i want to know how to process tissue for electron microscope
TEM protocols generally require fixation in buffered glutaraldehyde (+/- formaldehyde) followed by post-fixation in osmium tetroxide and then dehydration in ethanol and embedding in an epoxy resin. Once hardened, the blocks are trimmed and sectioned in an ultramicrotome. However there are many variations on this procedure, with occasionally added steps depending on your samples. As already mentioned there are many texts providing procedures, but you need to consider the sample carefully prior to starting. For instance no sample should have at least one dimension that is smaller than 1 mm to ensure adequate penetration of fixatives (particularly osmium). The pH of the buffers used need not be precise but should generally be around neutral and the osmolarity should be controlled.
The book, "Fixation for Electron Microscopy" edited by Hayat is an excellent resource. You can also look up papers in your field that have used TEM and follow their protocols.
I recommend first book Robards and Wilson (ed. Wiley), entitled Procedures in Electron Microscopy, Chapter 5 Basic biological preparation techniques for TEM.
Also Chapter 7 "preparative techniques for transmission electron microscopy and confocal laser scanning microscopy in lichen" written by Asuncion de los Rios and Carmen Ascaso in the book Protocols in Lichenology, editors Kranner, Beckett and Varma, and published by Springer 2002
I can personally give this book chapter.
The preparation of samples for TEM is in principle simple, but for optimum results the type of material must be taken into account with which you want to work
Yours sincerely
If you want I can send you the chapter entitled Preparative techniques for transmission electron microscopy and ....
my email is
I agree, Hayat is the most extensive source I know. Generally, TEM preparation aims at fixing proteins and lipids (membranes) as quickly as possible in place, with as few distortions and losses as possible. For any given sample, a perfect solutions is normally not possible, as different steps of the protocol exert different deletorious side effects. The optimal procedure for a given sample therefore may vary considerably according to the material ( structural density, pH value, Osmolarity, ....), and more often than not has to be assessed empirically, but a suitable starting point obviously helps a lot. Additionally, for TEM, you have to provide contrasting agents (usually heavy metal ions like Osmium, Tungsten, Uranium or Lead) at some point, as the biological material only has minimal contrast in the electron beam. In addition to the extensive compilation by Hayat, you might find the attached list of references useful.
In general, fixation using vitrification and freeze substitution (e.g. high pressure liquid propane jet freezing) are often superior to conventional chemical methods, but facilities are rare and expensive, and suitable material is limited. A good buffer is also important, and in recent years, PIPES and HEPES were used with better effect than PBS or Cacodylate. During aldehyde fixation, a mixture of freshly depolymerized paraformaldehyde with glutardealdehyde is often superior to pure glutardealdehyde (derivatives of Karnovsky's solution). A little Magnesium sulfate helped me to protect membranes for reasons I do not know. Dehydration and infiltration with embedding media are also crucial steps. The aim is to do it thoroughly, but without washing out to much material. The protocols therefore highly depend on material and media used.
You may also search for answers in the attached threads
It is also always helpful to scan the "Methods" sections of publications dealing with similar material and methods to get hints on suitable protocols. Also, I know of two good textbooks in german: "Romeis' Mikroskopische Technik" and "Der Semidünnschnitt"
kind regards
Paavo
https://www.researchgate.net/post/Has_anyone_had_experience_with_fixation_of_tissue_resulting_in_portions_of_mitochondrion_being_swollen_with_a_low_density_of_cristae/1
https://www.researchgate.net/post/Does_anyone_know_of_a_good_protocol_for_preparing_entire_spiders_abdome_for_histology
https://www.researchgate.net/post/4_paraformaldehyde_for_fixing_cells-4_degrees_or_room_temp
https://www.researchgate.net/post/Transmission_electron_microscopy_TEM_of_uterus_and_ovary_how_to_prepare_the_samples
https://www.researchgate.net/post/Can_anyone_help_with_protein_electron_microscopy_artefacts
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