Abstract:
An apparatus and a method for cryosubstitution and embedding of biological specimens is disclosed. The apparatus encompasses a container ( 6 ) for receiving at least one specimen carrier ( 2 ) having a specimen ( 4 ). The specimen carriers ( 2 ) are placed into troughs ( 12 ) of the container ( 6 ). Configured in each trough is a step ( 20 ) by which the specimen carrier ( 2 ), together with the specimen ( 4 ), is held in the container ( 6 ).

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority of the German patent application 10 2004 041 965.5. which is incorporated by reference herein. 
     FIELD OF THE INVENTION 
     The invention concerns an apparatus for cryosubstitution and embedding of biological specimens. The invention concerns in particular an apparatus for cryosubstitution and embedding of biological specimens that encompasses a container for receiving at least one specimen carrier having a specimen. 
     The invention further concerns a method for cryosubstitution and embedding of biological specimens. 
     BACKGROUND OF THE INVENTION 
     The Leica EM AFS discloses a device according to the existing art. A Dewar vessel is filled with liquid nitrogen, the Dewar neck having a chamber that is cooled to a specific temperature. The desired temperature is set via a control circuit and built-in heating elements. The substitution process usually begins at approximately −90° C. The frozen specimen is transferred into the chamber, for which purpose several different containers can be provided with which the specimens are immersed into a substitution medium, usually acetone or methanol. At this low temperature the slow process of substitution begins, in which the frozen water in the specimen is replaced by the solvent without the occurrence of recrystallization. During this process the temperature is then slowly raised, and the medium is exchanged and ultimately replaced with a low-temperature embedding medium. A UV lamp is placed onto the chamber for polymerization of the low-temperature embedding medium. The various containers for cryosubstitution and embedding are disclosed in the catalog for the Leica EM AFS. All the containers disclosed therein have the disadvantage that a loss of specimens can occur during the process of cryosubstitution and/or embedding. 
     There are a variety of methods and devices for effective freezing of specimens. The high-pressure freezing method yields the best results at present. After initial freezing, the frozen specimens are located in specimen carriers. These specimen carriers are illustrated on page 6 of the Leica EM PACT catalog. Detaching the small specimens (usually 0.2 mm thick and 1.2 mm in diameter) from the mount, manipulating these specimens in the substitution medium, and transferring them into an embedding mold are critical steps that often result in loss of a sample. 
     U.S. Pat. No. 6,516,620 discloses an apparatus for high-pressure freezing of specimens. Here the specimen carrier is clamped in on all sides into a holder, and is impinged upon by the corresponding pressure from one side. A cooling medium is sprayed from the outside onto all sides of the specimen holder in order to achieve the requisite low temperature. 
     U.S. Pat. No. 6,536,219 discloses an apparatus and a method for precise cryosubstitution of tissue samples. The apparatus possesses multiple depressions of different sizes and shapes for embedding. With this apparatus as well, however, the possibility that a loss of specimens may occur cannot be ruled out. 
     SUMMARY OF THE INVENTION 
     It is therefore the object of the present invention to make available an apparatus for cryosubstitution and embedding of biological specimens with which any possible specimen loss, both during transfer of the specimen into the apparatus for embedding and during the process of cryosubstitution and embedding, is considerably reduced if not entirely ruled out. 
     The aforesaid object is achieved by an apparatus for cryosubstitution and embedding of biological specimens comprising: a container for receiving at least one specimen carrier holding the specimen, wherein the container possesses the shape of a cylinder having a base and a sidewall and an opening opposite to the base, a plurality of troughs are embodied in the container, each for receiving one specimen carrier; wherein each of the troughs is configured with a tapering end and the tapered end of the troughs being arranged opposite the sidewall of the open cylinder, a step against which the specimen carriers rest being configured at the tapered end of each trough; and a supply conduit is provided with each of the troughs, so that a connection exists among the troughs. 
     A further object of the invention is to create a method for cryosubstitution and embedding of specimens with which specimen loss is almost ruled out. 
