Patent Abstract:
A sample retainer for a microscope is described, comprising a sample stage ( 32 ), a holder ( 34 ) arranged on the sample stage ( 32 ), a sample carrier ( 36 ), couplable to the holder ( 34 ), to which a sample is attachable, and an adjusting apparatus ( 44 ), engaging on the holder ( 34 ), with which with the sample carrier ( 36 ), together with the holder ( 34 ) to which the sample carrier ( 36 ) is coupled, is displaceable on the sample stage ( 32 ), relative to the objective ( 46 ), into a target position. A decoupling apparatus that decouples the sample carrier ( 36 ), arranged in the target position, from the holder ( 34 ) upon imaging of the sample through the objective ( 46 ) is provided.

Full Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is the U.S. National Stage of International Application No. PCT/EP2011/052797 filed Feb. 25, 2011, which claims priority of German Application No. 10 2010 009 679.2 filed Mar. 3, 2010. The present application claims priority benefit of International Application No. PCT/EP2011/052797 and German Application No. 10 2010 009 679.2 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to a sample retainer. 
       BACKGROUND OF THE INVENTION 
       [0003]    In the recent past, light-microscopy methods have been developed with which, based on a sequential, stochastic localization of individual point objects (in particular, fluorescence molecules), it is possible to display image structures that are smaller than the diffraction-limited resolution limit of classic light microscopes. Such methods are described, for example, 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 methods applied are known in the literature, for example, under the designations PALM, FPALM, (F)STORM, PALMIRA, or GSDIM. 
         [0004]    The new methods have in common the fact that the structures to be imaged are prepared with markers that possess two different states, namely a “bright” state and a “dark” state. For example, if fluorescent dyes are used as a marker, the bright state is then a fluorescence-capable state and the dark state is a non-fluorescence-capable state. In order for image structures to be imaged at a resolution that is smaller than the classic resolution limit of the imaging optical system, a small subset of the markers is then repeatedly prepared into the bright state. This subset is referred to hereinafter as an “active subset.” The active subset must be selected so that the average spacing between adjacent markers in the bright state is greater than the resolution limit of the imaging optical system. The luminance signals of the active subset are imaged onto a spatially resolving light detector, e.g. a CCD camera. A spot of light whose size is determined by the resolution limit of the imaging optical system is therefore acquired from each marker. 
         [0005]    The result is that a plurality of individual raw-data images are acquired, in each of which a different active subset is imaged. In an image analysis process, the center points of the spots of light (representing the markers that are in the bright state) are then determined in each individual raw-data image. The center points of the spots of light ascertained from the individual raw-data images are then combined into one overall depiction. The high-resolution image produced by this overall depiction reflects the distribution of the markers. For a representative reproduction of the structure to be imaged, a sufficiently large number of signals must be detected. But because the number of markers in the particular active subset is limited by the minimum average spacing that must exist between two markers in the bright state, a very large number of individual raw-data images must be acquired in order to image the structure completely. The number of individual raw-data images is typically in a range from 10,000 to 100,000. 
         [0006]    The time required for acquiring an individual raw-data image is limited at the low end by the maximum image acquisition rate of the imaging detector. This leads to comparatively long total acquisition times for a series of individual raw-data images that is necessary for the overall depiction. The total acquisition time can thus amount to as much as several hours. 
         [0007]    Movement of the sample being imaged, relative to the image-producing optical system, can occur over this long total acquisition time. Because all the individual raw-data images must be combined, after center-point determination, in order to create a high-resolution overall image, any relative motion between the sample and the image-producing optical system that occurs during the acquisition of two successive individual raw-data images degrades the spatial resolution of the overall image. In many cases this relative motion derives from a systematic mechanical motion of the system (also referred to as “mechanical drift”) that is caused, for example, by thermal expansion or contraction, by mechanical stresses, or by a change in the consistency of lubricants that are used in the mechanical components. 
