1/9/97
I would appreciate suggestions for examining 80 micron
eggs in seawater or distilled water by SEM where the user
wants to compare fresh egg surfaces (therefore cryo?) with
conventionally fixed, dehydrated and critically point dried
eggs.
The idea was to use cryoSEM but there are problems. I
have tried freezing (in ethane) on a thin smear of Tissue Tek
OCT - droplet on stub and then wiped off with cover slip (not
easy in itself?), but the eggs sank, never to be seen again!
I have also frozen them just mounted in a water film, but
some have simply sheared off the standard smooth stub -
should I try poly-L-lysine or super glue?
A separate problem seems to be that while the crustacean
in question is marine/estuarine and the eggs stand fresh
water for a while (therefore for rinsing), even after 3 or 4
distilled water changes I still see a fine, dried layer of
salt over everything. The rinsing is done in a watchglass
and the water was almost completely removed with an
autopipette before refilling.
One idea not tried yet is to place an egg on a millipore filter,
drain on filter paper, place on stub in cryoSEM airlock
cold-stage and freeze in situ. But with or without vacuum?
It seems a crazy world when it is easier to ask internet
friends than it is to search through a bucket a mud and then
do the experiments on the method to use!
Any comments would be appreciated. Take up gardening?
Thanks in advance - Keith Ryan
Plymouth Marine Laboratory, Citadel Hill,
Plymouth, Devon PL1 2PB, England
e-mail: k.ryan@pml.ac.uk
PML web site: http://www.npm.ac.uk/pml
To look at surfaces of small samples, roughening the stub surface
with fine sandpaper will help keep the ice on the stub during cryo, but we
went into overkill and machined a slight "well" in the surface of the stub
and then roughed it up. To get a cross sectional view, we drill several
small wells (1/16 in.dia x 1/8 in. deep) in the stub. Overfill the wells
(this can be tricky) with a high concentraion of sample in water leaving a
slight droplet (protruding meniscus) of sample protruding above the well.
Sometimes a little vaseline on the surface will help keep the droplets from
running together. Freeze and shear off the droplets.
For the dry eggs, we would use about a dozen (pelco #16079)
adhesive tabs stuck on the surface of a clean glass slide. Dissolve these
into 20 ml chloroform. Place a drop or two of this solution on the surface
of the stub (or coverslip for a smoother background surface), spread evenly
and drain the excess with the corner of a kimwipe. This usually produces a
very thin layer of adhesive strong enough for samples up to a few hundred
microns.
As for the salt on the surface after 3 or 4 washes, not much help
I'm afraid, just a thought. Is there a mucosal surface layer that the
distilled water is drawing the salt out of ?? If so, they might need an
EDTA treatment first to remove mucus. Then again, this mucus might be the
surface you need to see??? This might be a case of "a rock and a hard
place", but sucessful cryo should tell you what is on the surface if you
can get a fracture through an egg.
Hope this is helpful.
Cheers
:)
George
Department of Earth and Atmospheric Sciences
University of Alberta
Edmonton, Alberta T6G 2E3
Canada
ph: 403-492-5746
fax: 403-492-2030
george.braybrook@ualberta.ca
One way to examine your marine eggs would be to use the high-pressure
freezer to freeze a suspension of washed eggs in distilled water (a roughly
200 micrometer thick plug, 1000 or more micrometers in diameter). Then
fracture, etch, and coat as for a TEM replica but use the cryoSEM to look
at the rough surface of the fracture. By regulating the amount of etch you
could see the surface of the egg as well as cross-sections of the layers of
the walls. Paul Walther's articles on double layer coating may be
helpful.
Jacob
Jacob Bastacky, M.D.
Room 116 Donner Laboratory
Lawrence Berkeley Laboratory EMail: sjbastacky@lbl.gov
University of California Phone: (510) 486-4606
Berkeley, California 94720 Fax: (510) 486-4750
Excellent cryo-fracture images of single cells can also be produced,
using the Tissue-Tek/Carbon mixture. (My earlier note) It is also a good
adhesive, especially on the rough cryo-stub surface. George had very
good ideas on cryo-stub surface preparation.
This mixture also fractures well, exposing ALL inner surface of the
fractured eggs "embedded" in it. I hope it will work well for you.
Regard,
Laszlo
Laszlo J. Veto
Electron Microscopy & Image Analysis
Pacific Agri-Food Research Centre, AAFC
Ph: 250-494-7711
e-Mail: vetol@em.agr.ca
My recommendation would be to use a mixture of Tissue-Tek and carbon
dust (from carbon rod sharpener) paste on your cryo-stub. In this
mixture the eggs will not sink fast, therefore will have time to carry out
the freezing process.
