GB2110838A

GB2110838A - Transfer device for the removal of freeze-dried sections from the cooling chamber of a microtome for frozen sections, especially a cryo- ultramicrotome

Info

Publication number: GB2110838A
Application number: GB08232540A
Authority: GB
Inventors: Walter Bilek
Original assignee: C Reichert Optische Werke AG
Current assignee: C Reichert Optische Werke AG
Priority date: 1981-11-17
Filing date: 1982-11-15
Publication date: 1983-06-22
Also published as: DE3145505A1; FR2516652A1; FR2516652B1; SE8206494L; GB2110838B; SE8206494D0; JPS5892928A

Abstract

The transfer device comprises a vacuum transport head (10) connected to a source of vacuum and including a carrier plate (8) for the reception of the frozen sections (2) to be moved within or removed from the cooling chamber. The carrier plate is dimensioned to exceed the size of an opening of the transport head so that the latter can engage a seal (11) around the opening. The vacuum in the transport head prevents moisture being deposited on the frozen sections and also secures the carrier plate to the transport member by engagement with the seal. <IMAGE>

Description

SPECIFICATION Transfer device for the removal of freeze-dried sections from the cooling chamber of a microtome for frozen sections, especially a cryoultramicrotome The invention pertains to a transfer device for the removal of freeze-dried sections from a cooling chamber of a microtome for frozen sections, especially a cryo-ultramicrotome.

Biological objects for histochemical purposes, especially for the execution of an X-Ray microanalysis in an electron microscope, are often cut after a rapid freezing, "cryofixation", in the frozen state in cooling chambers, i.e. "cryomicrotomy" or"cryoultramicrotomy" chambers. Inasmuchas such kinds of hydrated cryo-sections can be irradiated and analyzed in the electron microscope only with difficulty, recourse is had at present to the method of freeze-drying in order to heighten the transparency of the sections in the electron beam, and to simplify the transfer of the sections from the cooling chamber into the electron microscope.The section on an object-carrier grid, for this purpose, for instance in a dry nitrogenous atmosphere of the cooling chamber, is heated from the temperature at which it was frozen to temperatures in the order of > -80 C. The greater part of the water molecules present in the section are thus transferred to the chamber cooling gas, which constantly flows over the sections and conducts the water molecules to the ambient roomatmosphere. U.S. Patents Nos. 3,828,571; 4,284,894; and 4,302,450 are exemplary of cryo-ultramicrotome apparatuses of the general structure referenced. U.S.

Patent No. 3,924,500 may be referred to for a showing of a conventional grid 10 for support of a section. However, it is difficult to remove the dried sections from the chamber after the freeze-drying has taken place without having condensates or sublimates of water occurring on the cold sectionsurfaces when entering the moist ambient roomatmosphere. Such water precipitates on the dried sections momentarily lead to an alternate distribution of ions and small molecules and thereby tend to invalidate any subsequent micro-analysis of the sections. Therefore, attempts have previously been made to enclose the sections mounted on objectcarrier grids in the cooling chamber in encapsulating holders, for instance small synthetic resin capsules, before removing the sections from the cooling chamber.Unfortunately this preparatory step requires very thick capsules, which in the cooling chamber at temperatures under -1 00 C, can be handled only with great difficulty. The same holds true for attempts to "wrap" the object-carrier grids in metal or plastic foils prior to removal from the cooling chamber.

Therefore the principal object of the present invention is to provide a transfer device of relatively simple structure which is safe and simple in its mode of operation which avoids many of the problems attendant the transfer of frozen-hydrated sections at cryogenic temperatures.

The device according to the invention is constructed in such a mannerthatthe object-carrier grids can be shifted from the heating plate provided for freeze drying, onto a carrier plate which forms the bottom of a vacuum transport head, which after the shifting of the grids onto the plate, can be evacuated to enable removal of the freeze-dried grid-carried section from the chamber space. The temperature of the transport head and the carrier plate during removal of the freeze-dried sections from the cooling chamber is maintained appreciabiy greater than the temperature of the cooling chamber.

Further, the transfer device in a preferred embodiment is made up at least partially of transparent material. A valve means is provided to vent the interior of the transport head, which precludes the risk of convection currents in conjunction with the ventilation of the head; Recesses are provided in the carrier plate for the reception and removal of the section carrying grids and a grid cover plate is also preferably provided. The carrier plate of the receptacle can be heated by means of a heating element during transfer or before the opening of the transport head, so that precipitation of water on the bio-colloids of the freeze-dried sections is obviated.

