Method in a microtome for creating the possibility that the slit between the knife edge and the specimen can be made extremely narrow

Method in a microtome, especially an ultramicrotome, in which a knife edge, turnable around its longitudinal axis, is used to cut sections from a specimen by making the specimen downwardly pass over the knife edge. The distance between the knife edge and the specimen before cutting is made extremely narrow without the specimen touching the knife. The slit is lit up from below by a light waveguide, one end of which is located under the knife edge and is turned with this and the other end of which is lit up by a light source movable in relation to the turning of the knife edge. The light from the waveguide is reflected against the surfaces of the knife as well as of the specimen which are turned towards the slit. The image of the knife surface reflected in the specimen surface, the size of which is proportional to the breadth of the slit, is viewed from at least one point located in a vertical plane outside of the vertical plane of the slit and on the same side of this as the knife.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention refers to a method in a microtome, especially an 
ultramicrotome, in which a knife edge which can be oriented around its 
longitudinal axis, is used to cut sections from a specimen by making the 
specimen pass downwardly over the knife edge, thereby making it the 
distance between the knife edge and the specimen before cutting extremely 
narrow without the specimen touching the knife. 
2. Prior Art 
Within microtomy, especially ultramicrotomy, there is a strong requirement 
for the monitoring and controlling the approach of the cutting knife to 
the specimen from which the sections are to be cut. The knives used 
generally consist of glass or diamond and the knife edges are extremely 
sharp and, therefore, are easy to damage if the knife, when approaching 
the specimen comes into contact with the specimen or if the first cut is 
too thick. On the other hand, if due to insufficient sensitivity of the 
control of the distance between the knife and the specimen surface, one is 
forced to feed the knife and the specimen in very small steps at a long 
distance between the knife and the specimen the process will take an 
unreasonable amount of time. According to an earlier known method, the 
approach between the knife and the specimen block is carried out in such a 
way that the slit between the knife and the specimen block is lit up from 
below by a separate lamp located in the knife holder under the specimen 
block and the knife. According to another method described in Swedish Pat. 
No. 7504111-1, the knife is lit up from below via a mirror. This latter 
method can, however, only be used for glass knives. In the former method 
the location of the light source under the knife holder leads to thermal 
disturbances and furthermore, a light beam which is too strong and too 
broad is obtained. This gives a dazzling effect which is especially 
disturbing in the subsequent cutting procedure in which so-called diffused 
light is used. In order to reduce the thermal disturbances and to decrease 
the dazzling effect, the lamp could be replaced by a fiber optic 
arrangement placed on the knife holder. The drawback is, however, that one 
has to take away the fiber cable before the cutting is started which 
easily gives rise to mechanical disturbances of the knife holder. Or, one 
has to let the cable remain during the cutting, which easily gives rise to 
mechanical tensions between the knife stage and the base plate and which, 
furthermore, contributes to the transfer of vibrations to the knife 
holder. 
BROAD DESCRIPTION OF THE INVENTION 
The object of the invention is, therefore, to provide a method in which the 
above mentioned disadvantages are eliminated and which is applicable to 
glass knives as well as to diamond knives. 
The invention involves a method in which a microtome, especially an 
ultramicrotome, wherein a knife edge is turnable around its longitudinal 
axis, is used to cut sections from a specimen by making the specimen pass 
the knife edge downwards, in order to make it possible that the distance 
between the knife edge and the specimen before cutting can be made 
extremely narrow without the specimen touching the knife. The slit is lit 
up from below by a light waveguide, one end of which is located under the 
knife edge and is turned with this, and the other end of which is lit up 
by a light source movable in relation to the turning of the knife edge. 
The light from the waveguide then reflects against the surfaces of the 
knife as well as of the specimen which is turned towards the slit. The 
image of the knife surface is reflected in the specimen surface. The size 
of the image is proportional to the breadth of the slit. The specimen is 
looked at from at least one point located in a vertical plane outside of 
the vertical plane of the slit and on the same side of this as the knife.

In FIG. 1, which schematically shows a side-view of ultramicrotome, 
reference numeral 1 denotes a glass knife located in a knife holder and 
reference numeral 2 denotes a specimen designed as a truncated pyramid. 
The specimen is fixed in a conventional way in torsional bow 15 which is 
supported by specimen arm 16. Knife holder 1 is placed in cradle 10 so 
that knife 1 can be turned around the axis of the knife edge in order to 
provide different so-called clearance angles .beta., which is the angle 
between the vertical front surface of the specimen and the plane of the 
knife facet which is turned towards the specimen. 
Reference numeral 3 denotes a filament bulb which via lens 4 and heat 
absorbing filter 5 lights up plane mirror 6 from which the light is 
reflected against light waveguide prism 7. Mirror 6 is placed in mirror 
holder 13, which is designed as a slide, and built-in a frame 17, which 
also holds up microscope 9 of the ultramicrotome. As mirror holder 13 is 
brought to its upper end position lamp 3 is connected to voltage source 20 
via switch 14. From mirror 6 the light is reflected against waveguide 
prism 7, in the opposite end of which the light beam is refracted upward 
and strikes light waveguide 8 at its lower edge. Via light waveguide 8 the 
light is conducted up toward the upper surface of the waveguide and 
strikes the very smooth and bright surface and the upper part of the knife 
at such angles that light reflections are obtained in microscope 9. The 
optical axis of microscope 9 leans in proportion to the specimen surface, 
so that the specimen surface functions as a mirror in which, except for 
the upper end of light waveguide 8, the upper part of the knife can be 
seen. This makes the whole specimen surface perceived as shining strongly 
when the knife is located relatively far from the specimen surface. Due to 
the fact that the light shines in towards the knife and the specimen 
similarly from a plane surface, the reflections are obtained in both beam 
paths when using a stereomicroscope. Furthermore, the location of the 
light source and the mirror holder in the frame of the movable microscope 
stand, as described above, means that the frame can be pushed freely 
toward or from the operator to the most suitable position for observation 
without the light intensity being changed in the light waveguides. 
