shrinkage of cells druing fixation

[Jeremy Adler-4]

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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.

[JOEL B. SHEFFIELD]

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Many years ago, I examined the effect of changing the osmolarity of my
fixative on the size distribution of dissociated retinal cells.  Although
this work was done with glutaraldehyde fixatives for electron microscopy,
it still seems to be valid.  We found that for those cells, the optimal
fixation was in 0.08M phosphate buffer, with 2.5% glutaraldehyde.  The
paper was published in Developmental Biology 23:36-61 (1970)

Joel



Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
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On Tue, Dec 9, 2014 at 5:52 AM, Jeremy Adler <[hidden email]> wrote:

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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.
>

[James Pawley]

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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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[Steffen Dietzel]

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Jeremy,

independent of the fixation itself, you should also have an eye on your
embedding medium. Hardening medium such as Mowiol or ProLong tends to
flatten the cells quite a bit, compared to non-hardening such as
Vectashield.

Steffen

Am 09.12.2014 11:52, schrieb Jeremy Adler:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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.
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

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[Mel Symeonides]

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One of my first experiments in graduate school was watching Drosophila
S2 cells bleb after fixation with PBS/4% PFA for 10 min, I highly
recommend it! From what I gather, the reason blebbing happens is the
decoupling between the fixed cortical actin network and the unfixed
(because it is mostly lipid) cell membrane (see
doi:10.1016/j.fob.2014.02.003). At least in these cells, I observed it
happening almost immediately after the fixative is washed off of the
cells. During the fixation there were no morphological changes, but as
soon as I washed it off and replaced it with PBS, the cells began to
swell evenly, and after 15-20 min, large blebs formed with a
simultaneous relief of the swelling.

I have observed the same effect in pretty much every cell line I've
used, including adherent and suspended cells. I have found that
introducing a 10 min permeabilization step immediately after fixation,
e.g. with 0.2% Triton X-100 or (even gentler) 40 ug/ml digitonin, serves
to prevent bleb formation, and makes for much nicer-looking cells. I
don't know whether there would still be loss of volume, though.

Most people I went to for help with this issue thought I was crazy, but
it turned out that they were all permeabilizing their cells for staining
purposes anyway, while I was not permeabilizing because I was using
cytoplasmic dyes and fluorescent protein tags exclusively, hence they
never noticed any blebbing.

Mel Symeonides

On 12/9/2014 8:49 PM, James Pawley wrote:

>
>>
>> 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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Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program
Department of Microbiology and Molecular Genetics
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95 Carrigan Dr
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