WO1987002130A1

WO1987002130A1 - Improvements in cryostatic microtomes

Info

Publication number: WO1987002130A1
Application number: PCT/GB1986/000595
Authority: WO
Inventors: Brian Laurence Jackson; Philip Parker
Original assignee: Brian Laurence Jackson; Philip Parker
Priority date: 1985-10-04
Filing date: 1986-10-03
Publication date: 1987-04-09
Also published as: DK284487D0; JPS63501820A; CN86107563A; EP0302054A1; GB8524530D0; KR880700257A; CN1008479B; DK284487A

Abstract

Cryostatic microtome in which the main structure of the microtome (1) is located outside the cryostatic chamber (2), thus reducing the thermal load on the cryostatic cooling system and greatly facilitating cleaning and decontamination of the apparatus. The invention also provides direct cooling of the specimen head (12) and includes a novel sample cassette system.

Description

IMPROVEMENTS IN CRYOSTATIC MICROTOMES

The present invention relates to a cryostatic microtome, that is to say a microtome which is designed and intended for use at temperatures below ambient temperature. Cryostatic microtomes have been manufactured for a number of years, and have consisted of a microtome which is mounted in a refrigerated chamber. The problem with this type of microtome is firstly that the temperature of the whole instrument has to be lowered, and secondly that the whole instrument has to be cleaned when, for example, infected pathological samples have been sectioned and decontamination procedures have to be followed. This involves very long decontamination times. According to the present invention, a cryostatic microtome comprises a refrigerated chamber provided with cooling means to lower the temperature of the chamber and its contents, a microtome having its base mounted externally of the chamber and its specimen head and knife located within the chamber, and means to reduce heat gain within the chamber via or around the parts of the microtome where they enter the chamber.

Either the knife or the specimen head, or both, may be carried directly or indirectly on corresponding thermally insulating members.

Either the knife or the specimen head, or both may be supported by corresponding members each of which may pass through an aperture in the wall of the chamber. Preferably, sealing means surround any member where it passes through an aperture in the wall of the chamber.

The sealing means may comprise one or more flexible membranes.

The invention also includes any microtome in which the reciprocating member, whether the specimen arm or the knife, moves in a plane offset from the main frame of the instrument so that it can be arranged with the knife and the specimen arm entering through two orthogonal sides of a generally square or rectangular cryostatic chamber. In one embodiment of the invention, the base of the microtome is "L"-shaped in plan view.

The advantages of the invention are firstly that the base of the microtome, and the bulk of its mechanism, are located outside the cryostatic chamber, secondly that with only the specimen carrier and knife located within the chamber the instrument is very simple to clean or decontaminate, thirdly that the relatively small size of the chamber reduces the load on the refrigeration plant and fourthly that deep-drawn seamless stainless steel tanks of a size suitable to accommodate the specimen carrier and knife are readily available commercial items which can be used as the cryostatic chamber in the invention.

Another aspect of the invention is the provision of a specimen holder which comprises a carrier adapted to accommodate a thin plate to which the specimen has been frozen. This enables ultra-rapid freezing of the specimen, with the corresponding reduction of frost destruction of tissue. The low cost of the plate enables frozen specimens to be stored on the plates in suitable cryostatic conditions.

The invention will now be explained in greater detail with reference to the accompanying drawings, in which: Figure 1 is a diagramatic plan view of a cryostatic microtome constructed in accordance with the invention,

Figure 2 is a diagramatic perspective view of the general arrangement of the microtome parts used in the embodiment of Figure 1 , Figure 3 is an elevation of the embodiment of Figure 1 looking in the direction of the arrow III in Figure 1 , partly broken away for clarity. Figure 4 is a fragmentary view of part of the reciprocating specimen arm of the microtome of Figures 1 to 3, Figure 4a is a diagrammatic view of a modified specimen arm provided with cooling means, viewed from a similar viewpoint to that of Figure 4, and

