Re: Vibratome (How thin?)
|From:||"J. A. Kiernan" <firstname.lastname@example.org>|
On Fri, 29 Sep 2000, Joan Yonchek wrote:
> I am interested in hearing from anyone versed in the use of a Vibratome.
> I have been asked to obtain 10-20 micron thick sections of small pieces (1mm
> cubes) of formalin fixed skin.
Ask the person who requested these thin sections to provide the
necessary technical instruction (from her/his practical experience)
or to provide references to published accounts of how it can be
I've had a vibratome in my lab for about 10 years and it has been used
by me (not enough to claim high-level skill) and by others (sometimes
daily for weeks on end, therefore skilled). It is possible to cut
sections of unfixed brain at 100-200 micrometres, and of fixed brain at
50 micrometres. I cannot believe that it would be possible to collect
more than occasional sections with this instrument set at a nominal
thickness of 10 or 20 micrometres. If you succeed in collecting a
section, it is probably much fatter or deeper than its nominal thickness.
The bumph that comes with a Vibratome tells you that the section
thickness setting is not a true indicator of the actual number of
micrometres. The mechanical advance of the block is not necessarily
the thickness of the next section, any more than it is with other
kinds of mocrotome.
If you haven't used a vibrating microtome before, you need to be warned
that this is an agonizingly slow way to cut sections. You have control
over the speed of advance of the razor blade and the amplitude of its
saw-like oscillation. (The frequency is that of the power supply: e.g.
60 Hz in N. America or 50 Hz in Britain.) A general rule of thumb for
getting the best sections is to have a wide amplitude and a slow speed
of advance. This can mean 30 seconds for one section.
For FIXED tissue, a vibrating microtome is the best instrument to use
if you must obtain thickish slices without freezing or embedding. Such
slices can be used for a few specialized staining methods, including
Golgi methods for the CNS, and they are useful for finding small areas
that can be cut out and processed for eventual transmission electron
microscopy. The vibrating microtome is the only instrument that will
cut sections of UNFIXED objects, allowing their study by organ culture
and other methods. Many cells (even adult neurons) survive and do their
stuff in a 100 micrometre (0.1 mm) slice for several days, if lovingly
If it's really possible to cut 10-20 micrometre sections of fixed
objects with a Vibratome, some HistoNetter reading Joan's question
or my answer will surely tell us how it can be done.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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