DE1962263U - MICROTOME. - Google Patents

Description

DE LA RtJE FRIGISTOR LIMITED, Langley, Buckinghamshire (England)

"M i k r ο t ο m"

The innovation concerns a microtome for cutting off Slices of a sample for microscopic examination.

Microtomes can be used below room temperature, but also within a cooled housing can be used. This housing is accessible through open walls with devices for operating and adjusting the microtome from outside of the housing as well as a transparent window for observation. Such housings are under the name "Cold regulator" (Cryostat ^ common article of trade. With such apparatus can of frozen organic Tissues or other objects can be cut off specimen slices which remain frozen in the freezing atmosphere and so that can be handled easily. The sample discs can thus be cut without chemical fixation. However, the different parts of the microtome are inside of the cooled.housing cannot be regulated in its temperature. Their temperature is approximately the same as the air temperature in the housing or fluctuates in an uncontrollable manner. Around furthermore, for example, the cutting knife to a certain extent

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To bring this low temperature, the entire apparatus must be cooled to this low temperature.

It is also known, the knife, the slide and possibly also to provide the damper for rolling movements of microtomes with thermoelectric cooling devices, so that frozen sample pegs in an unrefrigerated environment. can be cut. British patent specification 992 979 describes independent cooling and temperature control devices for these different parts, namely it had been found that the different parts of the Microtomes have different optimal temperatures for cutting samples of any given substance. However, since in this case the surrounding air is not cooled below freezing point, forms on the knife bis and it is difficult to actually get low temperatures on the knife and on the slide.

According to the innovation, the microtome with its knife and slide is arranged in a closed housing, which is equipped with cooling means for cooling the temperature within the housing, and wherein a first thermoelectric A cooling and control device for the temperature of the knife and a second thermoelectric cooling and control device for the temperature of the specimen holder are provided are, wherein the electrical current to the two cooling devices can be regulated separately and independently of one another.

IP "2" "

A conventional absorber or compressor cooler is preferably used to cool the interior of the housing. The However, the housing can also be cooled thermoelectrically or by expansion of carbon dioxide or by solid carbon dioxide. A thermoelectrically cooled cooling chamber for storing samples within the housing can also be provided being*

The thermoelectric coolants are as units of one Layering of semiconductor blocks formed between metal bridges, with the metal bridge on one side of the Semiconductor as the "hot connecting bridge" and the metal bridge on the other side of the semiconductor as "cold Connecting bridge ". Bie constituent parts of each Units are soldered together in an electrical series circuit, with a "cold connection bridge" each p-semiconductor block, a "hot connecting bridge", an n-semiconductor block etc. follows. The electrical current is supplied via "hot" connection pieces at the two ends of each such semiconductor sequence fed. Brine coolants are called "frigistor cooling units" common trade items. When the electric current passes through, a set of connections becomes "hot" and the other cold. When the electric current is reversed The hot and cold connecting bridges are also reversed. Conventionally, however, that is the object to be cooled

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facing connection set as "cold connection" and the other referred to as "hot joint" * Usually the connecting links are arranged in the sequence in which the hot connecting bridges appear, because this prevents the electrical leads from acting as heat conductors to the cold connections and thus to that too. have a cooling effect on the article or room.

On the surface of the cold connecting bridge is a disc thermally conductive but electrically insulating. Ceramic material (aluminum or beryllium oxide) or a metal disc, preferably made of aluminum, glued on or otherwise attached, the surface of which in contact with the cold connecting bridge has been oxidized (anodically oxidized) or made electrically non-conductive in some other way. It is also possible to use a thin sheet of polymerized material of the type commonly referred to as "plastic ¹ *". In the case of the slide, this sheet is the slide-holding plate onto which the sample is held, preferably by means of water, namely by thawing the back of the sample and freezing it In the case of the knife or, alternatively, the cooled storage chamber, the thermal contact between the cold connecting board and the knife (or the chamber) can be brought about by using a thin film of grease optionally be included in the walls of the chamber if they are anodic

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or otherwise electrically insulating aluminum is built, touching the cold connecting bridges of the thermoelectric cooling unit. From the hot connecting bridges the heat is by ^{ir ft} heat sink, preferably of aluminum, removed, which is rendered anodic or otherwise electrically insulated at least on the side adjacent to the hot junction surface. The heat sink is then preferably adhered to the unit by means of a epoxy resin. Each cooling unit with its anodized aluminum plate in thermal contact with the cold joint and the anodized aluminum heat sink in thermal contact with the hot joint can preferably be sealed by means of a layer of resin with which the entire cooling unit is coated and therefore protects it against moisture. ■ "■■■" .-

Other forms of heat sinks are also possible. So can E.g. cooling units are constructed, which are called Connections are flushed directly by a coolant.

