US3495490A - Microtome freezing attachment - Google Patents

Description

Fel 17, 1970 F.v|.. DoLLHoPF v3,495,490 Y mcno'rouf: FREEZING `ATTACHMENT Filed Feb' 19' 1968 A 2 Sheets-Sheet. 1

Feb.17,197o F.| .D1.LHPF 3,495,490

' MIGROTOM F-REEZING ATTACHMENT Enea Feb. 19, 196s z'sneejfs-sheet 2 OPERATED VALVE United States Patent O 3,495,490 MICROTOME FREEZING ATTACHMENT Franz L. Dollhopf, London, England, assigner to Shandon Scientific Industries Limited, London, England, a British company Filed Feb. 19, 1968, Ser. No. 706,364 Claims priority, application Great Britain, Feb. 17, 1967, 7,744/ 67 Int. Cl. B26d 7/08 U.S. Cl. 83-171 9 Claims ABSTRACT F THE DISCLOSURE The invention relates to a freezing attachment for a microtome, particularly an ultramicrotome, to enable sectioning to be accomplished with a specimen frozen below its glass transformation temperature in an atmosphere from which water vapour is excluded by displacement by gaseous coolant. The attachment consists of an enclosure with knife and specimen holders each with temperature controller that may comprise a trough for liquid coolant that vaporizes to displace atmosphere of enclosure and a heater element both under control of temperature sensing device that admits coolant or energizes heater as required.

This invention relates to microtomes, and is particularly, although not exclusively, concerned with an attachment for enabling an ultramicrotome to be used to provide ultrathin sections of a specimen in a frozen state.

Ultramicrotomes are used extensively to provide ultrathin :sections of a specimen requiring examination under an electron microscope. They are able to provide sections of the specimens of a thickness expressed in Angstroms. The sections are obtained by relatively moving two members, one of which carries a holder for thespecimen to be sectioned and the other of which carries a holder for the microtome knife. The microtome knife may comprise a glass block having a wedge-shaped portion terminating in an extremely sharp cutting edge, or a diamond-edged cutting surface of extreme sharpness. The sections slide off the knife edge onto a suitable liquid, which is held in a boat iitted behind the cutting edge of the knife. The preferred method of sectioning is to hold the knife holder stationary, and to move the specimen holder around a D-shaped path, in which sectioning occurs during the movement of the specimen along the linear portion of the D.

In order to obtain useful ultrathin sections it is necessary for the specimen to be completely rigid during the cutting process. To achieve this, it is customary practice to process and embed the specimen in a :suitable polymer. Amongst the drawbacks of this technique, however, is the likelihood of artifacts due to the penetration of the embedding material, and the relatively lengthy preparation which is necessary before sectioning can take place.

Previous experiments have proved it possible to achieve this hardening of the specimen temporarily by cooling it down marginally 'below its glass transition temperature. This temperature naturally varies from one type of specimen to another.

While a frozen specimen is being sectioned, it is constantly in contact with air, and, inevitably, Water vapour in the air tends to condense on the surface of the specimen, causing the formation of ice crystals. Such ice crystais can draw liquid from the boat onto the cutting face of the specimen. This liquid then acts as a lubricant between the specimen and the knife. In such a case ultrathin sections are unobtainable as, instead of cutting sections, the knife edge tends rather to slide over the exposed face of the specimen.

An object of this invention is the provision of a freezing attachment for an ultramicrotome.

According to the present invention, we provide a freezing attachment for a microtome comprising a thermally insulated 4box adapted to be secured to one of the members of the microtome and providing a compartment containing a knife holder and a specimen holder, one of said holders being xed to the box and the other having an attachment member passing through one wall of the box and adapted to be secured outside the box to the other member of the microtome, sealing means between said wall of the box and said attachment member arranged to allow sectioning movement of said other holder relative to said one holder, means for controlling the temperature of said holders, at least one bleed passage from the chamber and at least one conduit into the chamber through which a cold dry relatively inert coolant, such as nitrogen, can be introduced into the chamber in either a gaseous or liquid state to lill the chamber with cold dry gas and ush air and water vapour from the chamber through the bleed passage.

By sectioning movement of said other holder relative to said one holder is meant movement which with a specimen fitted to the specimen holder and a microtome knife fitted to the knife holder would result in the desired thin :sections -being cut from the specimen.

An advantage of the invention is that it enables ultrathin sections to be cut from a specimen without the customary artifacts caused by processing and embedding. This advantage stems from the fact that in a freezing attachment in accordance with the invention the cutting process is performed in an absolutely dry atmosphere, as the dry gas in the compartment displaces the air and water vapour from it. For this reason no ice crystals can form on the specimen.

