  12  on which a microscope stage  14  is screwed. The microscope stage  14  has a platform  16  on which a specimen holder  18  rests. On the platform  16 , there is a positioning device which is generally identified with the reference sign  20  and which is composed of a first slider  22  and a second slider  24 . The two sliders  22  and  24  are mechanically decoupled from each other and serve to displace the specimen holder  18  on the platform  16  in two orthogonal directions. For this, two handwheels  26  and  28  are mounted to the underside of the platform  16 , as shown in  FIG. 3 . 
         [0031]    The light microscope  10  further has a pair of eyepieces  30  mounted to the microscope stand  12  as well as an objective revolver  32  holding several microscope objectives  34 . In the illustration of  FIG. 2 , which shows the light microscope  10  with the microscope stage  14  lifted from the microscope stand  12 , the objective revolver  32  which is otherwise covered by the microscope stage  14  is visible. 
         [0032]    As shown in the bottom view of  FIG. 3 , an objective  38  is arranged on the underside of the platform  16  below a through hole  36  formed in the platform  16 . The objective  38  is screwed into a focusing drive  40  via an objective thread. The focusing drive  40  is, for example, a piezo-ceramic actuator which moves the objective  38  along its optical axis for focusing on the specimen to be imaged. The focusing drive  40  is mounted to a holder  42  which is pivotable below the platform  16 . By means of a pivoted lever  48 , the holder  42  together with the objective  38  can be swiveled into and out of the imaging beam path of the light microscope  10 . 
         [0033]    As a result of the direct mounting of the objective  38  to the platform  16 , the path over which the objective  38  is mechanically coupled to the specimen holder  18  is relatively short. The occurrence of a mechanical drift between the objective  38  and the specimen holder  18  can thus largely be prevented. 
         [0034]    To guarantee a high-resolution imaging of the specimen by the objective  38 , it has to be ensured that the microscope stage  14  supporting the objective  38  is accurately positioned on the microscope stand  12 . In particular, the objective  38  has to be precisely aligned to the optical axis of the microscope stand  12 . 
         [0035]    The adjusting device described in the following makes it possible to mount the microscope stage  14  to the microscope stand  12  in a precisely adjusted manner. In particular, by means of the adjusting device it is made possible to re-mount the microscope stage  14  to the microscope stand  12  without having to re-adjust this stage after it had been removed from the microscope stand  12  for example for repair purposes (see  FIG. 2 ). 
         [0036]    As shown in the view of  FIG. 4 , in which parts of the platform  16  are omitted, the adjusting device comprises two positioning bars  52  and  54  which are mounted to the microscope stand  12 . In the view of  FIG. 2 , the positioning bar  54  mounted to the microscope stand  12  is partially visible. The positioning bars  52  and  54  are fixed at standard screw holes which are already present on the microscope stand  12  anyway to mount the microscope stage  14  (without the inventive adjusting device) to the microscope stand  12 . 
         [0037]    The positioning bars  52  and  54  are each mounted to the microscope stand  12  via two screw/nut elements  56  which are screwed into the afore-mentioned standard screw holes. As can be seen in the enlarged view of  FIG. 6 , the screw/nut elements  56  each have a threaded shaft  58  which is screwed into the associated standard screw hole of the microscope stand  12 , and a nut  60 . The nut  60  has an internal thread into which a fixing screw  62  can be screwed to mount the platform  16  of the microscope stage  14  on the respective positioning bar  52  and  54 , respectively. For this, through holes  64  into which the fixing screws  62  can be inserted are formed in the platform  16 . 
         [0038]    The inventive adjusting device further has two positioning bases  66  and  68  which are allocated to the two positioning bars  52  and  54 , respectively. As can be seen in the bottom view of  FIG. 8 , the positioning bases  66  and  68  are arranged in base receptacles  70  and  72 , respectively, which are formed in the underside of the platform  16 . As the two positioning bases  66  and  68  have the same structure and function, only the positioning base  66  and the components interacting therewith are described in detail in the following. 
         [0039]    As can be taken from the enlarged view of  FIG. 6 , the positioning base  66  supports a mating pin  74  which can be engaged with a mating hole  76  formed in the positioning bar  54 . The mating pin  74  and the mating hole  76  engage with each other in an accurately fitting manner, i.e. largely free of any clearance. 
         [0040]    The positioning base  66  is mounted in the base receptacle  70  in a floating manner. This means that the positioning base  66  (in the unlocked state) is movable within the base receptacle  70  in a plane of adjustment that is parallel to the platform  16 . When the mating pin  74  and the mating hole  76  are engaged, thus the platform  16  can be moved in the plane of adjustment relative to the positioning base  66  and thus to the microscope stand  12 . This freedom of movement of the platform  16  is used to align the microscope stage  14  together with the objective  38  held thereon precisely to the optical axis of the microscope stand  12 . 
         [0041]    As soon as the microscope stage  14  is adjusted to the microscope stand  12 , then the previously movable positioning base  66  can be locked in the base receptacle  70  by means of two locking clamps  80  having the same structure to thus secure the platform  16  relative to the positioning bar  54 . As shown in  FIG. 6 , the two locking clamps  80  have the same structure. Therefore, merely the left-hand side locking clamp  80  of  FIG. 6  is described in detail in the following. 
