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Cells adhered to membrane imaged in a routine
tungsten source SEM. Cell processes are clearly visible
at the periphery of the cells. Micrograph width is 30 µm.
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Cells grown on silicon nitride membrane in an aqueous
environment. Note the nuclei and cellular processes.
Micrograph width is 100 µm.
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Several cells adhered to the membrane imaged in a routine SEM.
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Introduction:
The SPI SEM Wet Cell Kit™ is now available for those wanting to conduct back scatter electron
imaging on wet samples but not having the funds for purchasing a high cost SEM with low vacuum
capability. Indeed we believe the SPI SEM Wet Cell Kit™ could be used quite successfully on
any SEM, one that need not have LaB6 or FESEM capabilities.
The results shown on this page were done on an SEM that very definitely
was not new and was equipped with a heated tungsten filament capability only.
The concept:
Cells can be grown on the membrane just as they would on certain polymer film surfaces,
such as Aclar®, polycarbonate, etc. And once the cells are processed (e.g. fixed) and
washed, another cell can be glued to the first one creating a chamber that has the
volume of two membrane window grids. But since this is in fact more volume than is
actually needed, for the back side a blank silicon disc can be glued down instead.
The seal must be vacuum compatible of course.
The results:
Nothing but spectacular! The silicon nitride membrane window is thinner than a polymer membrane,
which is the reason for the results that are quite spectacular when compared to the results
obtained using systems that employ polymer membranes.
Experimental details:
All results shown on this webpage were from Cos 7 cells grown onto
100 nm thick silicon nitride membrane window grids, specifically, SPI #4122SN-BA.
The cells were grown overnight at 37°C (98.6° F) in an incubator in a chamber consisting of an
internally threaded aluminum cylinder epoxied to the membranes. Aluminum material was coated
in carbon prior to adhering the membrane. The cells were then fixed in
glutaraldehyde and OsO4
washing thoroughly with phosphate buffer after each step. Twenty micro litres of dextran loaded phosphate
buffer were then added and the silicon nitride membrane window cell sealed.
Selection of the right membrane thickness:
For this application, thinner is definitely better. The thinner the membrane, the higher will be
the resolution. The micrographs taken for the website presentation we done using 100 nm
membranes but there is no doubt in our minds that if thinner membranes were selected, that
far more information would be available from images that would be of demonstrably higher
resolution. The thinner the membrane the better the quality of the data but also,
the thinner the membrane the more likely one might be to rupture the membrane.
Therefore we offer the Wet Cell Kits in a selection of different membrane thickness
and we leave it to our customers to decide which is best for them. We also offer a
"sampler" kit that includes 2 each of four different membrane thicknesses
(e.g. 100 nm, 50 nm, 30 nm and 20 nm) plus 8 "blanks" so that there would be
enough material to make a total of 8 different Wet Cells.
Silicon disc "blanks"
Instead of using for the "backing" another membrane window grid, we recommend using a
silicon "blank". This is literally a piece of silicon from the same thickness wafer
but without the etch pit or hole. Hence when handling the cell, you need to worry
about not touching just one of the two sides. Using a blank results in a far more
robust system and one far less likely to rupture when handling or processing.
Special adhesive:
In theory, one can use a whole range of different adhesives for gluing together the two
different silicon "sides", one being the membrane window grid and the other, a silicon
"blank", including our own 5-minute epoxy glue.
But we would recommend a particular SPI # 6000WCG-AB, for this purpose since it is much more high
vacuum compatible. One unit of this adhesive, more than enough to glue ten cells, is included with each kit.
Wet Cell™ Kits
Each kit contains ten membrane grids of the stated membrane thickness plus 10 blanks to
make a total of 10 different SEM wet cells plus one pack of the special
3M® Brand DP 460 adhesive to glue the two sides of the cell together:
Replacement Wet Cell™ Kit Components:
- Replacement membrane window grids:
- Select what you need from our standard offering of
our TEM membrane window grids including silicon oxide as well as nitride.
Replacement silicon "blanks":
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Monday May 12, 2008
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