         [0008]    The effects described above will be illustrated below with reference to a conventional inverted light microscope, as depicted in  FIG. 1 . The microscope according to  FIG. 1  has a U-shaped stand  2  to whose limbs a sample retainer  4  is attached. Sample retainer  4  encompasses a sample stage  6  and a holder  8 , arranged on sample stage  6 , on which a sample carrier (not further depicted in  FIG. 1 ) having a sample is fastened. Located below sample stage  6  is an objective turret  10  having multiple objectives  12  that can be pivoted selectably into an imaging beam path that passes through a through hole  14  embodied in sample stage  6 . The imaged sample can be viewed through an eyepiece  16 . Also located on stand  2  is a port  18  at which a detector, e.g. a CCD camera, can be connected. 
         [0009]    In order to select the sample region to be imaged, holder  8  can be moved, together with the sample carrier fastened to it, laterally (i.e. perpendicularly to the imaging beam path) on sample stage  6 . A mechanical adjusting apparatus  20 , depicted entirely schematically in  FIG. 1 , is provided for this. One problem here is that adjusting apparatus  20  is usually not embodied in as drift-stable a manner as is necessary for acquisition of the above-described high-resolution overall image using localization microscopy. If a mechanical drift occurs in adjusting apparatus  20 , it is transferred to holder  8 , which ultimately results in a lateral relative motion between the sample and objective  12  arranged in the imaging beam path, and thus in drifting of the individual raw-data images assembled into the overall image. 
         [0010]    This kind of image drift in the individual raw-data images is also caused by the attachment of objective turret  10  to the U-shaped stand  2 . As a result of this conventional arrangement, for example, the image drift-relevant distance between the imaging objective  12  and the sample arranged on holder  8  is comparatively large, since the sample is coupled to objective  12  via sample holder  8 , sample stage  6 , the U-shaped stand  2 , and objective turret  10 . Because of this comparatively long distance, the microscope according to  FIG. 1  is susceptible to thermal instabilities and mechanical stresses that “add up” over the distance. The comparatively complex mechanism of objective turret  10  is also susceptible to drift. 
         [0011]    Reference is further made to US 2004/0051978 A1, DE 11 2005 000 017 B4, and DE 1 847 180 U regarding the existing art. 
       SUMMARY OF THE INVENTION 
       [0012]    The object of the invention is to describe a sample holder for a microscope that is sufficiently drift-stable even in long-term operation. 
         [0013]    The sample retainer described herein provides a decoupling apparatus that decouples the sample carrier, arranged in the target position upon imaging of the sample through the objective, from the holder. This decoupling of the holder and sample carrier prevents mechanical drift that occurs, upon imaging of the sample, in the adjusting apparatus engaging on the holder, from being transferred to the sample carrier and therefore to the sample itself. According to the present invention the sample carrier is thus “free” upon imaging of the sample, i.e. it remains unaffected by the drift-susceptible adjusting apparatus which serves to displace the sample carrier, together with the holder, into a target position in order to image the sample through the objective. 
         [0014]    The sample retainer according to the present invention is accordingly advantageously usable in particular in localization microscope as recited earlier, which of course is particularly susceptible to mechanical drifting. It is nevertheless self-evident that the sample retainer according to the present invention is also suitable for other applications in which it is important to minimize the influence on sample imaging quality of mechanical drift occurring in the sample retainer. 
         [0015]    The decoupling apparatus preferably encompasses at least one holding element, which is part of the holder and engages on the sample carrier upon displacement of the holder, and is disconnected from the sample carrier upon imaging of the sample through the objective. With this embodiment the decoupling apparatus is, as it were, integrated in the form of the holding element into the holder. The holding element has two functions here: on the one hand to position the sample carrier in positionally stable fashion on the holder upon displacement of the holder, and on the other hand to decouple the sample carrier and the holder from one another upon imaging of the sample. 
         [0016]    The holding element is, for example, an arm abutting laterally against the sample carrier and pivotable parallel to the sample stage. Alternatively, the holding element can also be a pin abutting laterally against the sample carrier and shiftable perpendicular to the sample stage. In the latter configuration, the holder preferably encompasses a frame, arranged above the sample carrier, on which the pin or pins is/are shiftably mounted. 
