Abstract:
An automatic loading apparatus ( 12 ) for specimens ( 1 ) for high-pressure cryosubstitution is disclosed. The automatic loading apparatus ( 12 ) is connected to a high-pressure freezing device ( 40 ). Specimens ( 1 ) are inserted in a holder ( 2 ). By means of a slider ( 4 ), the specimens ( 1 ) and holders ( 2 ) are conveyed to the automatic loading apparatus ( 12 ). The guidance element ( 26 ) allows exact positioning of the holder ( 2 ) in the clamping element ( 14 ). The automatic loading apparatus makes possible rapid transfer of the holder ( 2 ) having the specimen ( 1 ) from, for example, an optical microscope to the high-pressure freezing device ( 40 ).

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims priority of the German patent application 10 2004 053 073.4, filed Nov. 3, 2004, which is incorporated by reference herein.  
       FIELD OF THE INVENTION  
       [0002]     The invention concerns a high-pressure freezing device. The invention concerns in particular a high-pressure freezing device having a chamber in which the high-pressure freezing takes place, the sample with holder being retained in a clamping element, and having a reservoir of liquid nitrogen.  
         [0003]     The invention further concerns an automatic apparatus for loading specimens into a high-pressure freezing device.  
         [0004]     The invention additionally concerns a method for loading a high-pressure freezing device.  
       BACKGROUND OF THE INVENTION  
       [0005]     U.S. Pat. No. 6,269,649 B1 discloses a system for freezing samples under high pressure. The high pressure is necessary in order to prevent the formation of ice crystals in the sample. The sample is located in a sample holder and is clamped into a corresponding sample carrier. The sample carrier is manually transferred into the high-pressure freezing system by the user thereof.  
         [0006]     U.S. Pat. No. 5,493,865 discloses a method and an apparatus for high-pressure freezing of biological samples. Before high-pressure freezing, the sample is introduced manually into the sample holder.  
         [0007]     At present it is necessary, usually using a stereomicroscope, to fill the small specimen holders that are used in a high-pressure freezing device with a sample. That specimen holder is then installed into a holder for the high-pressure freezing device, and that holder is then inserted into the high-pressure freezing device. Insertion of the holder into the high-pressure freezing device is accomplished manually. The Leica EM PACT brochure discloses a high-pressure freezing device. This system operates with separate pressure and cooling systems. The consequence of this is that the specimen must be threaded in pressure-tight fashion into the sample holder. The Leica EM PACT brochure entitled “Microbiopsy Transfer System” and the Leica EM PACT brochure entitled “Flat Specimen System” disclose several installation aids for specimen holders and tools for manual transfer of the samples, secured in the sample holder, to the high-pressure freezing device.  
         [0008]     The brochure of the company styled BAL-TEC AG likewise discloses an apparatus for high-pressure freezing. In this unit, pressure buildup is ensured by way of the cooling medium. Although pressure-tight threading of the specimen holder is not necessary, a loading apparatus and a holder for the specimen carrier are necessary. The time for installation of the specimen carrier and introduction of the specimen carrier into the loading apparatus is also approximately one minute. In addition, the specimen carrier must once again be introduced manually into the high-pressure freezing device.  
         [0009]     The disadvantage of the existing art is that more than a minute is required for installation of the samples being examined into a holder provided for them, and for positioning of the holder in a clamping element. In addition, during the threading motion of the clamping element a specific torque must be observed so that the sample to be examined is not mechanically crushed and/or destroyed, which can result in a change in the morphology of the sample to be examined. The sample changes during the aforementioned loading time of approximately one minute, so that the states observed with the optical microscope are no longer present upon examination with an electron microscope. The state of the cells observed with the light microscope differs from the frozen cells. A comparison of the two cells is therefore possible only to a limited extent.  
       SUMMARY OF THE INVENTION  
       [0010]     It is the object of the invention to create a high-pressure freezing device which is configured so that it makes possible a rapid transfer of the samples into the high-pressure freezing device, and so that no morphological changes occur in the unfrozen state and in the frozen state in the sample to be examined.  
         [0011]     The aforesaid object is achieved by a high-pressure freezing device that encompasses a chamber in which high-pressure freezing takes place; a specimen with a holder being retained in a clamping element; a reservoir of liquid nitrogen; an automatic loading apparatus connected to the high-pressure freezing device, wherein the automatic loading apparatus is designed to transfer the clamping element into the chamber of the high-pressure freezing device.  
