Re: shrinkage of cells druing fixation

Posted by James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/shrinkage-of-cells-druing-fixation-tp7583101p7583106.html

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>Cells on coverslips tend to flatten during fixation.
>In LM the Z resolution is always poorer than in
>XY and shrinkage in Z exacerbates this mismatch.
>Does anyone have any suggestions for preventing or reducing shrinkage.

Dear Jeremy,

I have never liked to use the word "fixation" in
this way. It implies that it stops changes when
in fact it doesn't. It just stops the specimen
from rotting right away. How about chemical
treatment?

It should perhaps not surprise us that, on
encountering the fixative, living cells don't say
to themselves "Oh, hurray, I'm going to be in a
famous photo on the cover of Nature!". Instead
they react in a variety of ways (blebbing,
retraction etc.) in an attempt (one assumes) to
avoid being killed. When the cells in question
are only one cell-layer quick, diffusion is fast,
the struggle doesn't last long and the
rearrangements, if not always minor, are at least
usually fairly obvious (blebs on white blood
cells: they look like bubbles on the outside). In
fact, you can (and should?) watch this process on
the stage of a phase or DIC microscope.

However, it is not uncommon for "fixation" to be
followed by some sort of dehydration (into a
graded series of ethanol or acetone and then
perhaps into wax or a clear embedding liquid) and
as the structural shape of both lipids and
proteins is largely determined by their
interaction with the surrounding water, it is
this process that produces the greatest
distortions (a minimum of 60% volume shrinkage in
soft tissues such as embryos, according to the
most careful studies by Alan Boyde at UCL,
London.). When applied to cells attached to
glass, the glass acts as sort of a "drying frame"
(i.e., like those used to prevent the pelts of
fur-bearing animals from shrinking as they dry),
and by preventing the shrinkage in the x-y
direction, increases that in the z direction.

Fixation methods that start with a freezing step
show some improvement as long as they occur fast
enough to preclude the formation of ice crystals.
Impacting the cells on a Cu block cooled with
liquid He avoids ice crystals for the outer 10-15
µm; using a high-pressure freezer that
pressurizes the specimen to about 2,000
atmospheres before applying the LN2, can extend
this to over 100 µm. However, even going to these
tedious (and expensive) lengths will prevent only
some of the shrinkage attendant on replacing the
ice with some non-polar solvent
(freeze-substitution of the water by acetone
containing OsO4).

Which is why many folk replace the water with
glycerol, which is as hydrophilic as water,
although a bit lumpier.

Chapter 18 in the handbook of Biological Confocal
Microscopy (3rd edition) goes into some detail on
the subject.

Regards,

Jim Pawley
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