Hi, A fast and reliable technique is Low-temperature scanning electron microscopy. THe specimen is mounted, then frozen in liquid nitrogen and subsequently investigated in its frozen hydrated state on a cold stage in  the scanning microscope. This allows you to study hyphae in their natural turgid state. Please find a pdf attached.  

Freez drying and Osmium tetra oxide fixation

Hi, A fast and reliable technique is Low-temperature scanning electron microscopy. THe specimen is mounted, then frozen in liquid nitrogen and subsequently investigated in its frozen hydrated state on a cold stage in  the scanning microscope. This allows you to study hyphae in their natural turgid state. Please find a pdf attached.  

I agree with Christoph, however, it requires having the appropriate instruments. We routinely vapour fix with osmium tetroxide, dehydrate in -80C acetone, then critical point dry.

You are right that dehydration matters. Now my question is why you are so considered about this shrinkage? You just reduce the magnification and the shrinkage will not be prominantly visible.

Don't bother with dehydration - do a vapor fixation using only OsO4 for about a minimum of 1 to 48 hours, then do a mild vapor removal using a vacuum chamber of  some sort do not use a full vacuum, just mild. Do this for several hours  to clear out vapors. Use a 3 way valve to establish the mild vacuum, bleed in air through the three way valve. Put in an inline  cold trap to capture any vapors that might be coming from your sample towards your vacuum.Be sure to do the OsO4 with a full chemical hood, preferably at room temperature. Coat with a high resolution sputterer and examine to see if it worked. Fungi should not distort or shrink significantly if prepared properly.

I agree with all the respectable answerers, that treatment with osmium tetraoxide works but is the treatment protocol resembles with bacterial treatment. Please give any reference to read.

Thank you very much for the answers and advice everyone. I'll take note of that. 

By fixing it with gluteraldehyde, first you should grow you fungi on coverslip, the coverslip should be the size of stub than fix it gluteraldehyde 

Dear Candace,

your question seems to be taken a little bit „too easy“ with regard to (classical) specimen preparation for SEM.

There might be a lot of preparation methods which could be followed. Some of the foreposters have made hints on how to proceed, Christian Scheidegger pointed you to using “Low-temperature scanning electron microscopy”, providing an interesting article (do you have a SEM with cryo-chamber?).

Another point would be addition of some kind of special chemicals (to the fixative or during further processing) to prevent or at least decrease shrinkage/distortion of your delicate samples.

But unfortunately we don’t know yet what your previous specimen processing for your fungal mycelium specs was and which (instrumental & working) capabilities your lab can provide to facilitate your task. It perhaps woul help to direct you into the right direction.

If you are novice (we don't know either) you should “dig out” some literature, eventually googling for the huge amount of specific literature or even PubMed to see what has been (peer reviewed) published on the topic you are working…. and there might be surprise about excellent work in former days (1960ies up to 2013).

Just as some possibilities to think about are (not exhausting):

Perhaps read:

Ultrastructure Techniques for Microorganisms, Editors: Henry C. Aldrich, William J. Todd, therein the Chapter: by Alan Beckett and Nick D. Read,  1986 (Springer)[http://link.springer.com/chapter/10.1007/978-1-4684-5119-1_2 ] (unfortunately access only by PPV-Pay per view, available as e-book, 2 pages as preview visible/accessible).

Concerning think about [e.g. PDF: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC379999/pdf/applmicro00036-0252.pdf], Murphy JA, Campbell LL, Pappelis AJ, 1974 [Morphological observations of Diplodia maydis on synthetic and natural substrates as revealed by scanning electron microscopy]:

>

or consider alternatives for the primary fixative, as found in

MJ Talbot and White (‎2013): Methanol fixation of plant tissue for Scanning Electron Microscopy improves preservation of tissue morphology and dimensions

PDF: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853006/pdf/1746-4811-9-36.pdf

or Oliveira Junior E.N. et al (‎2012)   at:

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132012000500001,

or PDF: http://www.scielo.br/pdf/babt/v55n5/01.pdf, respectively,

with good, acceptable morphology, citing the method of Melo IS, Faull JL.: Scanning electron microscopy of conidia of Thichoderma stromaticum, a biocontrol agent of witches broom disease of cocoa. Braz J Microbiol. 2004; 35: 330-332.

An interesting, recent article, published 2012 in (A. Méndez-Vilas, Ed.):

by I. Ahmad and M.S.A Khan perhaps also widens your horizon for (not only fungal) specimen preps in multiple approaches (cf.: http://www.formatex.info/microscopy5/book/646-659.pdf ).

I wish you the best for reaching the goal of your work but – I guess -  as usual it might be worth some trial and error too…

Regards, Wolfgang

Try The Camphene technique

Hopefully one of the previous methods mentioned above work since mostly are standard and many of them tested However if nothing works you can try my technique which i developed and uses CAMPHENE substance   after sublimation to fix the specimen. we obtained good results with it on many cellular tissues .and I published a paper on the technique  entitled 

Application of Camphene technique in the examination of teeth for Scanning Electron Microscopy   by K.Galil & A.J.Gwinnett ,Arch.Oral biol 1973,Volume 18 page 925

 Cheers

K.Galil

Thanks a lot everyone for the answers and suggestions ! Thank you very much.

Give a treatment of MgCl2 and glyecrol for one minute then see waht will happen to shrinkage.