Abstract:
A Dewar vessel ( 1 ) for automated cryosubstitution or low-temperature substitution is disclosed. The invention further discloses an apparatus ( 10 ) for automated liquid transfer for cryosubstitution or low-temperature substitution. The apparatus ( 10 ) encompasses a container ( 50 ) that encompasses at least one specimen holder ( 2 ) and at least one reservoir holder ( 20 ); and wherein a movable transfer container ( 35 ) for automated exchange of at least one liquid ( 42 ) between the at least one specimen holder ( 2 ) and the at least one reservoir holder ( 20 ) is provided.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims priority of German patent application 10 2005 003 284.2, filed Jan. 25, 2005, which application is incorporated by reference herein.  
       FIELD OF THE INVENTION  
       [0002]     The invention relates to a Dewar vessel for automated cryosubstitution or low-temperature substitution. The invention relates in particular to a Dewar vessel for automated cryosubstitution or low-temperature substitution that is embodied with a neck and is filled with a liquid cooling agent, a chamber for receiving a container being provided in the neck.  
         [0003]     The invention further relates to an apparatus for automated cryosubstitution or low-temperature substitution.  
       BACKGROUND OF THE INVENTION  
       [0004]     Patent Application WO 94/05995 discloses an apparatus for the dewatering and/or embedding of preferably frozen specimens. The apparatus encompasses a Dewar vessel filled with liquid nitrogen and a metallic element, anchored at the base of the Dewar vessel, that is made of a material having good thermal conductivity. The metallic element possesses at its upper end, in the Dewar neck attachment region, a cover having a metallic cooling surface. Provided in the Dewar neck is a substitution (PLT) container in which the transfer of liquids for low-temperature substitution is performed manually.  
         [0005]     The brochure for the Leica EM AFS discloses a unit according to the existing art. A Dewar vessel is filled with liquid nitrogen, the Dewar neck comprising a chamber or a container that can be brought to a specific temperature. The temperature range extends from −140° C. to +65° C. The desired temperature is set via a control loop and built-in heating elements. Here as well, liquid exchange must be accomplished manually.  
         [0006]     German Utility Model DE 91 04 344.1 discloses a cooling device for specimen preparation for an electron microscope. The cooling device encompasses a holding insert that is subdivided into at least two segments. Both segments are equipped with orifices, of which the orifices in one segment serve to retain a container for the specimens to be freeze-dried. Reservoir vessels having reagents are retained in the orifices of the other segment. The specimens and the reagents are thereby cooled to the required working temperature. An automatic transfer of the reagents from one region of the container into the next is not disclosed.  
       SUMMARY OF THE INVENTION  
       [0007]     The object of the present invention is therefore to create a Dewar vessel that makes possible the automation of manual activities in the context of cryosubstitution or low-temperature substitution.  
         [0008]     Another object is, by way of the invention, to make possible a transfer of cold substitution media (solvents) and/or embedding media (synthetic resins), to enhance accuracy in the preparation of mixtures, and to reduce contamination susceptibility.  
         [0009]     The above object is achieved by an apparatus for cryosubstitution or low-temperature substitution that encompasses: a neck wherein the Dewar vessel is filled with a liquid cooling agent, a chamber formed in the neck for receiving a container wherein the chamber encompasses at least one specimen holder and at least one reservoir holder; and an apparatus for automated exchange of at least one liquid between the at least one specimen holder and the at least one reservoir holder is provided.  
         [0010]     A further object of the invention is to create an apparatus for automated cryosubstitution or low-temperature substitution with which a transfer of cold substitution media (solvents) and/or embedding media (synthetic resins) is made possible, accuracy in the preparation of mixtures is enhanced, and contamination susceptibility is reduced.  
         [0011]     The above object is achieved by an apparatus for cryosubstitution or low-temperature substitution that encompasses: a container that encompasses at least one specimen holder and at least one reservoir holder; and a movable transfer container for automated exchange of at least one liquid between the at least one specimen holder and the at least one reservoir holder.  
         [0012]     It is advantageous if the Dewar vessel for automated cryosubstitution or low-temperature substitution is embodied with a neck and is filled with a liquid cooling agent. A chamber for receiving a container is provided in the neck, the container encompassing at least one specimen holder and at least one reservoir holder. Also provided is an apparatus for automated exchange of at least one liquid between the at least one specimen holder and the at least one reservoir holder.  
         [0013]     An apparatus for automated cryosubstitution or low-temperature substitution is likewise advantageous if a container is provided that encompasses at least one specimen holder and at least one reservoir holder; and wherein a movable transfer container for automated exchange of at least one liquid between the at least one specimen holder and the at least one reservoir holder is provided.  
         [0014]     The apparatus can be immovably joined to the Dewar vessel. The apparatus can likewise be embodied removably from the Dewar vessel.  