Further advantages and features of the innovation emerge from the description of an exemplary embodiment using the Drawing.

Fig. 1 shows schematically the electrical and cooling circuit of an embodiment,

Fig. 2 shows in perspective the arrangement of the knife and its thermoelectric cooling units,

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Fig. 3 shows in perspective a microtome with slightly modified attachment of the knife and its cooling units and with a sample holder on one rotating arm,

Fig. 4 shows a section through an evaporator for circulating liquid and

Fig. 5 shows in perspective, partially sectioned, a Heat exchanger with a mixture that can be frozen and with evaporation line coils and line coils for guiding and circulating the liquid.

In Fig. 1, 1 designates the housing in which the microtome is arranged. It is schematically indicated that the electrical and cooling circuits through the wall 3 out of the housing are brought out. The housing 1 is provided with the means required to operate the microtome. The microtome has a knife 10 and an object holder with a heat conduction unit 11 and one of a thermoelectric cooling element 12 formed support plate for the sample 13. Outside the chamber is a main heater S with a relay or switch 21 is provided, which determines the current for the cooler depending on the thermostat 20 Controls temperature in the chamber. In addition, there is a cooling compartment 16 with a thermoelectric cooling unit 15 and a heat text unit 14 is provided * Furthermore, an energy source is provided 19 provided with three separate and independently controllable outputs, each of which leads to a thermoelectric Device 9, 12, 15 leads. Also is a thermometer

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intended for measuring the temperature in the chamber. The cooler S can be used as a compressor cooler, absorption cooler or also work thermoelectrically or carbon dioxide can be used. A cooling medium, such as carbon dioxide, which circulates through the pipe system 17, cools the interior of the housing 1. A further cooling circuit is connected to the heat exchanger 6, with which the hot connecting bridges of the thermoelectric »cooling units be cooled. The medium for the thermoelectric cooling units 9, 12, 15 can optionally be passed through the cooler 5 be cooled immediately. If the cooler 5 is, for example, a thermoelectric cooler, the medium flowing through the hot connecting bridges of the cooling units 9, 12, 15 will be on the cold side of this thermoelectric cooler 5 chilled. If a thermoelectric cooler is used as the main cooler, the housing 1 can also be used without an interposed one Cooling medium can be cooled by placing the insides of its walls either in thermal contact with the cold connecting bridges of the cooler 5 or the air inside the chamber 1 is led past the cold connecting bridges will. In an optional embodiment, the cooling medium for the thermoelectric coolers 9, 12, 15 and cooled for the chamber 1 by separate main coolers 5, which do not need to be of the same type. Through the pump 7 the cooling medium in the line system 22 for the thermo

electric cooler 9, 12, 15 circulated. This circulation can also take place in other ways, for example by gravity, if the cooling medium is highly volatile, especially if its boiling point is between ~ 30 ° C and +30 ^G C. In this case, the thermoelectric coolers 9, 12 can be cooled by evaporating the coolant, which then condenses again in the heat exchanger 6. The heat exchanger 6 can be accommodated inside or outside or in the walls of the cooling chamber 1, but in the preferred embodiment it is not arranged inside the chamber. The pump 7 is preferably located outside the chamber. The pump 7 and possibly a compressor for the cooler 5 are preferably arranged in such a way that no vibrations can reach the microtome. The liquid for cooling the hot connecting bridges preferably has a low freezing point. For example, an organic solvent with an oxygen atom, such as a lower alcohol, for example ethyl alcohol, or an antifreeze agent such as that used for automobile engines can be used for this purpose. This coolant runs through the heat conduction units 8, 11, 14, which are in thermal contact with the hot connecting bridges of the thermoelectric cooling units S, 12, 15. The other connecting bridges are each in contact with the knife 10, the sample 13 or the walls, the floor or the ceiling of the cooling chamber 16. However, the cooling chamber 16 and its accessories are not essential.

common parts of the apparatus. The cooling chamber can also be used directly be cooled by the coolant for the thermoelectric cooling units.