         [0017]    The adjusting apparatus preferably moves the holder, upon imaging of the sample through the objective, away from the decoupled sample carrier arranged in the target position. This allows the transfer of mechanical drift from the adjusting apparatus via the holder to the sample carrier to be avoided even more reliably. 
         [0018]    The alternative approach according to claim  7  provides a pressure apparatus that, upon imaging of the sample through the objective, presses the sample carrier, coupled to the holder and arranged in the target position, against the sample stage. The pressure force exerted by the pressure apparatus on the sample carrier is to be dimensioned so that it holds the sample carrier in positionally stable fashion in the target position even if drift forces act via the holder on the sample carrier as a consequence of a mechanical drift occurring in the adjusting apparatus. 
         [0019]    In a preferred embodiment, the pressure apparatus encompasses at least two elements interacting magnetically with one another, of which one is arranged on the sample holder and the other on the sample stage. For example, one of the elements is ferromagnetic while the other is a permanent magnet or electromagnet. Other configurations are, however, also conceivable, for example a pressure apparatus that presses the sample carrier onto the sample stage with a spring or a clamp. 
         [0020]    The sample retainer comprises an objective holder, attached to the sample stage, to which the objective is attachable. An objective holder of this kind makes it possible to keep the image drift-relevant distance between the objective and sample as short as possible, so that thermal instabilities and mechanical stresses occurring over that distance have less of an effect in terms of drift than is the case with a conventional arrangement as shown, for example, in  FIG. 1 . 
         [0021]    The objective holder is preferably part of a positioning apparatus which serves to focus the objective onto the sample. The positioning apparatus can form, for example, a substantially L-shaped arrangement that encompasses a first limb attached to the sample stage and arranged parallel to the optical axis of the objective, and a second limb, mounted shiftably on the first limb, to which the objective is attached. The L-shaped embodiment of the positioning apparatus makes it possible to bring the first limb of the arrangement as close as possible to the optical axis of the objective, and thereby to minimize the image drift-relevant distance between the objective and sample. 
         [0022]    In a further advantageous embodiment, a guidance apparatus is provided, with which the objective holder is guided movably on the sample stage and is thus removable from a working region in which the objective holder holds the objective in an imaging beam path. This makes it possible to provide in the microscope, in addition to the objective holder according to the present invention, an objective turret that is moved as necessary into the working region when the objective holder is removed from the latter. “Working region” means in this connection that region, usually located below the sample stage, through which the imaging beam path passes and in which the objective is focused onto the sample. This configuration thus offers the capability of working selectably with a conventional objective turret or, if the requirements in terms of image accuracy are particularly stringent, with the objective holder according to the present invention. 
         [0023]    The guidance apparatus preferably has a guidance groove embodied on the underside of the sample stage, and a carriage, guided in said groove, that is coupled to the first limb of the positioning apparatus. The positioning apparatus can thus be particularly easily removed from the working region along the sample stage. In a particularly simple embodiment, the carriage is embodied integrally with the first limb of the positioning apparatus. 
         [0024]    An elongated recess, in which the objective is movable upon removal of the objective holder from the working region, is preferably embodied on the underside of the sample stage. This configuration is provided for the case in which the objective is located inside a through hole, passing through the sample stage, over which the sample carrier is placed. In this case it is not necessary firstly to move the objective out of said through hole in order to remove the objective holder from the working region. The objective can instead simply be moved in the recess transversely to the imaging beam path. This makes possible a particularly compact construction. 
         [0025]    In an alternative embodiment, the positioning apparatus forms an arrangement, rotationally symmetrical around the optical axis of the objective, that encompasses an annular part which is attached to the sample stage and whose center axis coincides with the optical axis of the objective, and a circular plate, arranged shiftably on the annular part, to which the objective is centeredly attached. This rotationally symmetrical construction reduces disadvantageous influences that are caused by drifting of the mechanical components transversely to the imaging beam path. 
         [0026]    The positioning apparatus is preferably a piezoelectrically driven apparatus. As such, it has a priori comparatively little susceptibility to drift. 