Preferably the temperature control means for each holder includes an electrical resistance heater arranged to heat ,the holder.

Preferably the control means for both the holders includes an open trough in the holder disposed beneath the outlet of a conduit in the chamber and arranged to receive liquid coolant passed into the chamber from the conduit.

By using liquid nitrogen as the coolant, both the specimen holder and the knife holder can be cooled to a very low temperature, and, with the help of the heaters, any temperature from 0 to 150 C. can be achieved to suit all types of specimens.

The cutting process is performed in the closed thermally insulated compartment, and there is negligible interference from outside influences.

A freezing attachment for a microtome will now be described, by way of example, with reference to the accompanying drawings, in which:

FIGURE 1 is a perspective view of the freezing attachment,

FIGURE 2 is a view in vertical section of the freezing attachment, and

FIGURE 3 is a sectional view of part of the attachment taken along the line III-III in FIGURE 2.

The freezing attachment of the invention and illustrated in the accompanying drawings is designed to be fitted to an ultramicrotome incorporating one member comprising a horizontal platform with a slot into which a standard knife holder or block supporting a microtome knife is normally placed. Above this platform, another member comprising a specimen arm, which follows a D-path when moved, extends from the interior of the microtome. A specimen holder fitted ,to this arm is so arranged that during operation of the microtome the specimen face from which sections are to be cut is brought down across the stationary knife edge over the linear part of D. The specimen then travels upwardly over the arched part of the D-path, thereby by-passing the knife. During the upward movement, the specimen is advanced by a previously selected amount.

To use the freezing attachment both the standard knife block and specimen holder are removed from the microtome.

The attachment comprises a box made of plastics insulating material having a rib 12 formed in its underside for mounting on the microtome platform in place of the standard knife block. The rib 12 is shaped to lit within the slot normally occupied by the standard knife block. A locking screw is provided on the platform and, when tightened, holds the rib rigidly in the slot thus securing the box to the platform.

Inside the box is a compartment in which sectioning is carried out. The box has walls formed by sheets of Clear plastics material commercially known by the registered trademark Perspex. The walls insulate the compartment thermally from the surrounding atmosphere and enable sectioning to be performed at low temperatures within the compartment without large amounts of condensation forming on the outside walls of the box, thus allowing full observation of the sectioning operation at all times.

Such condensation as does take place is collected in a drip tray 4 below the compartment and fitted to the rib 12 as shown in FIGURE 2. The top of the compartment is provided by a removable lid 16 engaging a seal 17 around the upper edges of the walls of the box, and suitable releasable fastening means (not shown) are provided to hold the lid in place. The lid has a window 18 formed by a sheet of transparent material having an electrical resistance heating wire 20 embedded in it. Heating the window 18 prevents condensation on the window so that it remains clear to enable the operation of the microtome to be viewed through a stereomicroscope.

In the compartment is a specimen holder 22 comprising a specimen holding member 23 in a spigot 24 extending from an aluminum container 25 providing a trough 28. The container is accommodated as a clearance fit in a recess formed in a block shaped part of an attachment member 34 of an insulating material such as that known commercially by the registered trademark Tufnol and has one wall fixed to a wall 32 of the recess. The clearance space between the container 25 and the recess 30 allows the container to expand and contract freely with respect to the attachment member 39. The attachment member is provided with a pin 36 which projects through an aperture 37 in one wall of the box by way of a flexible polyethylene or silicone rubber sleeve or diaphragm 38 to the specimen arm of the microtome, the pin 36 being received in a socket in the specimen arm in place of the standard specimen holder, and being fixed by means of a clamping screw. The flexible sleeve 38 is sufficiently slack to allow the specimen arm to move the specimen holder around the D-path necessary to perform sectioning. At its inner edge the sleeve is tightly held around the pin, while its outer edge is sealed to the periphery of the aperture 37.

A platinum resistance thermometer 31 and an electrical resistance heater 34' are provided in the recess 30 outside the container and are connected to male connectors 3'9 extending from the pin 36 which engage in corresponding female connectors in the socket in the specimen arm which receives the pin 36.

Also within the compartment and secured to the bottom of the box is a knife holder comprising an aluminum block 42, provided with a socket 44 shaped to receive a standard metal clamp 46 in which is mounted a knife proper 48. A screw (not shown) in the side of the socket is provided with a shaft (not shown) extending through a bushing in a wall of the compartment to a knob which can be screwed up from outside the compartment to x the knife clamp 46 rigidly to the knife block 42. The knife clamp 46 is provided with a groove 50 to receive the knife 48 which is formed by a glass prism of right-triangular shape. The clamp 46 has two screws (not shown) for clamping the knife 48 in the clamp. The cutting edge of the knife is formed at the intersection of the hypoteneuse face of the glass prism and a vertical face, the angle between the two faces being 45. Alternatively a diamond knife can be tted. The cutting edge extends horizontally directly beneath the position occupied by a specimen carried by the specimen holder at the commencement of the vertical downward cutting stroke corresponding to the linear portion of the D movement.