The temperature in the chamber 1 can be at any desired level

can be set. Usually a main cooler 5 is sufficient, the chamber to -10 € or even only -6 ° G and that, Coolant for the hot connecting bridges can cool down to -12 ° C. The chamber should be at any temperature adjustable between -2 G and -SC. The cooling medium in circuit 22 preferably goes to the hot joints first the thermoelectric heat pumps of the knife and then its other functions. Usually the temperature fluctuates by 3 * 2 ° or even -2.5 C around the desired one Temperature. It is, however, a closer temperature control of the knife and the slide, reducing the need for finer temperature control of the air will. In certain circumstances it is necessary to prevent the air temperature from rising above 0 ° C. If there is a fluctuation

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of -2.5 G, it is desirable to set the temperature for the air to -3 ° C or below.

The medium for cooling the hot connecting bridges can be used on any suitable Temperature, preferably at a temperature between -10 G and -20 ° G are kept. Preferably the main cooler is working 5 constantly, while the thermostat 20 actuates a relay, which a shut-off valve in the cooling circuit 17

served. In contrast, in the exemplary embodiment shown

The thermostat 20 controls the energy supply to the main cooler 5 Optionally, two main coolers can be used, namely one for the chamber 1 and one for the thermoelectric cooler.

The temperature of the knife 10 and the cold connecting bridges of the unit 12 carrying the sample 13 can be measured and displayed directly, but if the air is inside of the chamber and the temperature of the cooling liquid flowing through the system 22 within fluctuations of -2.5 ° C are kept constant, this is generally unnecessary, since the temperatures are indirectly measured by measuring the the current supplied to the thermoelectric cooling units can be determined. An "anti-roll", device »of the microtome are thermoelectrically cooled, but this is not absolutely necessary because the Knife and air are cooled.

In the case of the microtomes according to FIGS. 2 and 3, the same parts are provided with the same reference numerals. Either of the two arrangements has a microtome knife 10, thermoelectric cooling units 9 along the knife and heat conduction units 11 in Contact with the cooling units 9. Opposite the knife (Fig. 3) is a slide plate from a thermoelectric Element 12 is formed, which is in contact with a heat conduction unit 11. The heat conduction unit 11 is on one

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50 arranged, which is pivotable at 52, so that the Slide plate is movable relative to the knife IG. The heat conduction device is rotatable on the arm 50 by means of Pin 54 arranged so that they are independent of the position of the arm in order to facilitate access to the sample can be twisted. Bas knife is from blocks worn in such a way that it is angle-adjustable with respect to the target plate is. According to FIG. 2, there is an inner block 30 provided, the angular position of which is adjustable within the block 33 by means not shown in order to adjust the angle of the knife 10 to change. The inner block 30 is held in place by screws 34. at In the embodiment according to FIG. 3, the knife 10 is held in its position by means of visual screws 34 which are placed on insulating plates 30a works. The thermoelectric cooling units 9 are supplied with power via lines 36, while the heat conduction units 8 are supplied with cooling liquid via lines 35. The block 30 is through a slot 32 (Figure 2) in To a certain extent designed to be flexible, so that the inner block 30 can rest against the outer block 33 when the Screws 34 are screwed through holes in block 33 and come to rest on knife 10. The Knife 10 is thermal by means of insulating washers 31 isolated (Fig. 2). With this holder, the knife can e.g. can be easily removed for regrinding without getting into the Intervene in the cooling system. The thermoelectric cooling

element 12 adjacent surface of the heat conducting unit 11 is anodized or in some other way with an electrical insulating layer provided.

Fig. 4 shows a cross section through a heat exchanger, in which a cooling medium via lines 17a, through a Evaporator space 40 is circulated, whereby a through the Line 22a flowing cooling liquid is cooled. This design can be used as a heat conduction device 11.

Fig. 5 shows a further heat conduction unit 3a, in which a Coolant is circulated through a ziekzag-shaped line 17a, while a cooling liquid to be cooled by a similar cooling coil 22a passes therethrough. Both line systems are immersed in a freezing mixture 40. This arrangement can be provided in the wall 3 of the chamber 1; it is denoted by 6 in FIG. 1.

