Cryogenic fixation apparatus

Cryogenic-fixation apparatus comprises a light source, a beam from which enter cooling liquid in a bath, through a window in the underside of the bath. The beam can be directed upwardly by means of a reflecting prism. When a lid of the bath is closed, the beam leaves the space defined by the path through an exit port in the lid, and is incident upon a specimen mounted on a specimen holder located above the exit port, the specimen holder being secured to an injector rod.

BACKGROUND OF THE INVENTION 
This invention relates to cryogenic-fixation apparatus. 
DESCRIPTION OF THE PRIOR ART 
Prior to the cryogenic-fixation of a small specimen, the small specimen, 
which may be a small drop of a suspension or a thin layer of a tissue 
culture, is applied to a specimen holder. Manipulating the specimen onto a 
holder, and also, in certain circumstances, connecting micro-electrodes to 
the holder, or modifying the holder in other ways, are important steps in 
successfully accomplishing a cryogenic-fixation operation. Accordingly, a 
stereoscopic microscope is commonly used when preparing the specimen for 
cryogenic-fixation. Following a cryogenic-fixation operation, the specimen 
is removed carefully from the cooling bath, which operation also requires 
care. 
Since the preparation and post-cryogenic-fixation operations call for 
careful manipulation of the specimen holder, these operations require good 
illumination of the specimen holder and the cooling bath. The cooling 
bath, commonly comprises a metal vessel, defining a cylindrical chamber 
for the cooling liquid, the chamber typically measuring approximately 20 
mm in diameter and 100 mm in height. The temperature of the cooling liquid 
in the bath is thermostatically controlled. Since the specimen-preparation 
procedure requires the use of both hands and the space is also required 
for the injection system, the stereoscopic microscope, and the cryogen 
supply line, installing an arrangement which is capable of providing the 
necessary illumination presents difficulties. It is particularly difficult 
to combine illumination of the specimen holder with overall illumination 
of the cooling bath. 
OBJECTS OF THE INVENTION 
It is an object of the invention to provide an arrangement which both 
illuminates the specimen holder and provides overall illumination of the 
interior of the cooling bath, in a manner which is both simple and 
generally applicable to cryogenic-fixation apparatus. 
BRIEF DESCRIPTION OF THE INVENTION 
According to the invention there is provided cryogenic-fixation apparatus 
comprising a cooling bath for a cooling liquid, a window on an underside 
of said bath, and a light source for directing a beam of light upwardly 
through the window to illuminate the interior of the cooling bath. 
When the apparatus is in use, the light beam enters the cooling bath from 
below, through the window which is made of glass, or of another 
cold-resistant and cryogen-resistant material, and which is sealed in the 
underside of the cooling bath. A specimen holder is normally located above 
the cooling bath and the light beam, rather than being directed to 
illuminate the whole interior of the bath, is directed towards the 
specimen holder and a specimen on the holder. Thus one light source only 
is required. The specimen holder can be viewed under a microscope when 
mounting a specimen on the holder, the apparatus imposing no restriction 
on a two-handed manipulation of the specimen holder and specimen. 
In one embodiment of the invention, the light source is disposed directly 
below the cooling bath. A heat-barrier filter may be provided to filter 
out the ultrared component of the light beam before the beam enters the 
cooling bath. The heat-barrier filter may be comprised by the window. 
In an alternative embodiment of the invention, the light source is disposed 
to one side of the cooling bath, and wherein the apparatus further 
comprises a reflecting means for directing the beam of light upwardly 
through the window. This arrangement facilitates replacement of the light 
bulb. The reflecting means may comprise, for example, a prism, a mirror, 
or a light guide. Advantageously the light source comprises a reflector 
lamp which emits light with a weak ultrared component, or alternatively a 
fibre-optic light guide may be provided, of a kind which eliminates the 
greater part of the ultrared component from a beam. 
The cooling bath may comprise a lid with a port for the transmission of the 
beam of light which has traversed the cooling bath. The port enables the 
cooling bath to be inspected and a specimen-preparation operation to be 
carried out on the specimen holder whilst the cooling bath is covered.

DETAILED DESCRIPTION OF THE INVENTION 
FIG. 1 illustrates a known form of cryogenic-fixation apparatus. A 
pot-shaped vessel 3 containing a cryogenic liquid in the form of nitrogen 
4 is surrounded by insulation 2 in a housing 1. The liquid nitrogen 4 
cools a bath in the form of a metal cylindrical body 5 directly, or 
indirectly when in a standby operating mode as illustrated. The body 
contains a cooling medium 6 into which a specimen 7 is immersed for 
cryogenic-fixation. The temperature of the cooling medium is maintained by 
thermostatic means. 
The specimen 7 is mounted on a specimen holder 8 which, in turn, is mounted 
on an injector rod 9 on a support 13. The injector rod can be lowered into 
the cooling liquid 6, upon operation of a lever 10. A stereoscopic 
microscope 11 is used to observe the mounting of the specimen 7 on the 
specimen holder 8, this operation frequently being extremely difficult. 
During the specimen-mounting operation, whilst the apparatus is in a 
standby operating mode, a thermally insulating lid of the vessel 3 which 
contains the cooling bath 5, is maintained in a closed position to limit 
consumption of nitrogen and to prevent damaging precooling of the specimen 
7 on its holding device 8. 
In this known arrangement, the mounting of the specimen is carried out with 
insufficient illumination. The stereoscopic microscope 11 occupies that 
side of the arrangement which faces the user, whilst the opposite, rear 
side is occupied by the support 13. Since, preparing, the specimen 
generally requires the use of both hands, the other two sides are also 
eliminated for the installation of a light source. Following immersion of 
the specimen 7 in the cooling bath 5, the specimen can be seen and handled 
only with difficulty in the cylindrical space defined by the bath 
containing the cooling liquid 6. 
In the arrangement of apparatus shown in FIG. 2 a glass window 14 forms the 
bottom of the cooling bath 5 containing the cooling liquid 6, the cooling 
bath, and the injection system being the same as the arrangement shown in 
FIG. 1 in other respects. A light source in the form of a high-output 
quartz-iodine lamp 15 is located immediately below the cooling liquid 6. 
The lamp is mounted in a holder 16 which, after tilting the appliance, can 
be removed, thereby enabling the light bulb to be changed without 
difficulty. A collector lens 17 focusses rays from the lamp. A 
heat-barrier filter is provided for filtering out or absorbing the 
ultrared component of light from the lamp, the filter being provided by 
window 14 or by a reflector of the lamp. The window is cooled by the 
cooling liquid 6. 
In the embodiment of apparatus shown in FIG. 3, a part in the form of 
transparent insert 18 in lid 12' permits a beam of light to pass out of 
the cooling bath, so that the specimen holder 7 can be illuminated when 
the lid is closed. 
A reflector lamp 19 is mounted externally of the apparatus, in a holder 20 
mounted on a rear wall. The holder can, alternatively, be mounted on one 
of the two side walls. The beam 21 emitted by the reflector lamp is 
directed by a reflecting means in the form of a prism 22 upwardly into the 
chamber defined by the cooling bath. 
Various modifications can be made to the apparatus shown in FIGS. 2 and 3. 
For example, various optical elements can be introduced into the optical 
path between the light source and the specimen holder and, in particular, 
the reflecting means can comprise alternatives to a prism such as a mirror 
or a light guide such as a fibre-optic light guide. Various forms of light 
source can be used. The port which enables the light to emerge from the 
cooling bath can be a solid transparent body, a tube with two transparent 
cover plates, a lens for further focussing of the rays, or a 
heat-absorbing glass.