         [0012]     A further object of the present invention is to create an automatic loading apparatus for specimens for high-pressure cryosubstitution which makes possible a rapid transfer of the sample into the high-pressure freezing device and likewise rules out mechanical changes in the morphology of the sample.  
         [0013]     The aforesaid object is achieved by an automatic loading apparatus for specimens for high-pressure cryosubstitution that encompasses a slider on which is retained a specimen located in a holder; and the loading apparatus comprises at least one guidance element with which the holder is positionable in a clamping element.  
         [0014]     A further object of the invention is to describe a method for loading a high-pressure freezing device that makes possible a rapid and reliable transfer of samples into the high-pressure freezing device, so that no morphological differences occur between an unfrozen sample and a frozen sample.  
         [0015]     The aforesaid object is achieved by a method that encompasses the steps of: 
        positioning a slider, having a holder for a specimen, in a guidance element of a loading apparatus for the high-pressure freezing device;     clamping the holder in a clamping element; and     displacing the clamping element, with the clamped holder, into the high-pressure freezing station by means of a pneumatic cylinder.        
 
         [0019]     The automatic loading apparatus according to the present invention for specimens for high-pressure freezing has the advantage that a sample that is first examined with an optical microscope, and is then introduced into an electron microscope for the observation of details that require a higher resolution, experiences little or no change in morphology. A confocal microscope is used, inter alia, to study proteins to which fluorescent markers are attached, in motion in the living cells. The insufficient resolution of the light microscope emerges as a disadvantage in this context. The proteins themselves cannot be identified with the light microscope. The electron microscope has the high resolution required for this, and the automatic loading apparatus according to the present invention thus makes it possible to freeze in place the dynamic state of the cells to be examined in a sample, and also to ensure that no morphological changes occur as a result of installation of the sample in the high-pressure freezing device. The automatic loading apparatus for specimens for high-pressure freezing furthermore has the advantage that, for example, transfer of a specimen from the confocal microscope to the high-pressure freezing device can be accomplished within five seconds. With this short time, it is possible to ensure that, for example, a protein observed with the light microscope is still in the same location when observed with an electron microscope. This time-optimized automatic loading system is implemented by the automatic loading apparatus for specimens for high-pressure freezing.  
         [0020]     The automatic loading apparatus for specimens for the high-pressure freezing device is advantageous because it encompasses a slider on which a fork is embodied. The fork comprises two oppositely located limbs, on each of which is embodied a recess in which a specimen, located in a holder, is retained. The loading apparatus possesses at least one guidance element with which the holder is positionable in the clamping element.  
         [0021]     The clamping element encompasses a pusher part and a counterelement. A shell that is embodied with two oppositely located openings partially encloses the pusher part and the counterelement.  
         [0022]     Also provided is a sensor that records the presence of the slider in the automatic loading station. Additionally provided is a motor that is connected via a shaft to the pusher part of the clamping element.  
         [0023]     A control system is provided which establishes, from the power consumption of the motor, a torque-limited threading motion between the counterelement, the holder, and the pusher part.  
         [0024]     The loading apparatus further possesses a pneumatic cylinder that introduces the clamping element, with the holder, into a high-pressure freezing station and uncouples the shaft of the motor from the clamping element.  
         [0025]     The clamping element possesses a connector tube for the high pressure. The pneumatic cylinder creates, in the high-pressure freezing station, the connection for high pressure to the connector tube of the clamping element.  
         [0026]     For examination of the sample or specimen with an optical microscope, a cutout is furthermore shaped into a stage support provided for the microscope. The slider is configured in such a way that the holder having the sample, inserted into the fork, comes to rest on a coverslip located on the stage support.  
         [0027]     A further advantage of the present invention is a high-pressure freezing device having a chamber in which high-pressure freezing takes place, the sample with holder being retained in a clamping element. The high-pressure freezing device furthermore possesses a reservoir of liquid nitrogen. The high-pressure freezing device is connected to an automatic loading apparatus, the automatic loading apparatus transferring the clamping element into the chamber of the high-pressure freezing device.  
         [0028]     A further advantage of the invention is a method for loading a high-pressure freezing device, in which a specimen located in a holder is placed into a slider. The slider is positioned in a guidance element of a loading apparatus for the high-pressure freezing device. The holder is then inserted, by way of the slider, into the clamping element. The clamping element is closed by way of a torque-limited motor, so that clamping of the holder in the clamping element occurs. The clamping element having the clamped-in holder is slid into the high-pressure freezing station with a pneumatic cylinder, the shaft of the motor being uncoupled from the clamping element. Lastly, a connection for high pressure is created, by the pneumatic cylinder, between the high-pressure freezing station and a connector tube of the clamping element.  