         [0015]     The apparatus encompasses a movable transfer container that transports the liquid between the at least one specimen holder and the at least one reservoir holder. The transfer container can be a syringe or a pipette. The liquid is taken into or ejected from the transfer container in motorized, pneumatic, or hydraulic fashion.  
         [0016]     The apparatus for automated exchange of at least one liquid between the at least one specimen holder and the at least one reservoir holder performs the liquid transfer in remotely controlled fashion between the at least one reservoir holder and the at least one specimen holder. The transfer container moves correspondingly back and forth, thus enabling the liquid transfer.  
         [0017]     An electronic control system or a computer software program is provided which makes possible programming of a chronological sequence of transfer steps between the at least one reservoir holder and at least one specimen holder.  
         [0018]     The container is embodied as a cup open at the top, the specimen holders and reservoir holders being arranged in the container on a circle about a central axis of the apparatus. The apparatus can furthermore have integrated into it one or more UV light-emitting diodes (LEDs) that polymerize embedding material (synthetic resin) for the specimen. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0019]     Further advantages and advantageous embodiments of the invention may be inferred from the dependent claims and are the subject matter of the Figures below and the descriptions thereof.  
         [0020]     In the individual drawings:  
         [0021]      FIG. 1  is a cross section through a Dewar vessel for cryosubstitution or low-temperature substitution;  
         [0022]      FIG. 2  schematically depicts a Dewar vessel onto which the apparatus for liquid transfer for automated cryosubstitution or low-temperature substitution is placed;  
         [0023]      FIG. 3  is an enlarged depiction of a first embodiment of the apparatus for liquid transfer for automated cryosubstitution or low-temperature substitution;  
         [0024]      FIG. 4  is an enlarged depiction of a second embodiment of the apparatus for liquid transfer for automated cryosubstitution or low-temperature substitution; and,  
         [0025]      FIG. 5  is a perspective view of the container for receiving at least one specimen holder and at least one reservoir holder. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0026]      FIG. 1  is a cross section through one possible embodiment of a Dewar vessel  1  for cryosubstitution or low-temperature substitution. In the description that follows, identical reference characters are used for identical elements. The cooling apparatus shown in  FIG. 1  serves for cryosubstitution or low-temperature substitution of biological and/or other water-containing specimens. The Dewar vessel encompasses an inner container  1   2  and an outer container  1   1 . The inner container is filled with a liquid cooling agent that is preferably liquid nitrogen  3 . A chamber  5  is inserted into neck  1   3  of Dewar vessel  1 . Chamber  5  is cup-shaped and possesses a heavy base  5   1 . Chamber  5  is open at the top and can be closed off with a cover  6  for insulation with respect to ambient temperature. Chamber  5  serves to receive multiple specimen holders  2  in which specimens  30  for cryosubstitution or low-temperature substitution are located. A first thermal conduction rod  7  is joined to base  5   1  of chamber  5 . A platform  8  is provided at the end of first thermal conduction rod  7  facing away from base  5   1  of chamber  5 . Platform  8  can be detachably joined to first thermal conduction rod  7 . It is also conceivable for first thermal conduction rod  7  and platform  8  to be embodied integrally. Above platform  8 , first thermal conduction rod  7  is surrounded by an insulator  12 . Insulator  12  serves to insulate first thermal conduction rod  7  with respect to liquid nitrogen  3  or cold nitrogen gas  3   1 . Insulator  12  causes the heat flux that cools chamber  5  or base  5   1  to be directed principally via platform  8 . The cooling power can therefore advantageously be determined by modifying the geometrical dimensions or selecting a suitable material for first thermal conduction rod  7 . The temperature in chamber  5  can be regulated by operating at least one heating element  14 . Also provided is at least one temperature sensor  15  that is used for temperature measurement. Temperature sensor  15  can be embodied as a thermocouple or a resistance temperature sensor. The temperature signal is used as feedback for an electronic regulating system  16  that controls the temperature of chamber  5  by adapting the heating output of heating element  14 . The length of first thermal conduction rod  7  is advantageously selected in such a way that platform  8  is immersed in liquid nitrogen  3  only when a certain fill level is reached. With a high fill level, platform  8  is immersed in liquid nitrogen  3 , and chamber  5  is coupled via first thermal conduction rod  7  directly to liquid nitrogen  3 . With a low fill level, platform  8  interacts with cold nitrogen gas  3   1 . Cold nitrogen gas  3   1  is heated by the heat flux from chamber  5  into inner container  1   2  of Dewar vessel  1 . By convection and by interaction with the walls of inner container  1   2 , this heat is fed back into liquid nitrogen  3  and causes an increase in the evaporation rate. The result is that platform  8  and chamber  5  arrive at an equilibrium temperature that is largely independent of the present fill level of liquid nitrogen  3  in inner container  1   2 . It is self-evident that the thermal coupling between chamber  5  and liquid nitrogen  3  is much greater at a high fill level than at a low fill level. Lower temperatures in chamber  5  can therefore be attained with a high fill level. On the other hand, the consumption of liquid nitrogen is lower with a low fill level.  