The innovation in a Cambridge microtome, the knife holder of which is shown in FIG. 2, has proven to be very advantageous modified and its slide has also been modified to accommodate a thermoelectric cooling unit was. The knife was from two F32 FRIGISTOR (trademark) cooling units and the slide block from one such unit cooled. The chamber was cooled between 0 ° and -5 ° C. in a conventional manner. This was done Alcohol circulated continuously at 0 to -S ° G through the heat sinks of the thermoelectric units. There was one

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Teflon-coated glass plate intended as a damper for rolling movements, as described by WTWest in "Stain Technology" I963 Vol. 37 page 5. At 12 amperes of current and a chamber temperature of -5 C, the lowest temperature on the center of the knife was -55 . With a current of 6 amps, the lowest temperature of the slide was -38 ° G. The heat conduction of frozen tissue is sufficient to ensure that the tissue is kept at the temperature of the thermoelectric cooling device * The temperature can be controlled up to -IC.

The following table gives the optimal temperatures of the knife »the knife block and the air inside the chamber again.

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ϋη solidified
tissue temperature
of
Knife temperature
of
Blocks Air temperature Hepatite
(Liver) -18 ° G -10 ⁰ G -5 ° C redder
Glass head -15 ° G - 8 ⁰ G -5 ° G skin -35 ° G -10 ° G -s ° c Fibrous tumor
(Keloid). -35 ° e -12 ° G -5 ° G Kneecap -40 ⁰ G -18 ° G -s ° c brain -18 ° G -15 ° C -5 ⁰ G thyroid -20 ^G G -10 ⁰ G -S ° G Solidified
tissue Hepatite
(Liver) -15 ° G - 8 ⁰ C ~ 5 ° G redder
Glass head -10 ⁰ G - 8 ⁰ C -s ° c skin -35 ° G - 5 ° G -5 ° G Fibrous tumor
. (Keloid). -35 ° G - 5 ° G -s ° c Kneecap -40 ° G -20 ° C -5 ° G brain -15 ° G - 8 ⁰ G -5 ° C thyroid -25 ⁰ C -12 ° C

The most significant observation, which can only be partially expressed in tabular form, is that each still soft tissues (mammalian or invertebrate) as standard

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4 microns or less can be cut. Using hard steel knife (Jung AG) can human skin, which is normally a problem with conventional gryostats represents can be cut without difficulty. Boneless tissue (e.g. helix, limnaea, patella, limax), which was previously very difficult to cut into fine slices, can be handled with similar ease will.

Observance of optimal temperatures for the slide and the knife reduces damage from pressure and breakage (if the slide is too cold), or from being stretched Melting zone (if the block is too warm) to an absolute minimum. Bs can therefore be more tissue-related Details can be obtained from a wide variety of tissues. This is for normal microscopes, but more than that of importance for electron microscopes which use the disk technique (cryostat), as first described by Tranzer in Journale de Microscopic I965, Volume 4 »pages 319-336 and 409.

As can finally be seen from the table above, it is now possible to determine the correct temperatures of the different parts of the microtome for tissue insert made in formalin, glutaraldehyde, ethanol or another Fixatives are solidified. So are means for

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Cutting thin slices of solidified tissue with large ones Advantages over the conventional technology created. This conventional technique (bench freezing microtome techniques) in all branches of research out of use, the solidified frozen slices of a sample need, even if the conventional technology in routine laboratory practice still exists for the time being. The lowest temperatures ever required are -SO C for the cutting knife and -35 C for the slide, and these temperatures can easily be obtained.

Normally both the cooling liquid for the thermoelectrisehen Coolant as well as the cooling liquid for cooling the air in the housing through the evaporator one conventional compressor cooler. This holds up advantageously even at a temperature of about -12 C at room temperatures of up to 35 6, so that the cooling liquid for the hot connecting bridges of the thermoelectric coolant to about the same temperature is cooled.

The evaporator is preferably of the shape that is capable of dry operation, as this provides preservation a constant temperature in the chamber is facilitated. However, when some of the following means, e.g. Cold reservoir) are used, this may be unnecessary.

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During normal operation, the compressor vibrates so small that they can easily be rendered harmless. When starting the compressor, however, the vibrations are livelier. To avoid this, both the compressor and the evaporator and the cooling coils are preferred for the coolant or for the coolant in a damper fluid (jelly) e.g. immersed in a water-salt mixture (Fig. 5) which has a melting point of approximately -12 ° G Has. Another cold reservoir can also be used, which compensates for temperature fluctuations and so after impairments the optimal temperature, for example by opening the chamber, this temperature is quickly set again, so that the compressor runs more smoothly.