     The object is achieved by a method for cryosubstitution and embedding of biological specimens, comprising the following steps:
         placing at least one specimen carrier, carrying a specimen, into a container that encompasses multiple troughs each for receiving one specimen carrier;   placing the specimen carrier against a step that is shaped into each of the troughs at a tapering end of the troughs;   delivering and extracting at least one medium for cryosubstitution and embedding through an introduction and extraction opening; and that each of the troughs is equipped with a supply conduit so that a connection of the troughs to the introduction and extraction opening exists;   polymerizing an embedding medium delivered to the troughs; and   ejecting each shape present in the troughs, the polymerized shape carrying the specimen.       

     The invention has the advantage that the apparatus for cryosubstitution and embedding of biological specimens encompasses a container for receiving at least one specimen carrier having a specimen. Multiple troughs are embodied in the container, each for receiving one specimen carrier. Each of the troughs is equipped with a supply conduit, so that a connection exists among the troughs. 
     The supply conduit and the connection among the troughs make possible an exchange of the various media that are required for cryosubstitution and embedding. The container encompasses an introduction and extraction opening for the medium. The introduction and extraction opening is connected to the supply conduits, so that the individual troughs can be furnished, through the supply conduits, with the medium that is currently required or being used. 
     It is particularly advantageous if the container possesses the shape of a cylinder open at one end. The container comprises a base and a sidewall. Opposite the base, the cylinder is open or possesses there the aforementioned opening. The at least one trough, and the introduction and extraction opening, are configured in the interior of the cylinder. 
     The introduction and extraction opening is arranged centrally. The multiple troughs are arranged radially around the introduction and extraction opening. Each of the troughs possesses a tapering end. The tapered end of the troughs is arranged opposite the sidewall of the open cylinder. 
     It is particularly advantageous if the troughs are configured in a separate trough plate. The trough plate is then insertable into the container, which has the conformation of a cylinder open at the top. The trough plate is embodied, at the tapered end of each trough, with a step against which the specimen carriers rest. 
     The method for cryosubstitution and embedding of biological specimens is embodied in particularly advantageous fashion in that at least one specimen carrier carrying a specimen is placed into a container that encompasses multiple troughs, each for receiving one specimen carrier. Once the specimen carriers have been placed into the respective troughs, cryosubstitution and embedding are accomplished by delivering appropriate media through the introduction and extraction opening. Each of the troughs is equipped with a supply conduit, so that a connection of the troughs to the introduction and extraction opening exists. Once cryosubstitution is complete, polymerization of an embedding medium delivered to the troughs occurs. After polymerization, ejection of each of the shapes present in the troughs occurs. The polymerized shape carries the specimen. 
     The specimen is arranged on an elevation of the polymerized shape. The specimen carrier falls away upon ejection of the polymerized shape from the trough. This is particularly advantageous because an automatic separation of the specimen from the specimen carrier occurs after polymerization. In addition, the step shaped into the trough plate ensures immobilization of the specimen carrier during cryosubstitution and embedding, so that after polymerization of the shape, a defined location of the specimen is stipulated. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Further advantages and advantageous embodiments of the invention may be inferred from the dependent claims and are the subject matter of the Figures that follow and their descriptions, in which specifically: 
         FIG. 1  is a perspective view of the specimen carrier for cryosubstitution and embedding; 
         FIG. 2  is a perspective view of the container for cryosubstitution and embedding, several specimen carriers being inserted into the container; 
         FIG. 3  is a cross section through the container along line A-A depicted in  FIG. 2 ; 
         FIG. 4  is a detail view of the container for cryosubstitution and embedding, showing the location of the sample carrier in the container; 
         FIG. 5  is a perspective depiction of the container for cryosubstitution; 
         FIG. 6  shows a UV-transparent annular disk that is placed into the container for cryosubstitution and embedding prior to polymerization of the embedding medium; 
         FIG. 7  is a perspective depiction of the container for cryosubstitution and embedding, the UV-transparent annular disk being placed into the container; 
         FIG. 8  schematically depicts an ejecting means with which the polymerized parts in the troughs are ejected from the container; and 
         FIG. 9  is a perspective view of the polymerized embedding medium, ejected from the trough, that carries the specimen at one end. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       FIG. 1  is a perspective view of a specimen carrier  2  that carries a specimen  4 . Specimen carrier  2  is annular in configuration, and carries specimen  4  in a depression  3 . Specimen  4  possesses a diameter of approximately 1.2 mm and a thickness of approximately 0.2 mm. 