         [0027]    In a further advantageous embodiment, a shield surrounding the arrangement made up of the objective holder and objective is provided, for example for protection from drafts. A shield of this kind can also be provided for the sample itself, so as to protect it, too, from drafts. 
         [0028]    According to a further aspect of the invention, a microscope in particular for use in localization microscopy, which is equipped with the sample retainer described above, is provided. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0029]    The invention will be described below on the basis of exemplifying embodiments with reference to the Figures, in which: 
           [0030]      FIG. 1  shows a conventional inverted microscope; 
           [0031]      FIG. 2  is a perspective view of a sample retainer according to the present invention according to a first exemplifying embodiment; 
           [0032]      FIG. 3  is a side view of the sample retainer according to the first exemplifying embodiment; 
           [0033]      FIG. 4  is a plan view of the sample retainer according to the first exemplifying embodiment; 
           [0034]      FIG. 5  is a side view of a sample holder according to the present invention according to a second exemplifying embodiment; 
           [0035]      FIG. 6  is a plan view of the sample holder according to the second exemplifying embodiment; 
           [0036]      FIG. 7  is a side view of a sample holder according to the present invention according to a third exemplifying embodiment; 
           [0037]      FIG. 8  is a plan view of the sample holder according to the third exemplifying embodiment; 
           [0038]      FIG. 9  is a side view of a sample holder according to the present invention according to a fourth exemplifying embodiment; 
           [0039]      FIG. 10  is a plan view of the sample holder according to a fifth exemplifying embodiment. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0040]      FIGS. 2 to 4  each show parts of a sample retainer intended for a microscope, according to a first exemplifying embodiment. 
         [0041]    As depicted in  FIG. 2 , the sample retainer encompasses a sample stage  32  and a holder  34  mounted laterally movably on the sample stage. Holder  34  serves to immobilize a sample carrier  36  on which a sample (not shown) can be arranged. 
         [0042]    Holder  34  comprises an approximately U-shaped frame  38  on which sample carrier  36  rests. An arm  40  is mounted on frame  38  pivotably around an axis  42 . When arm  40  is resting with its free end on sample carrier  36 , it presses the latter against that part of frame  38  which is located opposite it, with the result that sample carrier  36  is clamped in place on holder  34 . 
         [0043]    An adjusting apparatus  44  (depicted purely schematically in  FIG. 3 ) is provided for moving holder  34  laterally on sample stage  32 . Holder  34  is also depicted in simplified fashion (without sample carrier  36  and pivot arm  40 ) in  FIG. 3 . Adjusting apparatus  44  engages on holder  34 , in particular on its frame  38 , in order to move holder  34  into a target position in which the sample arranged on sample carrier  36  is arranged as desired in an imaging beam path that is defined by the optical axis of an objective  46  (see  FIG. 3 ). Objective  46  is held on a positioning apparatus  48 , L-shaped in the side view shown in  FIG. 3 , which serves to focus objective  46  onto the sample. Objective  46  projects, with its end facing toward the sample, into a through hole  50  that passes through sample stage  32 . 
         [0044]    Positioning apparatus  48  comprises a first limb  52  that is attached to sample stage  32  and is arranged parallel to the optical axis of objective  46 , and a second limb  54 , mounted shiftably on first limb  52 , to which objective  46  is attached and which is located perpendicular to the optical axis of objective  46 . Second limb  54  can be moved, via a piezoelectric drive (not shown), along first limb  52  in order to focus objective  46  onto the sample. 
         [0045]    In order to establish the region of the sample to be imaged, adjusting apparatus  44  moves holder  34  together with sample carrier  36  fastened to it, under the control of a precision control system (not shown), into a desired target position. In this context, arm  40  presses sample carrier  36  against frame  38 , with the result that sample carrier  36  is positioned fixedly in holder  34 . Once the desired target position has been reached, arm  40  is pivoted laterally (to the left in  FIG. 2 ) around axis  42  parallel to sample stage  32 , so that it detaches from sample carrier  36 . Adjusting apparatus  44  then moves holder  34  slightly away from sample carrier  36  so that sample carrier  36  is no longer resting on frame  38  of holder  34 . In this state, sample carrier  36  is thus lying freely on sample stage  32  (over passthrough hole  50 ). It is thereby possible to reliably avoid the transfer, via holder  34  onto sample carrier  36 , of any mechanical drifting motion that occurs in the mechanical components of the adjusting apparatus engaging on holder  34 . Imaging of the sample in a manner largely uninfluenced by mechanical drift is thus possible. 
