1/21/97

Does anyone have any experience with SEM of reconstituted rat tail collagen

matrix? The collagen is laid down onto glass coverslips and then cells are

plated on top of the matrix. I am interested in the collagen more than the

cells, but can't get any definition of fibers. In some areas there is some

definition, but the majority of the surface seems compacted with little or

no definition. I have tried a variety of preparation conditions including

different fixatives as well as cpd vs. freon for drying. The latter seemed

to produce the best results, but there is still little definition of the

matrix.

I have sputtered for different times, but I still seem to have a problem of

charging that eventually burns the sample no matter what kV I use. Does

this

sound like a preparation problem, an imaging problem, or both? Any help

would be greatly appreciated.

Thanks.

Bill Swaim

swaim@yoda.nidr.nih.gov

Hi Bill, I haven't done any collagen work but have found that

I can reduce charging of cultured cells by using a conducting

substrate instead of glass. We use Si discs and chips for growing

cultured cells. You might also try some metal impregnation such as the OTO

techniques reviewed by Judy Murphy some years ago. A dry tape fracture

method (after your drying step) might help uncompact the collagen fibers, if

that is what you want to look at.

good luck

Ed Basgall, PhD

Penn State Univ.

Surface Analysis Group

Dept. of Chemistry

University Park, PA 16802

edb@chem.psu.edu

Bill,

I have studied the conformation of fibronectin fibrils, which were formed either from fibroblast culture or in cell-free system, using a field emission SEM.

To succeed for high-resolution SEM, you need to preserve structures well in all specimen preparation steps and to reduce beam damage in the FESEM at high magnification. I use cryo techniques: fast-freezing, freeze-drying, cryo-transfer, cryo-coating, and cryo-SEM to achieve the goal.

The images obtained reveal the surface features of fibronectin fibrils and fibronectin molecule at a few nm resolution.

If you have any further questions or want to look at your samples, please feel free to contact me.

Here is a reference list:

Chen, Y., Centonze, V.E., Verkhovsky, A. & Borisy, G.G. (1995) Imaging of cytoskeletal elements by low temperature high resolution scanning electron microscopy. J.Microsc. 179(1), 67-76.

Chen, Y., Brummel, S. & Peters, D.M.P. (1996) High resolution cryo-scanning electron microscopy in studying of macromolecular structures and assembly of fibronectin fibrils. Mol.Biol.Cell 7(supplement),412a

Chen, Y., Brummel, S. & Peters, D.M.P. (1996) The structure and assembly of fibronectin fibrils studied by high resolution cryo-SEM. Scanning 18(3), 200-201.

Chen, Y. & Peters, D.M.P. (1997) High resolution cryo-scanning electron microscopy (cryo HRSEM) study of the macromolecular structure of fibronectin fibrils. Scanning , (In Press)

Peters, D.M.P., Chen, Y., Zardi, L. & Brummel, S. (1997) Conformation of fibronectin fibrils varies: discrete globular domains of type III repeats detected. J.Cell Biol. (submitted)

Hope this answers your questions.

Regards,

Ya Chen

My email address has been changed to: ychen14@facstaff.wisc.edu

Cryo/SEM Coordinator

Integrated Microscopy Resource (IMR)--

an NIH Biomedical Research Resource

University of Wisconsin, Madison, WI

1675 Observatory Drive #159

Madison, WI 53706, USA

TEL : 608-263-8481

FAX : 608-265-4076

Email:ychen14@facstaff.wisc.edu

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