Figure 5 is a sketch of a specimen holder suitable for use in microtomes according to the invention. The cryostatic microtome illustrated in the Drawings comprises a microtome indicated generally at 1 , with its specimen head and knife mounted within a cryostatic chamber indicated generally at 2. Only those parts which are necessary for a clear understanding of the invention are shown in the Drawings, it being clear to those skilled in the art how the details of non-illustrated items can be arranged to secure correct operation of the instrument. In the following description, the terms "front" and "rear" will refer respectively to that part of the microtome adjacent the cutting area and to the opposite end of the instrument.

As can be most clearly seen from Figure 1 , the microtome 1 is built on a generally elongated base 3 having an upstanding bracket 4 supporting a knife assembly generally indicated at 5. The upper surface of the end of the base remote from the bracket 4 carries a transverse platform 6 which projects from one side of the base and is rigidly secured thereto. A first pivotable member 7, equal in length to the platform, 'is hinged to the rear edge of the latter by means of strip hinges 8 of thin phosphor bronze. A second pivotable member 9 is secured to the top front edge of the member 7 by further phosphor bronze strip hinges 10. The front face of this member supports the reciprocating arm 11 of the micro¬ tome, which in turn supports the specimen head 12 through an intermediate thermally insulating member 13, fuller details of which can be seen in Figure 4. The action of the microtome is effected by pivoting of the two members 7 and 9 under the action of a drive motor. The member 7 is acted upon by a leadscrew (not shown) which drives a nut linked to an advance lever 14 which is hinged to the forward edge of the member 7 by phosphor bronze strip hinges 15 to overlie the main base 3 parallel to the latter. Forward movement of the lever 14 parallel to the length of the base 3 under the action of the leadscrew causes the member 7 to rotate about the strip hinges 8, thereby moving the upper edge of the member 7 in a forward direction and advancing the specimen head towards the knife assembly 5. The lever 14 is also provided with means (not shown) for causing retraction of the specimen between successive cutting strokes Reciprocating movement is imparted to the arm 11 is derived from a shaft 16 journalled in the base 3 and driven from a gear wheel 17. The shaft 16 is connected via an eccentric 18 to an "L"-shaped lever 19 which in turn is pivotally connected to the thermally insulating member 13. As can be seen from Figure 4, the member 13 consists of a hollow cylindrical portion 13a closed at its forward end and provided with a central shaft 13b of square section to which the end of the lever 19 is linked by a pin 20. Thus, as the shaft 16 is rotated, the arm 11 is caused to oscillate in a vertical plane about the line of the strip hinges 10 to cause the cutting action of the instrument. As will be readily understood, the movements of the members 7 and 9 are synchronised to cause the specimen head to be advanced before each successive cut, and retracted before each return stroke. Although not illustrated, the drive of the illustrated embodiment is effected through stepper motors under the control of a microprocessor.

The assembly of the microtome per se with the cryostatic chamber will now be described in greater detail. The chamber consists of a standard deep-drawn stainless steel sink unit 21 which has been formed with a drain at 22 in its base, a circular pressed-in section 23 in the side wall 24 adjacent the microtome base 3 which is so dimensioned that its circular portion lies in a vertical plane, and an elongated aperture 25 in the adjacent rear wall, with its long axis vertical. The unit 21 is supported in a cut-out 26 in a top panel member 27 of an enclosure (not shown). The rear end of the base 3 is slung from the underside of the top panel member 27 by means of two hangers 28, while the front end of the base is secured fast to the side wall of the unit 21 through the intermediary of a thermally insulating cylindrical boss 29 made of Delrin (Registered Trade Mark). The boss 29 is bored. from both end faces as indicated at 30 and 31 to receive set screws which pass through corresponding holes drilled in a circle through the section 23 of the wall 24 of the unit 21. The screws 30' and 31' which are located in the bores 30 and 31 serve to secure together the boss 29, located on the outer face of the wall 24 and a circular metal flange 32 located inside the unit 21, without establishing direct thermal contact between the bracket 4 and the interior of the unit 21. A blade carrier 33 is adjustably secured on the flange 32 to permit the usual adjustment of its angle of attack relative to a specimen mounted on the specimen head 12.