         [0046]    In this exemplifying embodiment arm  40  thus constitutes both a part of holder  34  and a decoupling apparatus which serves to decouple sample carrier  36  from holder  34  as soon as sample carrier  36  has reached its target position in which the sample is to be imaged. 
         [0047]    Such is also promoted by the attachment of objective  46  to the L-shaped positioning apparatus  48 , as is evident from  FIGS. 3 and 4 . The image drift-relevant distance between objective  46  and the sample to be imaged is thus shorter, as a result of the L-shaped attachment of objective  46  to sample stage  32 , than in the conventional configuration depicted in  FIG. 1 , in which this distance is defined substantially by the U-shaped stand  2 . It is evident in particular from the plan view in  FIG. 4  that first limb  52  of positioning apparatus  48 , attached to sample stage  32 , can be arranged comparatively close to the optical axis of objective  46 , so that the arrangement constituted from objective  46  and positioning apparatus  48  has a comparatively compact configuration. 
         [0048]      FIGS. 5 and 6  depict, as a second exemplifying embodiment, an embodiment modified as compared with  FIGS. 3 and 4 .  FIG. 5  shows a side view, and  FIG. 6  a plan view. In this modified embodiment, positioning apparatus  48  is attached displaceably to sample stage  32 . For this, sample stage  32  comprises on its underside a guidance groove  56  in which a carriage  58  is displaceably guided. In this embodiment, carriage  58  is embodied integrally with that end of first limb  52  which faces toward the sample. In addition, an elongated recess  60  that adjoins the through hole  50  which passes through sample stage  32  is embodied on the underside of sample stage  32 . 
         [0049]    In order to remove positioning apparatus  48 , together with objective  46  held on it, from the working region in which the end of objective  46  facing toward the sample is arranged inside through hole  50 , carriage  58  is moved in guidance groove  56  along sample  32  (downward in  FIG. 6 ). In that context, the end of objective  46  facing toward the sample moves in elongated recess  60 . It is therefore not necessary, before the shifting of positioning apparatus  48 , firstly to shift objective  46  sufficiently along the imaging beam path (downward in  FIG. 5 ) until the end facing toward the sample has moved completely out of through hole  50 . 
         [0050]    In order to immobilize positioning apparatus  48  in the working region, a fastening screw  62  that is screwed from above into sample stage  32  is provided in order to secure carriage  58  in guidance groove  56 . 
         [0051]      FIGS. 7 and 8  show a further modification as a third exemplifying embodiment.  FIG. 7  shows a side view, and  FIG. 8  a plan view. As may best be gathered from the side view according to  FIG. 7 , positioning apparatus  48  in this embodiment comprises an annular part  64  attached to sample stage  32 , and a circular plate  66  arranged shiftably on annular part  64 . Objective  46  is attached centeredly to plate  66 . The center axis of annular part  64  coincides with the optical axis of objective  46 . The arrangement constituted from objective  46  and positioning apparatus  48  is thus rotationally symmetrical around the optical axis of objective  46 . This arrangement ensures that drift motions of the mechanical components of the arrangement transversely to the optical axis, which are caused e.g. by thermal effects or mechanical stresses, are largely annulled. 
         [0052]    The side view in  FIG. 9  depicts a further modified embodiment as a fourth exemplifying embodiment. This embodiment differs from the exemplifying embodiment shown in  FIG. 2  by having a different configuration of holder  34 . Holder  34  according to  FIG. 9  thus comprises a frame  70 , arranged at a vertical spacing from the upper side of sample stage  32 , on which multiple vertically displaceable pins  72  are mounted. Pins  72  abut laterally against sample carrier  36 , with the result that sample carrier  36  is immobilized on holder  34 . In order to decouple holder  34  from sample carrier  36 , pins  72  are displaced upward in  FIG. 9  so that they no longer abut against sample carrier  36 . Once pins  72  have been shifted, sample carrier  36  is entirely free. Additional movement of holder  34 , as in the embodiment shown in  FIG. 2 , is not necessary here. 