         [0029]     Further advantages and advantageous embodiments of the invention may be inferred from the dependent claims and are the subject of the Figures below and their descriptions. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0030]     In the individual Figures:  
         [0031]      FIG. 1  is a perspective view of a slider for introducing a specimen, located in a holder, into a high-pressure freezing device, or for positioning the holder having the specimen on a microscope stage for optical examinations;  
         [0032]      FIG. 2  is a perspective view of a microscope stage having a slider positioned thereon, so that the specimen located in the holder can be examined optically;  
         [0033]      FIG. 3  is an enlarged depiction of a clamping element in which the holder having the specimen is clamped for high-pressure freezing;  
         [0034]      FIG. 4  is a perspective view of a portion of the automatic loading apparatus, the positioning aid for the slider likewise being depicted;  
         [0035]      FIG. 5  is a perspective view of the automatic loading apparatus, the slider already being slid sufficiently far into the automatic loading device that the holder having the specimen is positioned in the clamping element;  
         [0036]      FIG. 6  is an enlarged perspective depiction of the clamping element, the holder having the specimen being positioned in the clamping element;  
         [0037]      FIG. 7  is an enlarged perspective partial view of the automatic loading apparatus, the motor provided for closing the clamping element being uncoupled from the clamping element; and,  
         [0038]      FIG. 8  is a perspective overall view of a high-pressure freezing device to which the automatic loading apparatus for introducing the clamping elements into the high-pressure freezing device is connected. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0039]      FIG. 1  is a perspective view of a slider  4  for introducing a specimen  1 , secured in a holder  2 , into an automatic loading apparatus  12  (see  FIGS. 5 and 8 ). It is self-evident to one skilled in the art that the embodiment of the slider depicted here represents merely one exemplifying embodiment of several possible ones. The embodiment disclosed here is not to be construed as a limitation of the invention.  
         [0040]     Slider  4  is embodied at one end with a grip element  5  with which a user can transport slider  4 . A fork  3  is embodied at the end of slider  4  located opposite grip element  5 . Fork  3  possesses a first limb  3   a  and a second limb  3   b . A respective recess  7  is shaped into first limb  3   a  and into second limb  3   b  in the region of the open end  6  of fork  3 . Recess  7  is configured in such a way that the holder is retained in a clamping fit by first limb  3   a  and second limb  3   b . First limb  3   a  and second limb  3   b  are embodied elastically, so that a clamping force is exerted on holder  2  placed in recess  7 . This clamping force allows the holder to be firmly retained in and securely fitted into in the fork during transport by a user.  
         [0041]      FIG. 2  is a perspective view of a microscope stage  9  of an optical microscope (not depicted). The microscope can be, for example, a confocal microscope. Microscope stage  9  is embodied with a cutout  10  into which slider  4 , with a holder  2  having a specimen  1  and mounted in fork  3 , is positioned for optical examination with the microscope. Cutout  10  in microscope stage  9  and slider  4  are configured in such a way that when a slider  4  is inserted into cutout  10 , holder  2  and specimen  1  come to rest on a coverslip  11  provided on microscope stage  9 . Oil immersion can also be used for optical examination of specimen  1  inserted in holder  2 .  
         [0042]      FIG. 3  is a perspective view of a clamping element  14  in which holder  2 , with specimen  1  located therein, is clamped or immovably retained. Clamping element  14  possesses a pusher part  16  that is arranged opposite a counterelement  18 . Counterelement  18  is connected to a connector tube  20  for high pressure. Through the connector tube, high pressure is directed onto specimen  1  that is clamped between counterelement  18  and pusher part  16 . Pusher part  16  can be displaced via a threaded screw (not depicted) so that it moves toward counterelement  18  and thus clamps holder  2  having specimen  1  in the clamping element. A motor shaft  25  engages on pusher part  16  and thus moves pusher part  16  toward or away from counterelement  18 .  