The unit 21 carries on its outer surface a set of refrigerant tubes (not shown) which permit its temperature, and that of its contents, to be lowered as required. The outer surface of the unit is coated, over the refrigerant tubes, with a layer of rigid thermally insulating foam material some 4.0 cm. thick. To complete the thermal insulation of the cryostatic chamber so formed, a thermal barrier is provided where the specimen arm passes through aperture 25 its rear wall. A thermoplastic sleeve 34 makes a snug fit in the aperture 25, and is externally grooved at 36 and 37 to accept the outer edges of annular siliσone rubber boots 36' and 37' whose inner edges are clamped around the ends of the thermally insulating member 13. The flexibility of the boots 36' and 37' at the temperatures at which the microtome is operated, which may be as low as -80 degrees Celsius ensures free movement of the reciprocating arm 11 during operation of the instrument, but the thermal insulation provided by the member 13 and sleeve 34 and the double-walled seal provided around the arm by the boots 36' and 37' to prevent draught and convection losses, together with the other precautions against heat transfer via or around parts of the microtome, enable the low temperatures required within the cryostatic chamber to be rapidly attained and reliably maintained.

Figure 4a illustrates a modified specimen arm which is provided with cooling means to maintain a low and controlled temperature of the specimen. As far as possible, the reference numerals in Figure 4a correspond to those in the earlier Figures. In the arrangement of Figure 4a, the specimen arm comprises a solid copper bar 11a, carried in an insulating casing 11b made of rigid PVC, and which itself forms part of the linkage of the microtome via a portion 9a hinged to the block 7 of the microtome (see earlier Figures). The specimen head 12 is mounted on the bar 11a via a Peltier heat pump 44, which in use pumps heat from the holder 12 to the bar 11a. On its upper surface externally of the cryostatic chamber the bar 11a is further provided with two Peltier heat pumps 45a, 45b which pump heat away from the bar to a heat sink 46, whence it is removed by a forced draught of air ducted from a fan (not shown). By controlling the operation of the Peltier pumps in response to a platinum resistance thermometer (not shown) embedded in the specimen head 12, it is possible to exert good control over the absolute temperature of a specimen. In this embodiment, the PVC casing 11b and the gaskets 36', 37' reduce heat gain within the chamber 2 despite the thermal conductivity of the bar 11a.

Referring to Figure 5, a specimen holder designed for the microtome of Figures 1 to 4 comprises an aluminium body 38 which is designed to lock onto the specimen arm of the microtome, The upper edge of the free end of the body is formed with a projecting lip 39 which is slightly undercut, and the lower edge is provided with a spring loaded catch 40 movable via a knob 41. Specimens are frozen on to thin aluminium plates 42 which can be releasably secured on the front face of the body 38 between the lip 39 and the catch 40. The surfaces of the plate 42 are slightly roughened for adhesion of the specimen, and the plate itself is formed with a handle portion 43 covered with a dipped plastic coating. In use, a specimen may be very rapidly frozen to the plate 42, owing to the low thermal mass of the latter, thus minimising frost damage to the specimen, and the plates 42 are sufficiently cheap to use in quantity for the storage of specimens under low-temperature conditions.

