4/27/99

Question:

I am a relative novice at microscopy and have the task of

trying to freeze-substitute samples containing large amounts

of fat in the form of fat droplets (size range between

sub-micron to approx. 10 microns) for TEM. Although I can

produce reasonable results for semi-thin sections for LM, the

fixatives (combinations of glutaraldehyde, OsO4, RuO4, and

UA) and solvents (acetone and methanol) I have used do not

fix the fat sufficiently for ultra-thin sectioning. (I have

also varied the substitution and fixation periods from two

days to over a week with little success).

Can anybody suggest a fixative/solvent regime which will fix

and dehydrate, but not mobilize, the fat? I have good

indications that methanol and ethanol are redistributing the

fat considerably at -90C, even after three days osmication.

Sample preparation also requires the temperature to remain

below -25C until polymerization.

Any suggestions would be welcome!

Regards,

Nick Johnson



Nick.B.Johnson@Unilever.com



Dr. N. Johnson,

Structural Analysis,

Unilever Research,

Colworth House,

Beds, U.K.

Regarding TEM of fat, I might suggest two possible approaches. One would

be to use a hydrophobic resin like Lowicryl HM-20 or HM-11. It might even

serve as the substitution media.

Another thought is to freeze dry the samples and embedd them in resin

directly .

One could also expose the sample, after drying, to osmium vapors from the

crystals in order to keep things anhydrous. I would recommend a resin that

can tolerate a little water, like Epon or its equivalent, or a methacrylate

based resin.



Would using freeze-fracture EM give the results you are after??



Fats and lipids are a nightmare for EM.



Greg Erdos

Gregory W. Erdos, Ph.D. Ph. 352-392-1295

Assistant Director, Biotechnology Program

PO Box 110580 Fax:

352-846-0251

University of Florida

Gainesville, FL 32611

gwe@biotech.ufl.edu

Nick:

I am more of a novice than you, and would GREATLY appreciate any tips

you get on this. ( In fact would any *experienced* lab like to do this work

for me for a fee?) I am doing something similar, a suspension of 15

micron liposome aggregates containing vegetable oils.



I don't have the answer. I presume osmication or rutheniation should be

done extensively at room temp or preferably *above* (37C), before

dehydration. A method using imidazole was highly recommended to me,

as better than extended osmication, see reference below. I do expect

that freeze-substitution is the way to go for dehydration. Acetone is

known to extract fewer phospholipids than do the alcohols; it may be

better for nonpolar lipids as well. Just keep it well-chilled.



Angermuller S, Fahimi HD, Histochem J 1982 Sep;14(5):823-35,

Imidazole-buffered osmium tetroxide: an excellent stain for visualization of

lipids in transmission electron microscopy.

(nb: in PubMed just now I hit the "related articles" button and found a few

interesting articles

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&uid=6182131&dopt=m&dispmax=100 )



I hired a local EM tech to work on this and was disappointed with the

results, so hope you do find a method that works, and that you can let me

know. For your smaller particles, presumably you can use cryo TEM with

an energy loss filter and avoid fixing entirely, but I can't with my large

aggregates



Good luck

Richard

[Return to Tips & Tricks Menu]