Apparatus for cooling a biological or medical specimen

Apparatus for cooling a biological or medical specimen comprises a cooling bath, an injection device for injecting a specimen into cooling liquid in the bath at a velocity of between 5 and 15 m/sec, and a sleeve. The sleeve is movable between a lower position in the bath and an upper position for limiting the effects of splashing as a specimen enters the bath. The injection device comprises a closure plate for co-operating with the sleeve to further limit the effects of splashing. The sleeve is operatively associated with the injection device in a manner such that movement of a specimen carrier of the device towards the bath to immerse a specimen is initiated when the sleeve is moved from the lower position to the upper position.

BACKGROUND OF THE INVENTION 
This invention relates to apparatus for cooling a biological or medical 
specimen and, in particular, to apparatus for cryogentic-fixation of a 
specimen for microscope inspection. Cryogenic-fixation of a specimen 
involves the injection of the specimen at a rapid velocity, suitably 
between 5 and 15 m/sec, into a cooling liquid which may, for example, be 
pre-cooled to -190.degree. C. 
Cooling baths containing liquids at temperatures between -100.degree. C. 
and -190.degree. C., and with volumes between 5 and 100 ml, are used for 
numerous specimen-preparation operations, particularly for the process of 
shock-freezing or "cryogenic-fixation" of small biological or medical 
specimen for subsequent microscopic examination. Suitable preparation of 
the specimen which has been introduced into the cooling liquid can be 
achieved only when as much heat as possible is abstracted from the surface 
of the specimen within the shortest possible time. This applies 
particularly for biological or medical specimens which have not been 
pre-treated, that is, specimens which have not been subjected to a 
preliminary fixation and/or freezing-protection treatment. For such 
specimens, the cooling rate alone determines whether artificial separation 
of the water-rich plasmatic phases takes place (separation would render 
meaningful microscopical examination impossible) or whether the specimen 
freezes to a true-to-life, glassy form ("vitrification" takes place at 
cooling rates greater than 10,000.degree. C./sec). 
The cooling rates which are required for vitrification are obtained only in 
an edge zone of the specimen, this zone having a depth of 30 um at the 
most. The depth of the perfectly vitrified edge zone is determined 
essentially by the temperature and the specific properties of the cooling 
medium, and by the velocity at which the specimen enters the cooling 
medium. Liquified propane which has been cooled to a temperature only 
slightly above its freezing point (-190.degree. C.) offers the best known 
conditions for vitrification. Using this cooling medium, initial cooling 
rates of the order of 100,000.degree. C./sec are obtained, but only if the 
specimen is injected into the liquid at velocities exceeding 5 m/sec. 
The liquefaction, cooling, and storage of propane, under 
constant-temperature conditions, presents few problems. However, the 
injection of the specimen presents considerable problems and hazards. 
Since the specimen is injected in at a comparatively high velocity, up to 
15 m/sec, a considerable quantity of the cooling liquid may splash out of 
the cooling bath. Typically, the total quantity of cryogen is kept below 
100 ml in order to minimise the hazards associated with propane, i.e., 
fire, and the danger of explosion. The diameter of the specimen, and/or of 
the injector, typically exceeds 3 mm, and it is usually necessary that the 
specimen should travel a distance of 5 to 10 cm in order to achieve 
adequate cooling. While it is possible to use a non-flammable cooling 
media, such as halogenated hydrocarbons, (e.g. FREON 13, which has a 
boiling point of -185.degree. C.), these provide a 20% lower cooling rate 
and other problems still remain. The splashing of the cooling liquid will 
cause severe burns if the liquid strikes the skin. 
More than 90% of the cooling medium is often lost from the cooling bath 
when specimens are introduced at velocities greater than 10 m/sec, the 
specimens and specimen holders are of conventional diameter (approximately 
3 to 5 mm), and the injectors are of conventional diameter (approximately 
4 mm). Insufficient liquid usually remains in the cooling both to cool 
further specimens, even when the specimens are small, and the heat 
capacities of the specimen-holder and injection device are low. When a 
specimen and specimen holder is insufficiently cooled, the quantity of 
heat which remains in the interior of the specimen, and/or in the specimen 
holder/injector assembly, causes secondary heating of the surface of the 
specimen following the initial superficial cooling. Hence, an artifical 
separation of the initially perfectly vitrified surface occurs. In order 
to be able to enhance the quality of the cryofixation by means of greater 
injection velocities, as well as for safe operations, it is desirable to 
adopt precautions to prevent splashing of the cooling medium even when the 
injection velocities are comparatively high. 
