11/3/98

Here I am once again, hat in hand to ask for your help. This time

I have someone who wants to do SEM on a cell line grown in suspension (they

are completely non-adherent, so we can't grow them that way). What should

we do in terms of fixation, etc. and then how do we stick them to the stub?

Do we try to make them stick to something first & then run them up? Do

you run them up first & then stick them to something? Do you run up to

them & say "Stick 'em up"?

As you can tell, this is a new one to me. I've process little

things by wrapping them in lens tissue & then running the up & then shaking

the sample onto the sticky carbon dots. I don't think that procedure will

work for these because these cells are much tinier.

I thank you in advance for any suggestions you can give me. And I

thank you for all the help and advice you've given me in the past.

I'll stick by my computer until I hear from y'all,

Paula :-)

Paula Sicurello

UC Berkeley

Electron Microscope Lab

psic@uclink4.berkeley.edu

phone: 510-642-2085

fax: 510-643-6207

http://biology.berkeley.edu/EML

We can't leave you dangling like this. There are several approaches to

solve this suspensful situation. I base this on work I have done with

bacteria, fungi and tissue cultures grown in suspension.

In the first approach (easy but yielding few, isolated cells) , take a

scrupulously clean, glass microscope slide (use a good glassware detergent

and rinse it quite well - do not use acid cleaning agents as they may be

toxic). Treat the glide with a poly-L-lysine solution (1 mg/ml distilled

water) for 5 minutes, then rinse with distilled water. Place slide into a

petri dish containing a filter paper moistened with distilled water and

containing several pieces of applicator stick. The slide should be

suspended (like a bridge) over the applicator sticks. Gently layer on the

cell suspension on the slide so that it fills the slide but does not

overflow the edges (about 5 ml). Allow the cells to settle onto the slide

for about 1 hr. You should keep the cells "happy" during this time period

(out of the light, warm, perhaps in a CO2 incubator). Now, very carefully

aspirate away most of the liquid and very slowly add 5 ml of your primary

fixative (4% formaldehyde/2%glutaraldehyde) to again cover the slide. Allow

to fix for 15-20 min at room temp. Rinse by gently decanting and gently

adding buffer from one end of the slide. Post-fix in 2% osmium tetroxide

(buffered or in distilled water) for 30 min at room temp. Rinse in

distilled water and dehydrate the entire slide by overlaying with 50, 75,

95, ABS ethanol. They key here is "gently". After absolute ethanol,

critical point dry the slide (or portion of the slide), coat with

conductive metal and examine in the SEM. This technique will give you

beautiful black backgrounds with only a few cells per field of view. Some

people prefer this method since the cells are quite isolated and not piled

up.

A second approach (possibly yielding too many cells on a substrate), is to

pass the cell suspension through a micropore filter or silver membrane

(available through Structure Probe, for example) so that the cells are

retained on the surface of the membrane. This must be done very gently so

that the cells are not broken. We have best success by sucking the cells

down onto the membrane rather than forcing them onto the membrane from a

syringe. You will need to try several dilutions of cell suspension since

too many cells could pile up and create a mess. After filtering the cells,

you would then apply the various solutions (fix, wash, dehydrate) by

passing through the filter. In the end, you would remove the filter and

transfer it into the CPD device (keeping it wet, of course). Some holder

will be needed to help keep the filters from flip flopping in the CPD

device and to maintain the proper orientation (i.e., keep track of which

side the cells are on). Mount the filter on the stub (double sticky tabs),

coat with metal and examine in the SEM.

Good luck,

JB

John J. Bozzola, Ph.D., Director

Center for Electron Microscopy

Neckers Building, Room 146 - B Wing

Southern Illinois University

Carbondale, IL 62901

U.S.A.

Phone: 618-453-3730

Fax: 618-453-2665

Email: bozzola@siu.edu

Web: http://www.siu.edu/departments/shops/cem.html

For SEM studies of cells in suspension fixed with glutaraldehyde or

frozen-freeze-substituted, we attach them to silane treated (1% silane from

Aldrich in acetone for a few h in 50deg.C ) and carbon coated glass or mica

chips. If they are conducitively stained, they do not require coating.

The most important part of the procedure is to rinse off all the non-linked

aldehyde groups from the cells and free silane from the surface of the

chips.

We have developed this procedure with Hans Ris for SEM studies of human

blood normal and leukemic cells in which we were recovering ~95% of the

cells.

I will be happy to guide you through, provide more details, or literature

(containing also descriptions of our attempts to use other methods) if

within a sphere of your interests.

Marek.

Marek Malecki, M.D., Ph.D.

address: IMR, 1675 Observatory Drive, Madison, WI 53706.

cellular phone: 6084441680.

voice mail: 6082638481.

fax: 6082654076.

email: malecki@macc.wisc.edu

http://www.wisc.edu/cesip/

http://www.bocklabs.wisc.edu/imr/integrat/transg.htm