FR2516652A1 - TRANSFER DEVICE FOR THE EXTRACTION OF LYOPHILIZED CUTS OUTSIDE THE COOLING CHAMBER OF A MICROTOME FOR FROZEN CUTS - Google Patents

Abstract

THE DEVICE INCLUDES A HOLLOW TRANSPORT HEAD 10 THAT CAN BE COMMUNICATED WITH A VACUUM SOURCE AND BEING SIZED SO AS TO BE INSERTED INTO THE COOLING CHAMBER 5 OF A MICROTOME; HEAD 10 IS PROVIDED WITH A TEMPORARILY CLOSABLE OPENING BY A SUPPORT PLATE 8 ADAPTED TO SUPPORTING LYOPHILIZED CUTS, TO BE EXTRACTED FROM COOLING CHAMBER 5. AFTER PRESSURE REDUCED FROM TRANSPORT HEAD 10, LYOPHILIZED CUTS 2 EXTRACTED FROM THE COOLING CHAMBER WHILE BEING PROTECTED FROM THE AMBIENT ATMOSPHERE. EXAMINATION OF BIOLOGICAL CUTS.

Description

The present invention relates to a transmission device

  fertility for the extraction of freeze-dried sections from the cooling chamber of a microtome for sections

  frozen in particular of a cryo-ultramicrotome.

  Biological objects intended for hystochemical purposes

  Particularly for carrying out X-ray microanalysis by electron microscopy, they are often cut after a rapid freezing, called "cryofixation", in the frozen state in cooling chambers, that is to say

  "cryomicrotomy" or "cryo-ultramicro-

  Since such types of hydrated cryo-cups can only be irradiated and analyzed electron microscopically with difficulty, hitherto the freeze-drying process has been used so as to increase the transparency of the cuts in the microscope. electron beam and to simplify the transfer of cuts between

  the cooling chamber and the electron microscope.

  To do this, the cup which is deposited on a microscope slide, for example in the anhydrous nitrogen atmosphere of the cooling chamber, is heated from the

  temperature at which it was frozen at temperatures

  of around 8-30 CLa most of the

  water molecules present in the cup are thus trans-

  The US Pat. Nos. 3,328,571, 4,284,894, and 4,302,450 provide examples of cryovials in the cooling gas of the chamber, which constantly scavenges the cuts and entrains these water molecules to the surrounding atmosphere. Ultramicrotomes having the general structure referred to US Pat. No. 3,924,500 for the representation of a conventional grid 10 adapted to withstand a cut. However, it is difficult to extract the dry sections of the chamber after lyophilization without formation of condensates or sublimates of water on the cold surfaces of 3; the cut when it enters the ambient humid atmosphere Such precipitates of water on the dry sections momentarily lead to an alternating distribution of ions and small molecules and thus tend to distort any subsequent micro-analysis of the cuts. So far efforts have been made to enclose the sections deposited on grids in the cooling chamber, in the formulating supports, for example small capsules of synthetic resin, before extracting these sections of the Cooling chamber Unfortunately, this step requires very thick capsules that can only be handled with great difficulty in the cooling chamber at temperatures below -1 OO 0 C. This is also true with regard to the tests tending to envelop the grids in * sheets of metal or plastic prior to extraction

  out of the cooling chamber.

  The main object of the present invention is to provide a relatively simple structure transfer device which is safe and reliable in its mode of operation and which eliminates many of the difficulties associated with the transfer of

  frozen hydrated cups at cryogenic temperatures.

  The device according to the present invention is constructed in such a way that the microscope slides can be moved from the heating plate provided for lyophilization to a support plate which constitutes the bottom of a vacuum transport head, which head, after the displacement of the grids on the plate, can be removed to allow the extraction, out of the space of the chamber, of the lyophilized cup carried by the grid The temperature of the transport head and the support plate, during the extraction of lyophilized sections; out of the cooling chamber, is maintained at a value substantially

  greater than that of the cooling chamber.

  Furthermore, in a preferred embodiment, the transfer device is made at least partially of a transparent material. Valve means are provided for the re-establishment of the pressure inside the transport head, which eliminates the risk of convection current related to this restoration of pressure in the head Recesses are also provided in the support plate for receiving and extracting the grids bearing the cuts and, preferably, there is a cover plate of the grid The receptacle support plate

E 2 16652

  may be heated by means of a heating element for the transfer or before the opening of the transport head in such a way that the precipitation of the water on the bio-colloids of the freeze-dried sections is avoided In this respect, it is thermostatic control means of the heating element are provided for controlling the temperature of the support plate and preventing the heat from damaging the specimen.