OBJECT OF THE INVENTION 
It is an object of the invention to avoid the above-mentioned 
disadvantages; that is, to provide an arrangement which substantially 
prevents the cooling liquid from being expelled from a cooling bath even 
when the injection velocities exceed 5 m/sec. 
BRIEF DESCRIPTION OF THE INVENTION 
According to this invention, there is provided apparatus for cooling a 
biological or medical specimen, the apparatus comprising a cooling bath 
for containing a cooling liquid, an injector having a specimen carrier 
which is movable along an injection path for immersing a specimen mounted 
on the specimen carrier in cooling liquid in the bath. A sleeve is movable 
along a path aligned with the injection path, between a lower position 
disposed in the bath and an upper position in which the specimen carrier 
is positioned within the sleeve for immersing a specimen mounted thereon. 
The sleeve and the injection device cooperate when the sleeve is in the 
upper position so that cooling liquid splashed from the bath as a specimen 
is immersed strikes the sleeve or a baffle on the injection device to 
drain back into the bath. 
The specimen is shielded by means of the sleeve as the specimen approaches 
the cooling bath, the sleeve being pre-cooled by immersion in the cooling 
bath. Any cooling medium, which is displaced from the bath as the injector 
and specimen and specimen holder enter and penetrate the cooling liquid, 
is directed back into the cooling bath by the injection device and sleeve. 
The cooling medium drains back into the bath, for the most part along the 
cold walls of the sleeve, to become available again for continuing the 
process of withdrawing heat from the specimen and the specimen holder, 
which process is known as "aftercooling." All the hazards which are 
normally associated with splashing of the cooling medium in the laboratory 
are eliminated. 
In a preferred embodiment, the sleeve is in a close, sliding fit with the 
wall of the cooling bath. 
A tool receiver is mounted on or is integral with the sleeve, whereby a 
tool can be used to manually move the sleeve. The injection device 
comprises a generally horizontal closure-plate, or baffle, which has a low 
thermal capacity and which closes off the sleeve when the sleeve is in the 
upper position so that cooling medium is directed back into the cooling 
bath by the baffle, even if it would otherwise have escape through the 
space between the specimen holder and sleeve. The cooling medium quickly 
refills the bath without a significant temperature increase, which would 
interfere with the operation. The thermal capacities of the components of 
the apparatus, relative to the thermal capacity of the cooling medium, are 
preferable such that the rise in temperature of the cooling medium caused 
by the antisplash arrangement does not exceed 30.degree. C. (so that in 
the case of propane the temperature rises from -190.degree. to 
-160.degree. C.) The greater part of the cooling liquid thermal capacity 
remains available for cooling the specimen, the specimen holder, and the 
injector. 
In one development of the invention, the sleeve is spring-biased towards 
the upper position, the apparatus comprising releasable catch means for 
holding the sleeve in the lower position. 
Alternatively, the apparatus further comprises electrical or 
electromagnetic motor means for moving the sleeve between the lower and 
upper positions. These developments considerably reduce the time taken to 
lift the sleeve, so that the risk of premature cooling of the specimen by 
the cold sleeve wall is reduced. These developments also provide a simple 
and reliable means for triggering lifting of the sleeve. 
Preferably, the injection device comprises trigger means which, when 
activated, triggers movement of the specimen carrier towards the cooling 
bath, and wherein the sleeve or a member moutned thereon is adapted to 
activate the trigger means during the course of or at the completion of 
movement of the sleeve from the lower to the upper position. By this means 
injection is triggered at the earliest possible moment so that the risk of 
preamture cooling of the specimen is further reduced. The 
cryogenic-fixation operation therefore does not depend upon the skill and 
practice of the operator. Furthermore, premature injection without 
antisplash protection is prevented. 
Preferably, to facilitate entry of a liquefied cooling medium into the 
bath, the sleeve comprises upper and lower portions of reduced outer 
diameter whereby annular spaces are defined between the upper and lower 
portions and the inwardly facing surface of the bath when the sleeve is in 
the lower position, the sleeve further comprising one or more grooves 
extending between the upper and lower portions, and the lower portion 
defining an outlet from the lower annular space to the space defined by 
the sleeve, whereby cooling liquid can enter the space defined by the 
sleeve via the upper annular space, the grooves, the lower annular space 
and the outlet. 
