(Assignment Group 02) Histology Tissue preparation technique for TEM.ppt
1. Assignment # 02
Topic:
TISSUE PREPARATION TECHNIQUE FOR TRANSMISSION ELECTRON
MICROSCOPE
Submitted To:
DR. RIAZ HUSSAIN PASHA
Submitted By:
Abdul Rehman 23-Arid-2344
Muhammad Ali 23-Arid-1679
Zubair Hayat 23-Arid-1714
Ali Hassan 23-Arid-1653
Aqib Ameer Khan 23-Arid-1657
Tissue Preparation Technique for TEM 1
2. Transmission Electron Microscope
• In 1931 Max Knoll and Ernst Ruska
built the first TEM
• A microscopy technique in which a
beam of electrons is transmitted
through a specimen to form an image.
• Powerful imaging and analytical
technique to investigate the
morphology, structure, and chemical
composition of samples
• Use electromagnetic lenses to focus
electron beams
• Capable of extremely high
magnification up to 500000x may
higher
Tissue Preparation Technique for TEM 2
3. Tissue Preparation technique for TEM
Major steps in the preparation of specimens for diagnosis by TEM
are:
• Specimen handling
• Fixation
• Dehydration
• Infiltration
• Embedding
• Sectioning
• Staining
• Mounting
• Imaging
Tissue Preparation Technique for TEM 3
4. I. Specimen handling
• Samples are fixed as soon as possible after biopsy
• Most sensitive indicators of post-mortem change are
mitochondria and ER which show signs of swelling immediately
• Standard approach is to immerse the specimen in fixative
(precooled to 4°C) immediately
• Specimen size usually of the order of 1mm³
• Volume of fixative must be 10 times the volume of tissue
Tissue Preparation Technique for TEM 4
5. II. Fixation
• Fixative used in TEM generally comprising a fixing agent in
buffer and various additives to control iconic composition.
• Buffered within the range of pH 7.2-7.6
• Room temperature preferred generally for Osmium tetroxide
• Time required is depend on type of tissue, size of the sample,
fixative etc.
• Standard protocol involves:
i. Primary fixation (with Aldehyde)
ii. Secondary fixation (with Osmium tetroxide)
CONT…
Tissue Preparation Technique for TEM 5
6. • Glutaraldehyde is effective at a concentration of between 1.5
to 4% with 2.5% the simplest to prepare from 25 % stock
solutions available commercially.
Osmium tetroxide is used at concentration of 1 to 2 %
• Rinsing of specimen: After primary fixation excess
sample is rinsed to remove excess glutaraldehyde. So
for this purpose, sample washed in 0.1M Cacodylic
Acid buffer.
1 time for 10 min.
3 times for 20 min.
Tissue Preparation Technique for TEM 6
8. III. Dehydration
• Done by passing specimen through increasing conc. of organic
solvent
• The most frequently used dehydrants are ethanol and acetone
• Acetone should be avoided if en bloc staining with uranyl
acetate has been performed to prevent ppt. of uranium salts
• Ethanol overcome this difficulty but require the use of propylene
oxide(highly volatile and inflammable may form explosive
peroxides, so stored at room temperature)
CONT..
Tissue Preparation Technique for TEM 8
9. • Protocol:
This step allows water in sample to be slowly dehydrated
50% Ethanol 5 min
70% Ethanol 10 min
80% Ethanol 10 min
90% Ethanol 15 min
99.9% Ethanol 20 min
99.9% Ethanol 20 min
Tissue Preparation Technique for TEM 9
10. IV. Infiltration
• Removal or clearing of dehydrant from specimen
• Intermediate solvent which is fully miscible with ethanol and
paraffin wax, displace the ethanol in the tissue, then this, in
turn, will be displaced by molten paraffin wax
• A popular clearing agent is Xylene. Typically clearing sequence
for specimens not more than 4mm thick would be:
Xylene 20 min
Xylene 20 min
Xylene 45 min
CONT..