In conjunction therewith, means for thermostatic regulation of the heating element to control the temperature of the carrier plate is provided to prevent the heat from harming the specimen.

Reference is now made to the accompanying drawings, in which: Figure 1 is a schematic representation of a cooling chamber of known constructions showing a transfer device in accordance with the present invention before the removal of the freeze-dried sections from the cooling chamber; Figure 2 is a schematic representation of the closed, evacuated transfer device after removal from the cooling chamber and including a manually operable vacuum pump and valve system; Figure 3a is a schematic representation, in crosssection of a modified design of the transport device of the present invention with a thermostatically heatable carrier base-plate with recesses for the placement and removal of the object-carrier grids as well as a cover for the protection of the sections during transfer and ventilation; and Figure 36 is a top plan of the carrier base-plate of Figure 3a also showing the recesses for the reception of the object-carrier grids.

Further advantages and features of the invention will be seen from the following description of preferred embodiments.

In Figure 1 there is shown a generally conventional cooling chamber with a multilayered chamber-wall 1, which is cooled by liquid or gaseous nitrogen to a temperature substantially under 0 C, e.g.. as low as -170 C, which is kept frost-free by a continuous stream of dry, cold nitrogen gas in the interior. For the production of the frozen sections 2, to be analyzed, the frozen biological specimen 3 is traversed across the edge of the knife 4. The sections 2 removed by the knife-edge are transferred by means of suitable conventional devices, not shown, inside the chamber 5 onto circular disk-shaped object-carrier grids 6 conventionally used for the purpose.These grids 6 may have a diameter of 2.3 or 3 mm., and the grids with the sections are carried over onto a freeze-drying plate 7, on which they may be pressed flat with a suitable means, not shown, and dried at a temperature -80 C.

In accordance with the invention, the grids 6 with the pressed and dried sections thereon are shifted from the carrier plate 7 onto a plate 8 which due to its placement makes possible, in a simple manner, a shifting of the grids onto the carrier plate 7. For this purpose, the surface of the carrier plate 8 is arranged somewhat lower than the surface of the plate 7. The plate 8 is preferably introduced into the chamber 1 immediately before the transfer of the grids, whereby the plate 8 is more or less at room temperature.

Too rapid a heat exchange between the plate 8 and its support surface is avoided, for instance, by means of a thermai insulating foam-material layer 9, which in conjunction with rapid handling of the section-carrying grids assists in avoiding condensation on the sections. After the shifting of the section-carrying grids onto the plate 8, the likewise room-warm transport head 10 is placed on the plate 8 and evacuated to such a degree of vacuum that the plate 8 is releasably secured to the transport head 10 by a pressure differential which allows the removal of the plate 8, with the grids thereon, from the chamber-space without the risk of exposure to the ambient. Between the transport head 10 and the base-plate 8, there is located a seal means, which in an exemplary embodiment is an O-ring 11 for sealing of the interior space of the head 10.The head 10 is provided with an exhaust connection 12, on which a hose 13 is provided for connection to the pump.

In Figure 2 there is shown a complete transfer device according to the invention, with a simple hand pump 14with manometer 15 as well as air-inlet valve 16. The intake of air can take place through a drying agent, which is provided in a known manner, in a small tube 17 between the valve 16 and the hose-connection 13, to prevent the introduction of moist air to the freeze-dried sections when venting the interior of the transport head 10 to release the carrier plate 8. In accordance with the concept of the invention, the base-plate 8 with its planar under-side can be placed upon a heated plate 8a and in this manner can be heated to such an extent in excess of room temperature that the risk of an intake of moisture-laden air on opening the transport device can generally be precluded.An advantageous furth erstructural feature of the device according to the invention is that the transport head 1 0a may be constructed of transparent synthetic resin or glass which thereby makes possible a continuous observation of the position of the section-carrying grids 6.