Optionally, the coolant can also pass through the middle of the evaporator be guided. The cooling coils and evaporator coils can also be intertwined with one another.

Another possibility is to use a double channel in aluminum, such as an Alcanduct, with one channel for the evaporator and the other channel for the cooling liquid is provided.

The cooling liquid (for cooling the housing and the thermoelectric Cooling units) could be lost over time, and the device could be lighter without cooling liquid be transported. The initial entry of the cooling

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liquid or the replacement of loss is through use a self-feeding pump (reference number 7 in Fig.l) and a connected, located above the pump Tanks relieved "

The thermoelectric coolants on the knife make it difficult to remove the knife, so it is desirable to to remove the slide from the vicinity of the knife in order to be able to safely remove the specimen disc. This is done by articulating the slide block on the Arm 50 reached around pin 54. (Fig. 3) For cleaning this is Microtome preferably removable from the housing, for example it can be on the base plate of the chamber by means of a Snap connection that takes a pull of 5 pounds to trigger.

Since the optimal temperature in the housing is around -5 C compared to the previously usual temperatures of -30 C, fogging of the observation window can be avoided much more easily, for example by using a three-glass window, consisting of three parallel panes of glass with two evacuated rooms in between.

It is also possible at a temperature of -5 ° C that Samples without gloves using tweezers or the like. to attach or remove. The hands and fresh samples can be introduced through openings in the wall of the chamber, which close by themselves with elastic cuffs and

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form a seal around the operator's wrist and at the same time effectively preventing the penetration of steam-containing air into the housing. Such self-closing openings are well known of baby incubators, but modified materials can be used to make the to deal with low temperatures. Optionally, other known methods of air-blocking the service openings can be used to introduce and load samples without appreciably contaminating the air inside the enclosure and taking the samples can be done with the help of rubber gloves, which insertion openings in the Seal the chamber * Such service openings are made during operation covered with insulating material.

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Claims (14)

RA. 570 828 * - 2.11.6.6 Protection against proverbs 1. Microtome for cutting fine plates of a sample material for microscopic examination with a knife and a slide in a closed chamber and with operating devices for operating the knife and the slide from the outside, characterized in that a cooler (5) for cooling the interior of the chamber, first thermoelectric coolant (9) for cooling the knife and second thermoelectric coolant (12) for cooling the Slides are provided, these three cooling devices can be controlled separately and independently of one another. 2. Device according to claim 1, characterized in that the cooler (5) has a heat exchanger (6) for cooling a liquid which can be circulated by means of pipelines through the chamber for cooling its interior and which also cools the hot connecting bridges of the thermoelectric cooling units (9, 12, 15). 3. Apparatus according to claim 1 and 2, characterized in that the main cooling device (5) has an evaporator for Having evaporation of a freezing agent. -20- 4. The device according to claim 3, characterized in that the cooling medium circulated through the housing through the Evaporator is passed through. 5. Apparatus according to claim 2 and 3, characterized in that that the freezing medium and the cooling medium are passed through a heat exchanger (6). 6. The device according to claim 5 »characterized in that in the heat exchanger (6) a cold mixture (60) is included, which keeps the temperature of the coolant constant. 7. Apparatus according to claim 2, 4 or 5, characterized in that the passed through the heat exchanger (6) Liquid in good thermal contact with the cooling mixture (60) stands. 8. Device according to at least one of the preceding claims, characterized in that a pump (7) is provided for circulating the cooling medium. 9. Device according to at least one of the preceding Claims, characterized in that the cooling medium of an oxygen-containing organic solvent, for example a lower alcohol. 10 * Apparatus according to at least one of the preceding claims 2, 4, 5, 6 or 7, characterized in that the cooling medium is easily and liquid having a boiling point of -30 ° C to +30 ⁰ C. -21- 11. The device according to at least one of the preceding claims, characterized in that it has an "anti-roll ^w rod 12. Device according to at least one of the preceding Claims, characterized in that the specimen slide is arranged on a swivel arm (5Θ). 13 * Device according to at least one of the preceding Claims, characterized in that the microtome can be removed from the chamber surrounding it. 14. Device according to at least one of the preceding Claims, characterized in that the chamber (1) has an observation window. -22-