I have great LTSEM results using this mixture with unfixed single cell
culture and bacteria.
The separate problem about the fine layer of salt, (if the osmatic change
is all right!) use larger amount of distilled water to rinse for longer time.
Good luck,
Laszlo J. Veto
Electron Microscopy and Image Analysis
Pacific Agri-Food Research Centre
AAFC
vetol@em.agr.ca
It occurred to me that to be sure that your treatments did not introduce any
artefacts on the egg surface you might try a Wild (Leica) Elsam accoustic
microscope. I'm not very familiar with them but there is the potential (at
least) to look at them in seawater or other isotonic solution.
Just a thought,
Geoff Avern
Australian Museum
geoffa@amsg.austmus.gov.au
Here is my 2 cents.
1. Coating of poly-L-lysine do help the sample adhere to substrate.
2. Did you have tried freeze-substitution followed by either fast-freezing
again, freeze-drying, cryo-coating, and cryo-SEM observation, or
dehydration, critical point drying, and SEM observation at RT?
3. I agree with George Braybrook's comment that there is a mucous layer on
the egg surface.
With best wishes!
Ya Chen
My email address has been changed to: ychen14@facstaff.wisc.edu ***
Integrated Microscopy Resource (IMR)--
an NIH Biomedical Research Resource TEL : 608-263-8481
University of Wisconsin-Madison FAX : 608-265-4076
1675 Observatory Drive #159 Email1:ychen14@facstaff.wisc.edu
Madison, WI 53706 Email2:chen@calshp.cals.wisc.edu
Sticking and retaining the processed eggs onto a substrate prior to coating and
earlier, removing all traces of salt from the eggs
.
You could try poly-l-lysine. The old technique, which works well for
specimens of that size range, is a drop of a sticky solution onto a
substrate. After solvent evaporation a very thin "permanently" sticky layer
remains.
The solution is prepared by dissolving the gum of clear sticky tape
in chloroform overnight. Push a couple of meters of tape into a small jar
and add about 50 ml of chloroform. If you require a very clean background
the solution can be millipore filtered.
If some low power images are needed, than freshly cleaved mica gives
a very clean background. At zero tilt, the mica is very dark in secondary mode.
Removing salt from marine specimens can be very difficult. Leaving
the specimen for a lengthy period in water, even after fixation, may damage
structures. An appropriate concentration of ammonium acetate provides the
right molarity and the aqueous (or ethanol) solution leaves no residue.
Others have referred to mucous which may or may not be removed. EDTA
fell out of favour as a decalcifier some time ago. For the same reasons I
would prefer a broad spectrum enzyme. I used a snail enzyme many years ago
for removing mucous, but your local biochemist should be able to advise.
Hope this is some help with this difficult problem.
Happy New Year
Jim Darley
Probing & Structure (Microscopy Supplies & Accessories)
PO Box 111, Thuringowa QLD 4817 Australia
Phone +61 77 740 370 Fax: +61 77 892 313
A great microscopy site: http://www.ultra.net.au/~pns/
p&s@ultra.net.au
Tony King of VG Microtech in the UK (who produce a cryo-SEM, the Polaron
LT7400) had some additional comments which he asked me to pass along:
>> >My recommendation would be to use a mixture of Tissue-Tek and carbon
>> >dust (from carbon rod sharpener) paste on your cryo-stub. In this
>> >mixture the eggs will not sink fast, therefore will have time to carry out
>> >the freezing process.
Tony replies:
Try tissue tek smear (with carbon dust on a Dry colloidal graphite base;
this absorbs the tissue tek bulk and holds the samples in place.
>> >I have great LTSEM results using this mixture with unfixed single cell
>> >culture and bacteria. The separate problem about the fine layer of
salt, >> >(if the osmatic change is all right!) use larger amount of
distilled water >> >to rinse for longer time.
Tony replies:
>Use Osmium vapour to semi-fix the cells before plunging into water.
>Osmotic shock is then reduced to minimum.
>Regards,
>
>Tony King
>Product specialist
>VG Microtech/ Polaron range
>
>Tel: +44 (0)1825 746251
>Fax: +44 (0)1825 768343
>
>E&OE
>
Best regards,
steven E. Slap
Energy Beam Sciences, Inc.
Adding Brilliance To Your Vision
ebs@ebsciences.com
http://www.ebsciences.com/