In order to enable liquid to be removed from a bath, the apparatus 
preferable further comprises a container for insertion into the cooling 
bath, the container comprising a valve adapted for opening to permit 
cooling liquid to enter the bath as the container is inserted into the 
bath and for closing to retain cooling liquid in the container when the 
container is removed from the bath.

DETAILED DESCRIPTION OF THE DRAWINGS 
The apparatus which is illustrated in FIG. 1, corresponds to the present 
state of the art. A cylindrical metal cooling bath 2 is located in an 
insulating vessel 1, the cooling bath being connected to a cylindrical 
sleeve 4 via a thin-walled hollow sleeve 3, the sleeve 4 preventing direct 
contact between liquid nitrogen 5 which is used as the cryogen and the 
cooling bath during use of the apparatus. Liquefied propane or a 
halogenated hydrocarbon (e.g. FREON 13) is normally used as a cooling 
liquid 6. 
The freezing points of cooling liquids of the above-mentioned types are 
higher than the boiling point of liquid nitrogen (the freezing point of 
propane is -190.degree. C., that of FREON 13 is -185.degree. C., and that 
of FREON 22 is -155.degree. C.; and the boiling point of liquid nitrogen 
is -196.degree. C.). Unless preventitive measures are taken, the liquid 
cools until frozen. The liquid would lose heat via the sleeve 3 and via 
cold nitrogen gas 7 which completely surrounds the cooling bath 2, being 
present as a result of continuous boiling of the liquid nitrogen in the 
vessel 1. such cooling is prevented by means of a heating cartridge 8, the 
heat output from which is, as a rule, controlled in a manner such that the 
temperature of the cooling bath 2, and hence that of the cooling liquid 6, 
in continuously and thermostatically held at a value slightly above the 
freezing point of the cooling liquid. The thermostatic control is achieved 
by means of a temperature sensor 9 and an electronic control circuit which 
is not illustrated. 
The majority of cooling liquids are combustible and are also harmful to 
health so that it is necessary to prevent evaporation into atmosphere. To 
facilitate safe removal of the cooling liquid upon completion of the 
preparation of a specimen, a removable insert-sleeve 10 is located in the 
cylinder 2, for containing the cooling liquid. 
As shown in FIG. 1, a specimen 11 is located on a carrier 12 which is 
suitable for subsequent operations such as cryogenic-ultramicrotoming, 
freeze-etching cryogenic-substitution, and freeze-drying. The carrier is 
mounted on an injection rod 13. The rod is slidably movable in a 
cylindrical hole in the guide 14. 
To achieve high cooling rates when injecting a specimen, the injection rod 
13 is accelerated by a compression spring 15 (the velocity which can be 
attained by free fall of the specimen is too low for normal requirements). 
The injection operation is triggered by pulling a bolt 16 back, in the 
direction of the arrow, either manually, or by means of a triggering 
device 17 which is actuated by electrical or electromagnetic means. 
following triggering, the specimen 1 travels over a distance "L" of 
approximately 100 mm, in the course of which it enters the liquid 6 at a 
velocity of between 5 and 15 m/sec. A major disadvantage of apparatus of 
this type is the splashing caused by the immersion of specimens in the 
cooling liquid. 
The apparatus shown in FIG. 2 comprises a cylindrical sleeve 18 which 
replaces the insert-sleeve 10 illustrated in FIG. 1. A tool-receiving 
closure member 19 is secured to an upper rim of the cylindrical sleeve, 
the closure member being adapted to receive a suitable tool 20 with an 
insulating handle 21 to enable the sleeve to be lifted. The dimensions of 
the sleeve are such that the sleeve is a close sliding fit in the 
cylindrical bore of the cooling bath 2, to facilitate lifting of the 
sleeve. 
The cylindrical sleeve 18 can be lifted until the sleeve engages a thin 
plate 22, which is secured by means of bolts 23 to a guide 14' of an 
injection device. The plate comprises an aperture for permitting 
through-passage of the injector rod 13'. The raised sleeve (represented by 
broken lines), and the plate provide an antisplash enclosure which directs 
virtually all splashing cooling liquid back into the cooling bath 2. Prior 
to being lifted the sleeve and closure member are cooled to the 
temperature of the cooling bath. Furthermore the thermal capacity of the 
plate 22 is low, and the plate is spaced from the guide to minimise 
thermal contact. Consequently the antisplash enclosure does not cause any 
significant heating of the cooling liquid before the liquid draws back 
into the bath. Heating caused by the components of the antisplash 
enclosure is negligible, invariably remaining below 30.degree. C. and 
given suitable conditions remaining below 5.degree. C. 