         [0027]     This arrangement is advantageous in that in standard substitution processes, the lowest process temperatures (−90° C. and below) are needed at the beginning of the processes. The temperature is raised in the course of the substitution processes. Because liquid nitrogen  3  is also consumed during the process, the cooling power achievable by way of first thermal conduction rod  7  and platform  8  reflects the temperature profile of the substitution process. At the same time, insulator  12  also limits the coupling to liquid nitrogen  3  when the fill level is high. High temperatures can therefore be set even in this situation, with no need to exceed reasonable limits for nitrogen consumption and for the requisite heating output of heating element  14 .  
         [0028]      FIG. 2  schematically depicts a Dewar vessel  1  onto which apparatus  10  for automated liquid transfer for cryosubstitution or low-temperature substitution is placed. A chamber  5  is inserted into neck  1   3  of Dewar vessel  1 . Chamber  5  is cup-shaped and possesses a heavy base  5   1 . Chamber  5  is open at the top. Chamber  5  serves to receive a container  50  that encompasses at least one specimen holder  2  and at least one reservoir holder  20 . It is also conceivable for the at least one specimen holder  2  and the at least one reservoir holder  20  to be inserted directly into the chamber if the latter is embodied to be cup-shaped and open at the top. A movable transfer container  35  is provided for automated exchange of at least one liquid between the at least one specimen holder  2  and the at least one reservoir holder  20 . Apparatus  10  can be immovably joined to Dewar vessel  1 . It is also conceivable for apparatus  10  to be embodied removably from Dewar vessel  1 . Apparatus  10  is embodied, for example, as a module that can be placed as necessary, for example by the user, onto a Dewar vessel  1 . Transfer container  35  is a syringe or a pipette. The liquid is taken into or ejected from transfer container  35  in motorized, pneumatic, or hydraulic fashion. Apparatus  10  is equipped for that purpose with an actuation element  36  with which motorized, pneumatic, or hydraulic intake or ejection of the liquid into or from transfer container  35  is accomplished. A control unit  38  is provided with which apparatus  10  performs the automated transfer of at least one liquid between the at least one specimen holder  2  and the at least one reservoir holder  20  in remotely controlled fashion. Transfer container  35  moves correspondingly back and forth, thus enabling the liquid transfer. Transfer container  35  can move up and down along an axis  37 . Control unit  38  is what makes possible programming of a chronological sequence of transfer steps between the at least one reservoir holder  20  and the at least one specimen holder  2 .  
         [0029]      FIG. 3  is an enlarged depiction of a first embodiment of apparatus  10  for automated cryosubstitution or low-temperature substitution. Apparatus  10  can be placed onto neck  1   3  of Dewar vessel  1 . Apparatus  10  is embodied for that purpose with a flange  40  that fits around neck  1   3  of Dewar vessel  1 . It is also conceivable for the apparatus to be coupled to neck  1   3  of Dewar vessel  1  via mechanical coupling elements, e.g., a bayonet. Transfer container  35  can be moved up and down along axis  37 . The chronological sequence of transfer steps between the at least one reservoir holder  20  and the at least one specimen holder  2  is accomplished in co-action with actuation element  36  and control unit  38 . A plunger  41  of transfer container  35 , which makes possible the intake or ejection of liquid  42 , is likewise actuated by means of actuation element  36 . In the embodiment depicted here, transfer container  35  is equipped with a hollow needle  43  that makes possible accurate introduction and/or removal of liquid  42  into and/or out of specimen holder  2  and/or transfer container  35 . A specimen  45  for cryosubstitution or low-temperature substitution is placed in specimen holder  2 .  
         [0030]      FIG. 4  is an enlarged depiction of a second embodiment of apparatus  10  for automated cryosubstitution or low-temperature substitution. The difference as compared with the embodiment depicted in  FIG. 3  is that apparatus  10  for automated cryosubstitution or low-temperature substitution has integrated into it at least one UV diode  55  that polymerizes a liquid  42  serving as embedding material for specimen  45 .  
         [0031]      FIG. 5  is a perspective view of container  50  for receiving at least one specimen holder  2  and at least one reservoir holder  20 . According to the embodiment depicted here, container  50  is embodied as a cup  51  open at the top. Container  50  is subdivided into a first sector  52  and a second sector  53 . Specimen holders  2  are provided in first sector  52 . Reservoir holders  20  are provided in second sector  53 . Specimen holders  2  are suitable for receiving different types of specimen containers. Reservoir holders  20  are embodied as bottles and are inserted into second sector  53 . Second sector  53  has the shape of an annulus sector, so that each of reservoir holders  20  likewise possesses the shape of an annulus sector.