When the knife approaches the specimen surface the shining part is limited 
more and more by the very outermost border line of the knife edge and 
gradually forms a narrow line when the knife edge is brought to pass very 
close to the specimen surface. At a distance of about 1 .mu.m the color 
tints appear which are characteristic when the slit between the knife edge 
and the specimen surface through which the light is to pass becomes 
smaller than the wavelength of the light used. After further feeding, the 
light completely dies away and the specimen can be brought into its up and 
down cutting movement combined with a suitable feeding step between each 
cutting movement. After a few cutting movements, contact is obtained 
between the knife and the specimen and the cutting of sections starts. 
In waveguide prism 7 there is pin 11 and in cradle 10 there is located 
carrier 12, by means of which prism 7 of the cradle can be brought away 
from its left end position against which it is fixed by means of stop 21 
and spring 22. Waveguide prism 7 is focused in such a way that the rear, 
left limitation line of the lit up field comes approximately in line with 
the rear plane of waveguide 8, when .beta.=0. When the cradle is turned in 
such a way that the clearance angle increases, waveguide prism 7 is forced 
to follow, which results in the lower end of the waveguide 8 being equally 
lit up at all of the present values of the relief angle. At the same time 
the strongly shining front upper side of the prism giving disturbing side 
reflections in the microscope at great values of the clearance angle, is 
avoided, which is not the case if the light source is fixed. Furthermore, 
a good so-called dark field background is provided, i.e., the area under 
and on the sides of the specimen block remains dark irrespective of the 
value of the angle of clearance and, therefore, a maximum contrast between 
reflection and background is obtained. 
The above mentioned focusing of light waveguide 7 allows its front peak to 
bulge out in front of the base of the knife holder cradle enough to give 
light directly to the specimen surface without the assistance of waveguide 
8. The cradle is set in such a way that the angle of clearance is 
approximately as large as the angle of the microscope axis towards the 
specimen surface. Thus, a reflection is obtained in the microscope 
directly from the peak of waveguide 7 via the specimen surface. According 
to the description above, carrier 12 is designed in such a way that the 
waveguide stops in a pre-determined end position even if the value of the 
angle of clearance is further increased or if the cradle is completely 
taken away from its slide. This end position, determined by stop 21, is 
possible to trim so that errors of tolerance of the microscope angle and 
the location of the knife stage can be eliminated. By means of this 
arrangement the very important basic adjustment of the vertical 
orientation of the specimen surface is simplified. Especially, when having 
very small specimen block faces (sizes of 0.3.times.0.3 mm), it is very 
difficult to adjust the vertical orientation of the surface so that it 
catches the reflection desired when the knife approaches the surface if 
orientation bow 15 of the specimen is not suitably set. Locating the knife 
edge at a distance of about 0.5 to 1 mm from the specimen surface and with 
the specimen arm fixed in a horizontal position, the bow is adjusted until 
an optimal reflection is obtained in the microscope from the specimen 
surface, which means that the surface is vertical. The knife holder cradle 
can then be set at a value (usually lower) for the relief angle for 
cutting and the knife can be brought closer to the specimen by means of 
the reflection which then substantially comes from waveguide 8. Finally, 
the end of the waveguide prism 7 which is turned towards the mirror 6 can 
be provided with a concave end surface in the horizontal plane so that the 
knife holder cradle can be turned at least .+-.10.degree. in the 
horizontal plane without shadings arising. This is necessary in the cases 
where the front surface of the specimen block does not subtend at a right 
angle with its symmetry line. 
In FIG. 2 a part of the ultramicrotome described in FIG. 1 is shown, in 
which glass knife 1 has been replaced by a diamond knife and holder 19 
belonging to it. As to the rest of the device, the same reference numeral 
are used as in FIG. 1. Diamond knives are generally fixed in metal frames 
and then the edge facet often has an angle which is disadvantageous to the 
generation of suitable light reflections at the approach of the knife to 
the specimen. This is especially valid when the diamond knife is refaced 
after having been damaged. This means that the reflection surfaces 
obtained by the use of a diamond knife edge facet have quite variable 
angles as compared to when glass knives are used, the facet surface of 
which, turned towards the specimen is always vertical at an angle of 
clearance of 0.degree.. When using diamond knives the light waveguide is, 
therefore, bent in an angle of about 5.degree. which brings it into the 
cavity wherein the diamond is embedded. The light falls then from the 
upper end of the light guide towards the surface of the specimen in such 
angles that a good reflection from this surface is obtained in the 
microscope. In order to enable the use of a glass knife and a diamond 
knife, alternatively, without the exchange of the waveguide 8, waveguide 8 
is formed of flexible plastic which permits straightening out the above 
mentioned angle of 5.degree. in the case of a glass knife waveguide 8 
returns to its original position when a diamond knife is again inserted.