       FIG. 2  is a perspective view of a container  6  for cryosubstitution and embedding. Container  6  is cylindrical in shape and possesses a sidewall  8  and a base  16 . The container is open at the end opposite base  16 . Multiple troughs  12  are configured in the interior of container  6 . It is particularly advantageous if troughs  12  are configured in a trough plate  10  that is removable from the inner region of container  6 . In the embodiment depicted here, trough plate  10  possesses a central introduction and extraction opening  14  through which the chemicals or media necessary for cryosubstitution and embedding can be delivered and extracted. In this embodiment, troughs  12  are arranged radially around introduction/extraction opening  14 . Toward side wall  8  of container  6 , troughs  12  possess a tapered end  18 . Tapered end  18  is configured with a step  20  with which the specimen carrier is held in the trough. One specimen carrier  4  is placed into each trough  12 , and slid onto the tapering end  18  until it comes into contact against step  20 . 
       FIG. 3  is a cross section through container  6  along line A-A drawn in  FIG. 2 . Container  6  is embodied rotationally symmetrically about an axis  23 . Introduction/extraction opening  14  is arranged about axis  23 . Below introduction/extraction opening  14 , base  16  of container  6  possesses a depression  25 . In addition, trough plate  10  does not extend all the way to base  16  of container  6  in the region of introduction/extraction opening  14 , so that as a result of the coaction between depression  25  and trough plate  10 , which does not extend all the way to the base of container  6 , a supply conduit to the individual troughs  12  is formed. The supply conduit is shown as item  22  in  FIG. 4 . 
       FIG. 4  is an enlarged view of container  6  in order to elucidate the retention of specimen carrier  2  in trough  12 . As already mentioned in the description of  FIG. 3 , trough plate  10  does not extend all the way to base  16  of container  6  in the region of introduction/extraction opening  14 . A supply conduit  22  from introduction/extraction opening  14  to trough  12  is thereby formed. Toward sidewall  8  of container  6 , the trough terminates in a tapering end  18 . Configured in the region of tapering end  18  is step  20  that partially radially wraps around specimen carrier  2 . 
       FIG. 5  is a perspective view of container  6  into whose opening  9  trough plate  10  is inserted. Several specimen carriers  2  are inserted into troughs  12 . 
       FIG. 6  shows a UV-transparent annular film  30  that can be placed onto trough plate  10 . Annular film  30  possesses a central opening  32  which possesses a larger diameter than introduction/extraction opening  14 . Opening  32  of annular film  30  is dimensioned in such a way that in addition to access to introduction/extraction opening  14 , access is also maintained to each trough  12  in trough plate  10 . UV-transparent film  30  is put in place in a final step, i.e. prior to polymerization. A UV lamp (not depicted) is placed onto container  6  in order to begin polymerization of the embedding medium. If film  30  were not placed onto trough plate  10 , the embedding medium would polymerize more slowly and more unevenly. Because the embedding medium contracts upon polymerization, access to each trough  12  is still necessary. 
       FIG. 7  is a perspective depiction of container  6  with UV-transparent annular film  30  in place. Opening  32  of annular film  30  permits access to introduction/extraction opening  14 , and partial access to each of troughs  12 . 
       FIG. 8  shows an ejection means  34  that serves to eject the fully polymerized shapes in trough  12 . The ejection means engages through base  16  of container  6  and acts directly on the fully polymerized embedding-medium shapes present in the individual troughs  12 . 
       FIG. 9  is a perspective view of polymerized body  40  made of embedding medium. Polymerized body  40  made of embedding medium is substantially a negative impression of trough  12  in trough plate  10  in which the embedding medium was polymerized. The polymerized body comprises an elongated oval part  42  that corresponds substantially to trough  12 . Adjoining elongated oval part  42  is a round part  44  that corresponds to tapered portion  18  of trough  12 . Round part  44  carries specimen  4 . Step  46  configured in elongated part  42  has the shape of a circle sector. Step  46  is produced upon ejection of body  40  from trough  12 . Ejection causes specimen carrier  2  to fall away from fully polymerized body  40 . Specimen  4  thus remains behind on round part  44  of polymerized body  40 .