         [0053]    The plan view according to  FIG. 10  depicts a modified embodiment as a fifth exemplifying embodiment. Whereas in the exemplifying embodiments shown in  FIGS. 2 to 9  the sample carrier  36  is decoupled from holder  36  upon imaging of the sample (labeled  74  in  FIG. 10 ), the embodiment according to  FIG. 10  provides for a pressure apparatus that presses sample carrier  36  against sample stage  32  upon imaging of sample  74 . In this exemplifying embodiment, the pressure apparatus is constituted by two permanent magnets  76  arranged on sample carrier  36 , and by two ferromagnetic regions that are arranged on sample stage  32  and are associated with permanent magnets  76 . The magnetic interaction between permanent magnets  76  and the respective ferromagnetic regions associated with them ensures that sample carrier  36  is pressed sufficiently strongly onto sample stage  32  to avoid the transfer, via holder  34  onto sample carrier  36 , of a mechanical drift occurring in adjusting apparatus  44 . To ensure this, permanent magnets  76  are arranged on sample carrier  36 , and the ferromagnetic regions associated with them are arranged on sample stage  32 , in such a way that the intended magnetic interaction for image drift-avoiding securing of sample carrier  36  in its target position is possible. 
         [0054]    Permanent magnets  76  can also be arranged on sample stage  32 , and the ferromagnetic regions on sample carrier  36 . Permanent magnets  76  can also be replaced by electromagnets. The latter can be switched on and off in defined fashion by way of the precision control system (not shown) in order to achieve the desired effect. It is thus conceivable, for example, to leave the electromagnets switched off at first so that sample carrier  36  held on holder  34  can more easily be moved on sample stage  32 . Only once the target position has been reached are the electromagnets then switched on in order to press sample carrier  36  against sample stage  32 . 
         [0055]      FIGS. 2 through 10  explained above each depict only those components of the sample retainer that are necessary for an understanding of the respective set of facts being illustrated. In the plan view according to  FIG. 10 , for example, various components of the sample retainer are omitted in order to simplify the depiction, in particular holder  34 , adjusting apparatus  44 , and objective  46 . A holder of the kind shown in  FIG. 2  can be used, for example, in the embodiment according to  FIG. 10 ; instead of the pivotable arm  40 , a stationary holder component, for example an additional part of frame  38 , can be provided. 
         [0056]    It must also be pointed out that the different embodiments depicted in  FIGS. 3 to 10  can usefully be combined with one another. For example, both the embodiments shown in  FIGS. 2 and 8  that are directed toward decoupling of sample carrier  36 , and the embodiment shown in  FIG. 10  that is directed toward securing sample carrier  36 , are each of themselves combinable with the embodiments according to  FIGS. 3 to 8 . 
         [0057]    Because of the compact configuration of positioning apparatus  48 , it is further possible to protect it, together with objective  46  held on it, from drafts by way of a shield (not shown in the Figures) that can be attached, for example, to sample stage  32 . 
       PARTS LIST 
       [0000]    
       
         
           
               2  Stand 
               4  Sample retainer 
               6  Sample stage 
               8  Holder 
             Objective turret 
               12  Objectives 
               14  Through hole 
               16  Eyepiece 
               18  Port 
               20  Adjusting apparatus 
               32  Sample stage 
               34  Holder 
               36  Sample carrier 
               38  Frame 
               40  Arm 
               42  Axis 
               44  Adjusting apparatus 
               46  Objective 
               48  Positioning apparatus 
               50  Through hole 
               52  First limb 
               54  Second limb 
               56  Guidance groove 
               58  Carriage 
               60  Elongated recess 
               62  Fastening screw 
               64  Annular part 
               66  Plate 
               70  Frame 
               72  Pin 
               74  Sample 
               76  Permanent magnets

Technology Classification (CPC): 6