Bilal,

Given that the fixatives must penetrate the tissue for suitable preservation (and your root cells should have cell walls), I would carefully dissect the 3 mm pieces in the primary fixative as a first step.  I would also follow the ethanol dehydration series with HMDS chemical drying.  You can also do short pulse microwave irradiation to accelerate the penetration of the fixative.  I would do this in a ice bath with two 400 ml beakers filled with water as heat traps.  Using a digital thermometer afterwards to ensure that your fixative temp does not exceed 30 degrees.  You may want to incorporate a secondary fixation of osmium or permaganate  Good Luck.

Dear Fabienne, Thanks for your kind response. It seems that It took pretty much time of yours to write that elaborate answer.

Regarding the suggestion in option 1 and in publication of yours; embedding of the root in a resin is required only for TEM analysis, that you have also used for TEM visualisation, as mentioned in your publication.

Coming to the option 2, we have tried the critical point drying (CPD) of root samples

Thanks Michael,

But as you got it, the fixation is not a problem. It is the distortion of the sample due to drying after Gold sputter coating that creates hindrance otherwise samples retain the normal architecture and I have observed this under light microscopy.

Dear Bilal,

I realize you have probably considered this and may not have access.  One could view the unfixed sections fully hydrated in an ESEM.  Root hairs would collapse very shortly on exposure to the electron beam.  I have tried to attach an ESEM image of cucumber.

Thanks Dr. Fabienne, I'll definitely go for the sectioning after embedding in resin block. Hope it will work..

Bilal,

I have attached an image using a method similar to the one you describe. Differences are: Initial fixing in glutaraldehyde and sodium cacodylate buffer. An extra step was post-fix with 1% aqueous osmium tetroxide 1hr. The dehydration used an extra 95% and a second 100% dried ethanol over molecular sieve.  Then after dehydration critical point drying. This is the method that I was taught at the Natural History Museum, London. It is perfectly adequate for imaging roots of grass. No embedding or sectioning required, only cutting with a blade. As you can see there is no distortion.

Its perfect....

Dear Ian,

Thanks

Can I go for this dehydration without using molecular sieve, simply in eppendroff tubes? bcoz I dont have this now..

I don't know! because we would only used dried ethanol. It is a straightforward process, see link.

http://ccc.chem.pitt.edu/wipf/Web/Solvent_Drying.pdf

@Faizan Abul Qais

Ho gaya daac saab.

@Bilal Ahmad can we have the protocol??