The hypoteneuse face of the knife is provided with a boat 52 formed by a strip of inert plastics material or metal tape sealed by inert wax to the glass prism adjacent the knife cutting edge, the boat extending across the hypoteneuse face for some distance from the cutting edge. During normal operation a triangular cavity formed between the strip and the sloping hypoteneuse face is filled with suitable liquid, so that sections sliced from a specimen slide on to the surface of the liquid in the boat, where they stay afloat.

The block 42 is formed with a trough 54 and has a platinum resistance thermometer and a resistance heater, indicated together at 56 embedded in the block and connected to suitable leads (not shown) extending from the box 10.

Upper and lower conduit pipes v58 and 59 respectively extend into the compartment through one wall of the box, to which they are sealed. At its end within the compartment the pipe S8 is bent downwards to provide an outlet directly above the trough 28 in the specimen holder. The pipe 59 is bent downwardly at its end within the compartment over the trough 54.

In use, the conduit pipes 58 and 59 are connected through solenoid operated valves (valve 58' for pipe 58 being shown) and silicone rubber hoses to the bottom of a pressurised vessel containing a coolant such as liquid nitrogen. The solenoid valve connected with each conduit pipe, and the heater in the associated knife or specimen holder are connected to a suitable automatic electrical control unit and are controlled by the associated platinum resistance thermometer so that when the temperature of the holder rises above a predetermined limit the solenoid valve is opened to allow liquid nitrogen to pass into the trough to cool the holder and when the temperature of the holder falls below a predetermined limit current is passed to the heater. The above predetermined limits are ararnged to lie as closely as possible on either side of a selected temperature between O and C. set on a dial on the control unit so that the holder is maintained substantially at the selected temperature.

The box is formed with a bleed passage 10 extending through one of the walls, and which vents the compartment to the outside atmosphere.

A syringe 53 has a collar portion 55 removably sealed into one wall of the box above the level of the knife assembly. The plunger 57 of the syringe extends on the outside of the compartment and is movable from outside of the `compartment while the needle of the syringe, comprising a capillary tube 60, extends within the compartment and has its end portion turned downwardly into the boat 52. The syringe is filled with liquid, such as alcohol with which the boat is to be replenished from time to time during operation of the microtome.

The freezing attachment is tted to the microtome and used as follows.

The locking screw holding the standard knife block assembly to the platform is slackened and the knife block assembly as well as the standard specimen holder which is fitted to the specimen arm are removed. The box is placed on the platform so that the rib 12 in its underside enters the slot formerly occupied by the standard knife block assembly. The locking screw is then tightened to fix the box rigidly to the platform. The conduit pipes 58 and 59 are connected through their respective solenoid valves and silicone rubber hoses to the bottom of the coolant vessel which contains liquid nitrogen. The vessel is closed so that partial evaporation of the liquid nitrogen pressurises the vessel. The vessel is provided with a blow-off valve which limits the pressure in the vessel to about 4 p.s.i.

The specimen holding member 23 is fitted to the attachment member which is then inserted into the compartment. The stem 36 is passed through the centre hole of the flexible sleeve or diaphragm 38 which corresponds in position approximately to the movable microtome specimen* arm. The stem 36 is then fitted to the microtome arm and the inner marginal edge of the dexible sleeve ris clamped tightly to the stem 36 passing through it. The sleeve is sufficiently slack to allow the stem of the trough block to move freely along the D- shaped sectioning path.

The knife clamp 46, to which the knife 48 carrying the boat 52 has already been fitted, is mounted on the knife block 42 and is held in place by screwing up the knife block locking screw from the outside of the compartment. The knife is rigidly held by its own locking screws in the knife clamp and the parts are so arranged that the cutting edge of the knife blade is situated horizontally directly beneath the specimen at the commencement of its downstroke.

The syringe 53 has already been charged with suitable liquid, such as alcohol, for the boat. The lid is placed on the compartment and fastened to the box.

The temperature for the specimen and the knife holders are selected on the temperature selection dials 0n the control unit. The control unit is connected to a suitable power supply and switched on. Since the temperatures of both the specimen and the knife holders are originally above the selected temperatures, the control unit causes the solenoid valves to open automatically.

Liquid nitrogen travels through the solenoid valves, the silicone rubber hoses and the conduit pipes 58 and 59 into the two cooling troughs 28 and 54 where at first it boils-off very rapidly.