Tissue Preparation Technique for TEM 10
11. • Wax Infiltration:
• Wax may be mixture of purified paraffin wax and various
additives may include resins as styrene or polyethylene
• Allow tissues infiltrated with the wax to be sectioned thickness
at least 2 micrometre
• Sequence would be:
wax 30 min
wax 30 min
wax 45 min
Tissue Preparation Technique for TEM 11
12. V. Embedding:
• The standard practice following dehydration and if required
treatment with a transitional solvent
• This usually require introduction of resin beginning with a 50:50
mix of transitional solvent(propylene oxide) and resin followed
by 25:75
• An hour in each of the preliminary infiltration steps is usually
adequate but preferable to leave sample in resin for 24 hours.
• Once infiltrated tissue sample are places in an appropriate mold
which is filled with resin and allowed to polymerize using heat
• Some resins are Epoxy resins and Acrylic resins
• Various shaped and sized molds are available
Tissue Preparation Technique for TEM 12
13. • Capsule made from polyethylene are recommended as
• They are unreactive with resin.
• Easily removed from latter by bending the mold
• Can be reused
• Can be cut away from block using a razor or scalpel blade.
Tissue Preparation Technique for TEM 13
14. VI. Sectioning
• Refer to clean and consistent cutting paraffin embedded into
thin section
• Tissue slide’ thickness around 50 to 100nm
• Ultra microtome equipped with a glass or knife used in
sectioning
• This step require precision and skill for uniform slices
• Then slices are mounted onto grid
Tissue Preparation Technique for TEM 14
16. VII. Mounting
• In this step, ultrathin tissue sections are carefully transferred
onto support grid typically made of metal such as Nickel or
Copper
• This is done using fine tipped tools to avoid damaging the
delicate section
• Once mounted the grid undergo additional treatment such as
staining
Tissue Preparation Technique for TEM 16
18. VIII. Staining
Uranyl Acetate Staining:
• Prepare a solution of uranyl acetate, typically at a concentration of 1-
2% in water or buffer.
• Apply the uranyl acetate solution to the TEM grids, covering the
sections completely.
• Duration usually around 15-30 minutes.
• Rinse the grids with distilled water to remove excess stain and dry
• It stains nucleic acids, ribosomes, and other acidic structures
• It provides a negative contrast
Tissue Preparation Technique for TEM 18
19. Lead Citrate Staining: Prepare a solution of lead citrate.
• Apply the lead citrate solution to the TEM grids, covering the sections
completely.
• Duration typically around 5-10 minutes.
• Rinse the grids with distilled water to remove excess stain and air dry
• It stains basic structures such as membrane, proteins and other basic
components
• It provides a positive contrast
Tissue Preparation Technique for TEM 19
Lead Citrate solution
Step#1: 2.5g lead nitrate in 10ml distilled water
Step#2: 3.5g trisodium citrate dehydrate in 10ml
distilled water (In separate container)
Step#3: Combine lead nitrate solution from step
1 with trisodium citrate solution from step 2 and
mix thoroughly
Step#4: Adjust pH around 12 by adding 1M
NaOH solution
Step#5: Adjust final volume of the solution to
100ml with distilled water
21. IX. Imaging
• In TEM imaging the mounted section on grid is placed inside
the vacuum chamber of TEM
• A beam of electron then directed at sample and interaction
between tissue and electron produce signals that are captured
by detectors
• Signals convert into image, revealing detailed information about
ultrastructure of tissue at much higher magnification
• Recent detail allowing visualization of subcellular and molecular
interactions
Tissue Preparation Technique for TEM 21
23. Conclusion and Recommendation:
• Sample preparation for TEM involves more and some diff. steps
than those for SEM
• Most of chemicals used are dangerous
• Every step can effect the quality of final electron micrograph
• A single mistake in one step can affect remaining steps
• Must be aware of potential hazards as chemical, electrical or
physical
Tissue Preparation Technique for TEM 23
24. References:
• Theory and Practice of Histological techniques by John D.
Bancroft and Marilyn Gamble
• Methods in Molecular Biology by Parastou Tizro, Cecilia
Choi and Negar Khanlou
• Techniques in Electron Microscopy of Animal Tissue by N.F.
Cheville and J. Stasko
Tissue Preparation Technique for TEM 24
