Biomedical Engineering Reference
In-Depth Information
3. Put the resin block into the holder of an ultramicrotome and
examine it under a stereomicroscope.
4. Place the holder with the sample under the stereomicroscope,
and using a steel needle and rotating the sample in the holder,
make two small cavities in such a way that they form a horizon-
tal line with our cell appearing in the center of the sample.
5. Introduce the holder into the ultramicrotome in such a way
that the segment arc of the ultramicrotome is in the vertical
position and the two cavities form a horizontal line.
6. By rotating the glass knife stage, align the bottom edge of the
pyramid parallel to the knife-edge. Using the segment arc, ori-
ent the plane of the sample vertically.
7. Bring the sample as close as possible toward the glass knife.
8. Adjust the gap (which is visible as a bright band if all three of
the lamps of an ultramicrotome are switched on) between the
knife-edge and the surface of the sample. The gap has to be
identical in width between the most upper and lower edges of
the sample during the up and down movement of the resin
block. This ascertains that every point of the sample surface
containing the cell of interest is at the same distance from the
knife-edge.
9. Slowly moving the sample up and down, continue its approach
until the knife begins to cut one of the edges of the sample.
The sectioning begins from either the upper or the lower part
of the sample; the middle part of the sample where the cell of
interest is situated will be unaffected because the length of the
radius passing through the cell is shorter than the radii passing
through the upper and the lower edges of the sample.
10. If the sectioning is to begin from the upper part of the sample,
tilt the segment arc to approach the lower edge toward the
knife. If the sectioning is to begin from the lower edge of the
sample, tilt the segment arc and approach the upper edge of
the sample toward the knife. A vertically oriented sample
should produce equal sections from both the upper and lower
edges of the samples (Fig.
4b
). Note down precisely all of
parameters relating to the position of the sample in the ultra-
microtome, i.e., the degree of rotation of the sample in the
holder, the degree of tilting of the segment arc, and the degree
of rotation of the knife in its stage. Do not take the sample
from the holder and do not rotate the sample inside the holder.
11. Take the sample and trim it to provide a narrow horizontal
pyramid. The pyramid should be as narrow as possible, and the
cell of interest should be at the center of the pyramid. The
length of the pyramid should be smaller than 0.9 mm and its
height smaller than 0.1 mm. In this case, you could place on
the slot 18 serial sections. An experienced person can trim a
pyramid directly with a razor blade.