The dry, cold gas produced by this rapid vaporisation of the liquid nitrogen is discharged into the compartment and rapidly scours out the air and moisture in it by way of the bleed passage.

The nitrogen vapoursing in the compartment provides a cold dry inert atmosphere which is ideal for carrying out sectioning of the specimen.

The temperature of each holder falls until it reaches the lower limit below the selected temperature. The respective solenoid valve is then closed by the control unit and the respective heater is energiscd to restore the temperature to the selected value. The valve re-opens and the heater is cut off as soon as the temperature reaches the upper limit above the selected value.

The control unit is provided with two further dials showing any deviation from the selected temperatures due to a malfunction.

When the specimen and knife are at the optimum temperatures for sectioning, the microtome is operated to cause sections of the desired thickness to be sliced from the specimen, and deposited one by one in the boa't where they float on the liquid which is so selected that it does not freeze at the temperature of sectioning.

Periodically, the boat may require replenishing with liquid which can be performed by means of the syringe.

After the required number of sections has been cut, the compartment lid is removed and the sections are lifted olf the liquid surface in the boat by a mesh grid disc, and transferred to an electron microscope for examination.

In a simpler version of the freezing attachment (not shown) the automatic control unit is dispensed with and the box also carries two tanks for liquid nitrogen one of which is connected Iby a vent to the compartment to supply vapourised nitrogen to the compartment and the other of which is closed and provided with a blow-off valve so as to supply liquid nitrogen under pressure via a conduit pipe to the trough on the specimen holder through a manually controlled needle valve. In this version of the freezing attachment, the trough on the knife block is dispensed with, as is the corresponding conduit pipe for supplying liquid nitrogen and the block is thermally connected to said one tank by a copper bar extending beneath lthe compartment. Both holders are provided with platinum resistance thermometers and electrical heaters as before but the thermometers are merely arranged to provide a temperature reading on a dial on a manual control unit and the heater and needle valve are adjusted manually to maintain the desired temperature.

The above decribed freezing attachments enable ultrathin sctions of frozen materials to be taken under relatively ideal conditions of temperature and atmosphere and without the danger of ice crystal formation on the surface of the specimen. Also the sectioning is performed under absolutely dry conditions.

In consequence, specimens can be cut satisfactorily without the prior processing and embedding previously considered essential.

I claim:

1. A freezing attachment for a microtome having two members, comprising:

(a) a thermally insulated box adapted to be secured to one of the members of the microtome, providing;

(b) a compartment containing;

(c) a knife holder;

(d) a specimen holder;

(d) a specimen holder; one of said holders being fixed to the box and the other having an attachment mem- Iber passing through one wall of the box and adapted to be secured outside the box to the other member of the microtome;

(e) sealing means between said wall of the box and said attachment member, arranged to allow sectioning movement of said other holder relative to said one holder;

(f) means for controlling the temperature of said holders;

(g) at least one bleed passage from the chamber;

and

(h) at least one conduit into the chamber for introducing a cold dry relatively inert coolant into the chamber to `fill the same with gas phase coolant with displacement of air and water vapour from the chamber through the said bleed passage.

2. The freezing attachment of claim 1 in which said means for controlling the temperature of each said holder includes an electrical resistance heater 'arranged to heat the holder.

3. The freezing attachment of claim 2 in which each said holder includes temperature sensing device arranged to control the current supply to the said heater so as to prevent the temperature of the holder from falling below a predetermined value.

4. The freezing attachment of claim 3, in which means for controlling the temperature of each said holder includes an open trough in the holder disposed to receive liquid coolant from a said coolant-introducing conduit.

5. 'Ihe freezing attachment of claim 4 in which said specimen holder includes a specimen holding member formed separately from the said attachment member and said trough comprises a container fixed to said specimen holding member and accommodated as a clearance t within a recess in said attachment member.

6. The freezing attachment of claim 5, in which said conduit serving a coolant trough includes a valve controlled by said temperature sensing device to `admit liquid coolant to the trough associated therewith when the holder temperature exceeds a predetermined value.

7. The freezing attachment of claim 1, in which said Wall of said box has an aperture and said sealing means comprises a flexible impermeable sleeve sealed to the attachment memebr and to said wall around said aperture.

8. The freezing attachement of claim 1, in which said box has a removable transparent lid.

9. The freezing attachment of claim 8, in which said lid has an electrical resist-ance heating element.

References Cited UNITED STATES PATENTS 3,236,133 2/1966 De Pas 83-915.5 X

JAMES M. MEISTER, Primary Examiner U.S. C1. X.R. 10 82-915.5

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