In Figure 3 there is shown a schematic representation of another embodiment of the transfer device in accordance with the invention. The base-plate 8a supports, in this case, three feet 19 having pointed ends, only 2 of which may be seen, which reduce solid-body contact between the plate 8a and a given support surface to a minimum and in this manner minimizes the heat-exchange therebetween. Likewise, in accordance with this embodiment of the invention, the heat-exchange between the plate 8a and the chamber ambient gas can be reduced by means of an insulating layer 9a. In the base-piate 8a are preferably located a thermostatic heating means, for instance comprising a cartridge-type heater and a temperature probe 21, which are linked over a multi-conductor cable 22 with a control device, not shown.The multi-conductor cable 22 is provided with a thermal insulating holding-handle 23 which projects out of the cooling chamber. Afurther advantageous embodiment can consist in having the grids 6 sized so as to be receivable within complementary sized recesses 8b, which are linked by means of a groove so that removal of the grids with the jaws of tweezers is readily possible. An advantageous further feature resides by providing the individual recesses with indicia, i.e., numerals and/or letter designations, for the different grids or slides located therein. Another advantageous feature is the provision of a grid cover plate 24, so that jarring in conjunction with the transfer or convection currents occurring when ventilating the head 10 will not alter the position of the sections or the position of the grids.In so doing, the spatial position of the cover 24 on the base-plate 8a as seen in Figure 3 can be determined and localized by means of pegs, not shown.

The embodiments shown and described in Figures 1 through 3 merely show a few preferred variants for the implementation of the concept of the invention.

The invention, moreover, can be implemented in numerous ways not represented or described. Thus it is possible, for instance, to effect the seal between the head 10 and the base-plate 8 through precision ground surfaces on their opposed surfaces in place of an O-ring. The type, installation and attachment of the vacuum system, of the ventilation equipment as well as type and arrangement of a heating and of the insulation of the base-plate, can be modified within the scope of known technical variants, without thereby affecting the concept of operation of the device according to the invention. Likewise, materials employed in constructing the device of the invention may be varied as may operating temperature ranges. The pump, heating transport head, carrier plate etc. comprise integrated elements of the invention, but can be realized in the most varied manner according to the given state of the technology and operative connection. The embodiments shown and described thus merely represent exemplary embodiments of the transfer device according to the invention.

Claims

1. Transfer device for the removal of freezedried sections from the cooling chamber of a microtome for frozen sections comprising a generally hollow generally closed transport head adapted to be placed in communication with a source of vacuum, said transport head being sized so as to be capable of being introduced into the cooling chamber, said transport head being provided with an opening and a sealing surface surrounding the opening, a carrier plate adapted to support freezedried sections to be removed from the cooling chamber, said carrier plate being sized so as to be capable of closing the opening in fluid tight sealing relation thereto to shield the sections from the ambient atmosphere and whereby after evacuation of the generally hollow transport head by a source of vacuum the freeze-dried sections can be removed from the cooling chamber while being shielded from the ambient atmosphere.

2. The transfer device according to Claim 1 wherein the transport head is bell-shaped, and the carrier. plate forms a bottom plate closing the transport head opening.

3. The transport device according to claim 1 or 2 in combination with a cooling chamber comprising a pressure and heating plate and a juxtaposed support for the carrier plate, said support and heating plate being positioned so that a top surface of the carrier plate is at generally the same height as a top surface of the pressure and heating plate.

4. The transfer device according to Claim 1,2 or 3 and including vacuum pump means operatively connected to the generally hollow interior of the transport head, and conduit means for communicating the vacuum pump and the transport head comprising a handle for the carrier plate.

5. The transfer device according to Claim 1, 2,3 or 4 including means for venting of the hollow interior of the transport head.

6. The transfer device according to Claim 5, including means for drying fluid used to vent the transport head.

7. The transfer device according to any preceding claim wherein the transport head is constructed, at least in part, of a transparent material.

8. The transfer device according to any preceding claim wherein the top surface of the carrier plate is provided with at least one freeze-dried section supporting grid receiving recess, and including indicia for identification of the grid supported section.

9. The transfer device according to Claim 8 wherein at least one recess is sized to complement the size of the object support grid and includes at least a laterally extending recess to facilitate the grasping of the object support grid by means of a tweezer.

10. The transfer device according to Claim 8 or 9 including a cover for said at least one recess, said cover being sized so as to fit within the transport head and being further sized so as to generally not permit shifting of the cover during movement of the transport head.

11. The transfer device according to any preceding claim wherein the carrier plate is heat-insulated on its underside.

12. The transfer device according to Claim 3, wherein the carrier plate support provided in the cooling chamber is heat-insulated.

13. The transfer device according to any preceding claim wherein the carrier plate includes a thermostaticaliy contriollable heating means.

14. The transfer device according to any preceding claim wherein the carrier plate includes an insulating handle having a portion manually engageable from the exterior of the cooling chamber.

15. A transfer device for the removal of freezedried sections from the cooling chamber of a microtome for frozen sections, the device being constructed substantially as herein described with reference to Figures 1 and 2 or 3a and 3b of the accompanying drawings.