  The invention is hereinafter described in detail by reference

  FIG. 1 is a schematic representation of a cooling chamber of known construction showing a transfer device according to the present invention prior to the extraction of lyophilized sections from the cooling chamber; Figure 2 is a schematic representation of the closed transfer device, under reduced pressure, after the extraction of the cooling chamber and comprising a manually operable vacuum pump and a valve system; FIG. 3a is a diagrammatic cross-sectional representation of a modified embodiment of the transfer device according to the present invention with a thermostatically heatable support base plate and including recesses for placement and removal. removing grids and a cover for cutting protection during transfer and pressure recovery; and FIG. 3b is a top view of the base plate 3 of FIG. 3a also showing the recesses

  for the reception of grids.

  Referring to Fig. 1, there is shown a conventional cooling chamber comprising a multilayer chamber wall 1 which is cooled by liquid or gaseous nitrogen at a temperature substantially below 00 ° C., for example, such as that -1700 C, and which is kept free of frost by a continuous stream of nitrogen gas cold and dry through the interior For the production of frozen sections 2 to be analyzed, the frozen biological sample 3 _ is sliced by l- The edges 2 removed by the knife edge are transferred, by means of suitable conventional devices, not shown, towards the inside of the chamber 5 on grids 6 in the form of circular disks. commonly used for this purpose These grids 6 may have a diameter of 2.3 or 3 μm and the grids provided with the cuts are fed to a freeze-drying plate 7 on which they can be flattened using a suitable means. the unrepresented and dried at a temperature) -800 C. According to the present invention, the grids 6 provided with the pressed and dried cuts are moved from the support plate 7 on a plate 8 whose location makes it possible, in a simple manner, a For this purpose, the surface of the support plate 8 is arranged slightly lower than the surface of the plate 7. The plate 8 is preferably introduced into the chamber 5 immediately before the transfer of the grids. Plate 8 is more or less at room temperature. It avoids a too rapid thermal exchange between the plate 8 and its support surface, for example by means of a layer 9 made of a honeycomb material having thermal insulation properties. which layer contributes, with the rapid manipulation of the grids, to prevent the condensation 2-5 on the cuts After the displacement of the grids on the plate 8, the transport head 10 which is also at ambient temperature, is placed on the plate 8 and subjected to a reduced pressure such that the plate 8 is momentarily glued to the transport head 10 by a pressure difference which allows the extraction of the plate 8 on which the grids rest from the space of the chamber without risk of exposure to the ambient atmosphere Between the transport head 10 and the base plate 8, is disposed a sealing means which, in the form of Execution is given by way of example, is an O ring insulating the interior space of the head 10 The head 10 is provided with a discharge pipe 12 on which is

  mounted a pipe 13 for connection to the pump -

  FIG. 2 shows a complete transfer device according to the present invention, with a simple manual pump 14 provided with a pressure gauge 15 and an air inlet valve. The air inlet can be passed through a dehydrating agent which is provided in a manner known per se, in a small tube 17 between the valve 16 and the pipe 13 to prevent the introduction of moist air into the cups

  freeze-dried when air is allowed to enter the

  In accordance with the present invention, the baseplate 8 with its flat underside can be placed on a heated plate 18 and thereby be heated to a higher temperature. at room temperature such as the risk of introduction of moist air when opening the transport device -can generally be avoided An additional advantage of the structure of the device according to the invention is that the transport head 10 has can be made of glass or transparent synthetic resin, which makes possible the continuous observation of the position

grids 6.

  FIG. 3 shows a schematic representation of another embodiment of the transfer device according to the present invention. The base plate 8 has, in this case, three feet 19 provided with pointed ends and only two of which are visible. these feet minimizing the contact between the plate 8a and a given support surface and, in this way, the heat exchange between

  Similarly, according to this form of execution of the invention,

  In that case, the heat exchange between the plate Sa and the ambient gas of the chamber can be reduced by means of an insulating layer 9a. Thermostatic heating means consisting of a cartridge type heater 20 and a probe 21, which are connected by a multiconductor cable 22 to a control device, not shown, are arranged in the plate 8a. The multi-conductor cable 22 is provided with an insulating handle 23

  thermal energy that projects from the cooling chamber

  Another advantage may lie in having grids 6 dimensioned so as to be able to be received in recesses 8b of complementary size which are

  together by means of a groove so as to facilitate the extrac-

  Another advantage lies in providing the individual recesses with indicia, for example numbers and / or letters for the different grids that are received. Preferably, a plating is provided. grid cover 24 so that disturbances related to the transfer or convection currents occurring during the circulation of air in the head 10 does not change the position of the cuts-or 1.0 those of the grids In this way, the spatial position of the cover 24 on the base plate 8 a-, as shown in FIG. 3 a, can be determined and

  localized by means of gougeons not shown.

