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(Assignment Group 02) Histology Tissue preparation technique for TEM.ppt
- 1. Assignment # 02 Topic: TISSUEPREPARATION 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 techniquefor 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 • Fixativeused 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 iseffective 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
- 7. Tissue Preparation Techniquefor TEM 7
- 8. III. Dehydration • Doneby 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 stepallows 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 • Removalor 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: • Thestandard 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 madefrom 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 • Referto 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
- 15. Ultra microtome Tissue PreparationTechnique for TEM 15
- 16. VII. Mounting • Inthis 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
- 17. Tissue Preparation Techniquefor TEM 17
- 18. VIII. Staining Uranyl AcetateStaining: • 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
- 20. Tissue Preparation Techniquefor TEM 20
- 21. IX. Imaging • InTEM 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
- 22. Tissue Preparation Techniquefor TEM 22
- 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 andPractice 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