         [0043]      FIG. 4  is a perspective partial view of automatic loading apparatus  12 , housing parts of automatic loading apparatus  12  having been omitted so as thereby to provide insight into the manner of operation of the automatic loading apparatus. Automatic loading apparatus  12  is substantially linear in construction. Associated with automatic loading apparatus  12  is a guidance element  26  with which holder  2  is positionable in clamping element  14 . Clamping element  14  is positioned in automatic loading apparatus  12 . Slider  4 , which holds holder  2  having specimen  1  in fork  3 , is inserted into guidance element  26 . As a result of the linear displacement of slider  4  toward clamping element  14 , holder  2  is positioned in the clamping element between counterelement  18  and pusher part  16 . The linear displacement of the holder toward clamping element  14  also starts the automatic loading operation. For this, a corresponding switch (not depicted) is actuated. Automatic loading apparatus  12  encompasses a motor  24 . From motor  24 , motor shaft  25  extends toward clamping element  14 . As already explained above, pusher part  16  can be moved with motor  24  toward counterelement  18  so as thereby to clamp the holder between pusher part  16  and counterelement  18 . Automatic loading apparatus  12  likewise encompasses a sensor  28  that ascertains the presence of slider  4  in the automatic loading apparatus. A control system  31  is also connected to the motor and establishes, from the power consumption of motor  24 , a torque-limited threading motion between counterelement  18 , holder  2 , and pusher part  16 . This ensures that holder  2  is not crushed to an unnecessary extent by counterelement  18  and pusher part  16 . Unnecessarily severe crushing could result in a change in the morphology of specimen  1  to be examined.  
         [0044]      FIG. 5  is an enlarged perspective partial view of automatic loading apparatus  12 , in which specimen  1  located in holder  2  has already been introduced into clamping element  14 . Slider  4 , which carries holder  2  having specimen  1  and is placed on guidance element  26 , has been advanced toward clamping element  14 . Motor shaft  25  is in contact with pusher part  16  of the clamping element. Pusher part  16  is then moved in threading fashion, with motor  24 , toward counterelement  18 . Holder  2  is thereby clamped between pusher part  16  and counterelement  18 . Once clamping is achieved, the slider is pulled back.  
         [0045]      FIG. 6  is an enlarged depiction of clamping element  14  in which fork  3  with holder  2  is positioned between pusher element  16  and countermember  18 . Before pusher part  16  is moved in threading fashion with respect to counterelement  18 , sufficient clearance is present between pusher part  16  and counterelement  18  that fork  3 , along with the holder retained in the fork and the specimen, can be introduced into this clearance between pusher part  16  and counterelement  18 . Clamping element  14  comprises a shell  22  that possesses two oppositely located openings  30 . Because of openings  30  embodied in shell  22  of clamping element  14 , the fork can be positioned between pusher part  16  and counterelement  18  so that holder  2  is clamped when pusher part  16  is moved in threading fashion with respect to counterelement  18 .  
         [0046]      FIG. 7  is an enlarged perspective partial view of automatic loading apparatus  12 , in which clamping element  14  has been slid into high-pressure freezing device  40 . Motor  24  is uncoupled from clamping element  14 . Clamping element  14  is slid into high-pressure freezing device  40  by means of a pneumatic cylinder  35 . In high-pressure freezing device  40 , the connection for high pressure is made to connector tube  20  of clamping element  14 . Pressure on the specimen can be built up via connector tube  20  of clamping element  14 . Pneumatic cylinder  35  exerts sufficient pressure to ensure the connection for high pressure, and so that no pressure leakage occurs. A slider  4  is no longer inserted on guidance element  26 . After high-pressure cryosubstitution is complete, automatic loading apparatus  12  is once again ready to receive a slider  4  with a holder  2 , having a specimen, secured therein.  
         [0047]      FIG. 8  is a perspective view of a high-pressure freezing device  40  having an automatic loading apparatus  12  mounted thereon. A display  41  is embodied on the housing of high-pressure freezing device  40 . Display  41  can be embodied as a touch screen with which the user can enter parameters for the high-pressure freezing that is to be carried out. A reservoir  42 , containing liquid nitrogen for cooling clamping element  14 , is also connected to high-pressure freezing device  40 . An electronic system or control system  43  that controls and regulates execution of the high-pressure freezing operation is also accommodated in the housing of high-pressure freezing device  40 . Control system  43  additionally serves to synchronize the high-pressure freezing procedure. As already mentioned, the specimen is pressurized via connector tube  20  of clamping element  14 . Immediately thereafter, liquid nitrogen is sprayed onto the clamping element in order to achieve appropriate cooling. The intensity of the pressure can be adjusted within a range from 1 to 2100 bar. High-pressure freezing device  40  is embodied with a chamber  44  in which high-pressure freezing takes place. An outlet  46  for high pressure is embodied on one sidewall  45  of chamber  44 . The connection for high pressure to connector tube  20  of the clamping element is made at this outlet  46 . This is accomplished, as already mentioned, through the pneumatic cylinder.