Injection of the specimen 11 is triggered by moving a bolt 24 upwardly, in 
the arrowed direction, the bolt being moved upon engagement with the 
closure membe 19 when the sleeve is raised. It will be appreciated that 
the bolt can be moved in various ways, for example, by means of mechanical 
intermediate elements, or by electrical or electromagnetic means. 
Accidental triggering which could occur (for example by pushing in the 
bolt 24 while attaching the specimen 11, or the specimen carrier 12, to 
the injector rod 13') can be prevented by means of an additional unlocking 
device which is operated manually. 
When the apparatus is in use and the protective sleeve 18 has been lifted, 
the injection operation takes place immediately, so that damage to the 
specimen 11, which can occur when the specimen remains in close proximity 
to the refrigerated sleeve, is minimised. The operative connection between 
the lifting of the protective sleeve and the triggering of the injection 
operation in conjunction with the additional locking mechanism, ensures 
that injection does not take place until the sleeve is raised. 
In the embodiment of apparatus shown in FIG. 3, the sleeve 18 is lifted by 
means of compression springs 25 and 26, the springs being released upon 
movement of triggering elements 27 and 28. Injection of the specimen 11 is 
triggered when the sleeve interrupts a light beam, light from a source 29 
being focused by a lens 30 on a photosensitive device 31. Downward 
movement of the specimen carrier can be commenced either during upward 
movement of the sleeve, or after an appropriate delay. As a result of the 
movement in opposite directions of the sleeve and the specimen carrier, 
the duration of the operation is minimised, and the effect of premature 
low temperatures on the specimen is reduced to a minimum. 
In the embodiment of the apparatus shown in FIG. 4, the sleeve 18' is a 
close sliding fit in the cooling bath but has upper and lower end portions 
of reduced diameter outer "d.sub.2 ". The diameter of the end portions 
differs from the diameter "d.sub.1 " of the cooling bath by an amount 
which is sufficient to enable the cooling liquid to move in the annular 
spaces between the cylindrical wall of the cooling bath and the outwardly 
facing surfaces of the end portions of the sleeve. Grooves 33 in the 
outwardly facing surface of the sleeve extend generally vertically to 
enable the cooling liquid to flow downwardly between the upper and lower 
end portions. An outlet into the space defined by the sleeve from the 
space between the lower end portion of the sleeve and the wall of the 
cooling bath is provided by one or more cutouts 34 at the lower end of the 
sleeve. The cooling medium can be liquefied without removing the sleeve 
18', the risk of overpressure, caused by blocking of the downward flow 
during an attempt to achieve liquefaction being eliminated. A gas (for 
example propane or FREON gas) enters liquefier 32 through tube-connection 
35, the liquefied medium entering the cooling bath through the outlet 36. 
In the cooling bath, the liquid flows downwardly along the grooves 33 and 
fills the cooling bath from below, through the cutouts 34. 
The apparatus shown in FIG. 5 is adapted to facilitate disposal of the 
cooling liquid. The sleeve is removed from the cooling bath 2 following 
completion of the specimen preparation procedure. In its place, a 
container 10' which comprises a ball valve is inserted, in the direction 
indicated in the left hand sectional diagram, using a suitable tool 37. A 
ball 38 is spaced from an aperture in the base of the container as the 
container is inserted to enable cooling liquid 6 to enter the container. 
Once the container has been fully inserted into the cooling bath, the 
container is raised, the ball blocking the aperture so that the cooling 
liquid is retained in the container. The ball valve can comprise a 
compression spring to facilitate operation thereof. The container enables 
cooling liquid to be transferred into a pressure vessel which is suitable 
for storing combustible media, or to be reused or to be flared off in a 
known manner. 
It will be appreciated that various combinations and modifications of 
features of the embodiments which have been described by reference to 
FIGS. 2 to 5 can be made which are within the scope of the invention. The 
sleeve may, for example, be other than cylindrically shaped. Instead of 
the grooves 33 in the sleeve 8' illustrated in FIG. 4, the through-flow of 
the liquefied cooling medium can be provided for by means of recesses in 
the wall of the cooling bath 2'. the cooling liquid removal sleeve 10' can 
be dimensioned for introduction into the bath without removing the sleeve.