Steps of specimen preparation for electron microscopes- TEM and SEM

1. Sample preparation for SEM
Clening the surfce of specimen
Stbilizing the specimen
Rinsing the specimen
Dehydrating the specimen
Drying the specimen
Mounting the specimen
Coating the specimen

2. Cleaning the surface of the specimen
proper cleaning of surface is essential to ensure the speciemen is
free of dust, mud or soil.
Stabilizing the specimen- Fixation
Fixation is usually performed by incubating the sample in a solution of buffered chemical
fixative, such as gluteraldehye, sometimes in combination with formaldehyde and other
fixatives.
Fixatives that are commonly used are Aldehydes, Osmium tetroxide, Tanic acid,
Thiocarbohydrazide etc.
These fixatives form covalent bonds with biological molecules to stabilize thiear structursl
organization.

3. Rinsing the specimen
Sample must be rinsed to remove excess fixatives.
Dehydrating the specimen
Water must be completely removed
Air drying of the specimen causes collapse and shrinkage and therefore is
fixed by a graded series of treatment with organic solvents like acetone or
ethanol.

4. Drying the specimen
The specimen must be completely dry since the specimen chamber is at high vacuum and
sample would be destroyed at such a condition.
Mounting of the specimen
Specimen has to be mounted rigidly on the holder called specimen stub.
Mounted using adhesives such as epoxy resin.

5. Coating the specimen
• Mounted specimens are normally coated with a thin layer of heavy
metal which serves as a source of secondary electron .
• It also prevents high electron build up and increase conductivity.
• Normally coated with thin layer (20nm- 30nm) of conductive metals
lke gold, gold-palladium or platinum.
• Non metals can be used by making them conductive with the help of a
device called ‘’ Sputter coater’’.
• Coated sample is kept inside specimen stub and image is finally
formed on the computer monitor.

6. • This form of image processing is
only in gray scale and therefore
the images can only be viewed
in black and white.
• These images can be colourized
through the use of editing
softwares or graphic editors for
realistic effects and clarifying
the strucures.

7. Sample preparation- TEM
• Cleaning the specimen
• Primary fixation
• Rinsing
• Secondary fixation
• Dehydration
• Infiltration with resin
• Sectioning
• Staining

8. Cleaning the surface of the specimen
• To remove unwanted deposits such as dust,slit and detritus .
• Specimen is rinsed carefully, three times for 10 mins. in 0.1 M
cacodylic buffer at room temperature.
Fixation
•Chemical processing of sample to stabilize its molecular organizations.
•specimen is immersed in chemical preservatives called fixatives that denature and
precipitates cellular macromolecules.

9. Commonly used fixatives :-
• GLUTARALDEHYE : 5-Carbon with aldehyde group at each
ends of the molecule. The aldehyde group reactes with
amino group and cross links with the proteins to form an
insoluble network.
• OSMIUM : Heavy metal that react primarily with fatty acids
leaading to the preservation of cellular membranes.

10. Rinsing- To remove the excess fixative, the sample is
rinsed with 0.1M cacodylic acid buffer again for 2-3
times.
Secondary fixation
Specimen is post fixed with 1% Osmium tetroxide
prepared in 0.1M cacodylic acid buffer for 1.5 hrs at
room temperature (immersion fixation).

11. Dehydrating the Specimen
• Specimen is dehydrated in a graded series of ethanol/
acetone.
• Dehydrated in 50% ethanol for 5 mins, 70% ethanol for 10
mins, 80% for 10 mins, 90% ethanol for 15 mins and 99.9%
ethanol for 20 mins twice.
• This allows water in the sample to be slowly exchanged
through liquids with lower surface tensions.

12. Infiltration
• As ethanol is not miscible with plastic embedding medium, another
intermediary solvent must be used.
• It is actually an alochol substitution process.
• Propyylene oxide is used for the process.
• Sample is Immersed in propylene oxide twice for 20 mins at room
temperature.

13. Embedding
• Conventionally, Epoxy resins are used for embedding the specimen.
• Epoxy resins are polyaryl ethers of glycerol with terminal epoxy group.
• Unpolymerised liquid epoxy resins make the specimen hard like solid
block.

14. Cutting
• The embedded specimen is cut into ultra thin sections of 50-100 nm
using a glass or diamond knife of special instrument called
ultramicrotome.
• These thin sections are taken on small copper discs called grids for
support.
• These sections are then stained with Uranium acetate followed by
lead citrate to enchance contrast of many cellular structures.
• Specimen mounted grid is then placed into specimen stage and the
image is viewed either on a fluorescene screen or a photographic
plate.

15. SPECIMEN PREPARATION
FOR ELECTRON
MICROSCOPE
BY,
SREEDEVI SAJEEV

16. Introduction
• Electron microscopes are very powerful tool in visualising biological
samples. They allow us to view cells, tisues and small organisms
clearly and specifically with great detai.
• Biological samples are subjected to high energy electron beams and
vacuum conditions and therefore cannot be viewed whilst alive .
• They need to undergo certain preparations to withstand
environments inside microscope.

17. Inside a Transmission Electron Microscope (TEM), electrons beams are of much higher energy to pass
through the sample and out the other side. The temperature can rise upto 150℃ where the beam hits the
sample and may cause damages to sample. This temperature is far too high for living cells to survive.
Scanning electron microscope (SEM) uses a lower energy beam but it can still damage the sample.
In TEM, images are viewed when electrons pass right through the sample and need the sample to be cut to
very thin cross sections . Samples are coated with heavy metals to increase the level of contast .
In SEM, samples aren’t cut to thin sections as it visualises the surface of 3-D objects .
It is also coated with thin layr of metal to make it more conductive