  The embodiments shown and described in FIGS. 1 to 3 merely show some of the preferred embodiments of the concept of the invention. Other embodiments are of course possible. For example, sealing between the head 10 and the support plate 8 can be obtained by giving to their opposite surfaces a precision finish instead of using an O-ring. The type, the installation and the method of attachment of the vacuum system. the equipment and pressure recovery, as well as the type and arrangement of the heating means or the insulation of the support plate can be modified using technically equivalent variants known without affecting the mode of operation according to the invention Similarly,

  can vary the materials used for the construction

  this device and the temperature ranges.

  The pump, the heating means, the transport head, the support plate, etc. comprise elements belonging to the invention but may be made in any suitable manner. In any state of the art, the embodiments

  described and represented were only examples.

Claims (10)

  1 Transfer device for extracting freeze-dried sections from the cooling chamber of a microtome for frozen sections, characterized in that it comprises a generally hollow and closed conveying head (10) adapted to be placed in communication with a source   vacuum valve (14), said transport head (10) being   it is capable of being introduced into the cooling chamber (5) and provided with an opening and a sealing surface surrounding said opening, a support plate (8) being adapted to support freeze-dried sections to be extracted. the cooling chamber (5), said support plate (8) being dimensioned in a manner   to be able to close, in a fluid-tight way, the opening   transporting the transport head (10) to protect the cuts (2) from the ambient atmosphere, whereby after the reduced pressure of the generally hollow transport head (10) by the vacuum source, the freeze-dried sections (2) can be extracted from the cooling chamber while   by being protected from the ambient atmosphere.   2 transfer device according to claim 1, characterized in that the transport head (10) is bell-shaped and in that the support plate (8) forms a bottom plate closing the opening of said head of transport (10).   3 transfer device according to claim 1, combined with a cooling chamber (5) comprising a pressure plate and heating (7), characterized in that it comprises a support base juxtaposed for the support plate (8) said pressure and heating plate (7) being arranged in such a way that an upper surface of the support plate (8) is generally at the same height as an upper surface of the pressure plate and heating (7).   Transfer device according to claim 1, characterized in that it comprises vacuum pump means (14) operatively arranged inside the generally hollow interior of the transport head (10) and conduit means (13) for the communication between the vacuum pump (14) and the transport head (10) which comprises a   handle (23) for the support plate (8).   Transfer device according to claim 1, characterized in that means (16) are provided for restoring the pressure in the hollow interior of the head of the transport (10).   Transfer device according to claim 5, characterized in that means (17) are provided for drying the fluid used to restore the pressure in the chamber. transport head (10).   Transfer device according to claim 1, characterized in that the transport head (10) is   constructed, at least partially, of transparent material rent.   8 transfer device according to claim 1, characterized in that the upper surface of the support plate (8) is provided with at least one recess (8 b) receiving grids (6) of freeze-dried section support and in that it has clues (1-2-3-4) for   identification of the cut supported by the grid.   9 transfer device according to claim 8, characterized in that at least one of the recesses (8 b) is dimensioned in a manner complementary to the size of the object-carrier (6) and in that it comprises at least a lateral groove to facilitate the gripping of the grid   holder (6) by means of forceps.   Transfer device according to claim 8, characterized in that it comprises a cover (24) for at least one of the recesses (8b), said cover (24) being dimensioned so as to be able to penetrate into the transport head (10) without being able to be moved during the   movement of said transport head (10).   11 transfer device according to claim 1, characterized in that the support plate (8) is isolated on its underside.   -12 Transfer device -according to claim 3, characterized in that the base-support for the support plate (8) provided in the cooling chamber (5) is thermally insulated. Transfer device according to claim 1, characterized in that the support plate (8) comprises   heating means (20) thermostatically controlled.   Transfer device according to claim 1, characterized in that the support plate (8) comprises an insulating handle (23) having a portion that can be grasped   manually from outside the cooling chamber (5). I