         [0042]    The locking clamp  80  comprises a clamping screw  82  and a clamping nut  84  screwed thereto. The clamping screw  82  is seated in an insertion hole  86  which is formed in the platform  16  and leads from above into the base receptacle  70 . The clamping nut  84  is arranged in the base receptacle  70 . 
         [0043]    On both sides of the mating pin  74 , the positioning base  66  has two recesses  90 . In the cross-sectional view of  FIG. 6 , the recesses  90  are each formed in a stepped manner. Each recess  90  is composed of a connecting portion  92 , which is connected with the associated insertion hole  86 , and a contact portion  94  facing the positioning bar  54 . In these two portions  92  and  94  of the recess  90 , the clamping nut  84  is arranged. Thus, the clamping nut  84  has a cylindrical coupling part  96  which is arranged in the connecting portion  92  of the recess  90  as well as a contact part  98  which substantially has the form of a rectangular cuboid and is seated in the contact portion  94  of the recess  90 . The contact part  98  of the clamping nut  84  is expanded relative to the coupling part  96 . 
         [0044]    The coupling part  96  of the clamping nut  84  has an internal thread into which the clamping screw  82  can be screwed. When the clamping screw  82  is screwed into this internal thread, then it tightens the contact part  98  of the clamping nut  84  on the contact portion  94  of the recess  90 . As a result thereof, the positioning base  66  is locked to the platform  16 . 
         [0045]    In the embodiment shown in  FIG. 6 , the clamping nut  84  has a clearance in the plane of adjustment in the unlocked state, whereas the clamping screw  82  is seated in the insertion hole  86  in a largely clearance-free manner. However, this embodiment is only exemplarily. It is likewise possible to provide the clamping screw  82  with clearance by way of a suitable dimensioning of the insertion hole  86  and to arrange the clamping nut  84  fixedly on the positioning base  66 . For adjusting purposes, it must only be guaranteed that the positioning base  66  has a clearance in the plane of adjustment in the base receptacle  70  so that the platform  16  can be displaced relative to the positioning base  66  which is spatially fixed to the positioning bar  54  in that the mating pin  74  engages with the mating hole  76  in an accurately fitting manner. 
         [0046]    As can further be seen in  FIG. 6 , the fixing screw  62 , too, is arranged in the through hole  64  such that there is clearance in the plane of adjustment. This clearance has to be dimensioned such that the fixing screw  62  can be screwed into the nut  60  within the entire area in which the platform  16  can be moved relative to the microscope stand  12  for adjusting purposes. 
         [0047]    The afore-explained relations are once again illustrated in  FIG. 7  where different adjusting states are shown. Here, the arrows each indicate the displacement of the platform  16  on the positioning bar  54  and thus on the microscope stand  12 . These displacements are reflected in corresponding movements of the locking clamps  80  within the base receptacle  70  relative to the positioning base  66 . 
         [0048]    For mounting the microscope stage  14  in an adjusted manner to the microscope stand  12 , at first the platform  16  is placed on the positioning bars  52 ,  54 , which are firmly mounted to the microscope stand  12 , such that the mating pins  74  present on the positioning bases  66  engage with the associated mating holes  76  formed in the positioning bars  52 ,  54 . Since the mating pins  74  and the mating holes  76  engage with one another largely free of clearance, the positioning bases  66  and the positioning bars  52 ,  54  (and thus the microscope stand  12 ) have a fixed spatial arrangement to one another. Then, the platform  16  is displaced on the positioning bars  52 ,  54  in the plane of adjustment until the objective  38  mounted to the platform  16  is precisely aligned to the optical axis of the microscope stand. Thereafter, the locking clamps  80  are tightened to fix the positioning bases  66  to the platform  16 . As a result, the mating pins  74  are fixed relative to the platform  16 . Finally, the fixing screws  62  are screwed into the screw/nut elements  56 . 
         [0049]    When the microscope stage  14  which is mounted to the microscope stand  12  in an adjusted manner is again removed later on for example for repair purposes by releasing the fixing screws  62 , then it does not have to be re-adjusted when it is mounted the next time as the mating pins  74  are fixed to the platform  16  in a correctly positioned manner. 
         [0050]    The invention is not to be limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the invention. 
       LIST OF REFERENCE NUMERALS 
       [0000]    
       
         
           
               10  light microscope 
               12  microscope stand 
               14  microscope stage 
               16  platform 
               18  specimen holder 
               20  positioning device 
               22  first slider 
               24  second slider 
               26 ,  28  handwheel 
               30  eyepiece 
               32  objective revolver 
               34  microscope objective 
               36  through hole 
               38  objective 
               40  focusing drive 
               42  holder 
               48  pivoted lever 
               52 ,  54  positioning bar 
               56  screw/nut element 
               58  threaded shaft 
               60  nut 
               62  fixing screw 
               64  through hole 
               66 ,  68  positioning base 
               70 ,  72  base receptacle 
               74  mating pin 
               76  mating hole 
               80  locking clamp 
               82  clamping screw 
               84  clamping nut 
               86  insertion hole 
               90  recess 
               92 ,  94  portions 
               96  coupling part 
               98  contact part