The advantages of the cryostatic microtome of the invention can be readily appreciated from the illustrated embodiment, with particular reference to Figure 1. The space within the cryostatic chamber can be seen to be uncluttered, giving ample access through the open top of the chamber for loading and unloading the specimen head and adjusting or replacing knives, although the size of the unit 21 is only 32 by 30 by 20 cm. deep. For the same reasons, the chamber is very easy and quick to clean or decontaminate since, as none of the drive components of the microtome is within the chamber,and strong anti- septic or disinfectant sprays can be used within the cryostatic chamber without damage to the instrument. It will be appreciated that the invention is capable of a wide variety of implementations, and that the illustrated embodiment is not to be regarded as in any way restrictive. For example, although the instrument illustrated is a histological microtome, the invention could be applied to ultramicrotomes operating under cryostatic conditions.

Claims

1. A cryostatic microtome assembly of the kind comprising a cryostatic chamber provided with cooling means to lower the temperature of the chamber and its contents, characterised by a microtome (1) having its base (3) mounted externally of the chamber (2) and its specimen head (12) and knife (33) located within the chamber, and means to reduce heat gain within the chamber via or around the parts of the microtome where they enter the chamber.

2. A cryostatic microtome assembly according to Claim 1 characterised in that the knife (33) is carried on a thermally insulating member (29).

3. A cryostatic microtome assembly according to Claim 1 characterised in that the specimen head (12) is carried on a thermally insulating member (13).

4. A cryostatic microtome assembly according to Claim 2 or Claim 3 characterised in that the or each item carried on a said thermally insulating member is directly carried thereon.

5. A cryostatic microtome assembly according to any one of the preceding Claims characterised in that either the knife ( 33 ) or the specimen head (12) is supported by means (30' ,31 ' ;13) which pass through an aperture (23,25) in the wall of the chamber.

6. A cryostatic microtome assembly according to Claim 5 characterised in that both the knife (33) and the specimen head (12) are supported by respective members (30' ,31 ' ; 13) which pass through one or more apertures (23,25) in the wall of the chamber.

7. A cryostatic microtome assembly according to Claim 5 or Claim 6 characterised in that sealing means (36* ,37') surround any member where it passes through an aperture (25) in the wall of the chamber.

8. A cryostatic microtome assembly according to Claim 7 characterised in that the sealing means comprises one or more flexible membranes (36',37').

9. A microtome for use in a cryostatic microtome assembly according to any one of the preceeding claims characterised in that the reciprocating member moves in a plane offset from the main frame of the instrument.

10. A microtome according to Claim 9 characterised in that the base (3) of the microtome (1) is "L"-shaped in plan view.

11. A microtome assembly or a microtome according to any one of the preceeding claims, characterised in that the specimen head is provided with cooling means.

12. A microtome assembly or a microtome according to

Claim 11 characterised in that the specimen head (12) is carried on a thermally conducting member (11a) which is arranged to extract heat from the sample head (12) but is in turn thermally insulated so that in use the entry of heat into the cryostatic chamber is minimised.

13. A microtome assembly or a microtome according to Claim 12 characterised in that a heat pump (44) is located between the specimen head (12) and the thermally conducting member (11a).

14. A microtome assembly or a microtome according to Claim 12 or Claim 13 characterised in that a further heat pump (45a,45b) is provided to extract heat from the thermally conducting member (11a).

15. A microtome assembly or a microtome according to Claim 13 or Claim 14 characterised in that the or any of the heat pumps are thermoelectric devices.

16. A microtome assembly or a microtome according to any one of Claims 11 to 15 characterised by the provision of a heat sink (46) to assist in the extraction of heat.

17. A microtome assembly or a microtome according to any one of Claims 12 to 16 characterised in that the thermally conducting member (11a) is a metal bar.

18. A microtome assembly or a microtome according to Claim 17 characterised in that the metal bar (11a) is encased in a thermally insulating casing (11b).

19. A microtome assembly or a microtome according to Claim 18 characterised in that the thermally insulating casing (11b) forms an operational part of the linkage of the microtome ( 1 ) .

20. A specimen holder for a microtome assembly or a microtome according to any one of the preceeding claims, characterised by a carrier (38-41) adapted to accommodate a thin plate (42) to which a specimen may be frozen.