Histology & Cell biology
Microscopes & Microtechniques
Prof. D. Azza Saleh Embaby
Intended learning outcomes (ILOs)
By the end of the lecture, the students will be able to:
• Outline the principles of light & electron microscopes and the difference
between them.
• Define resolution and magnification of microscopes.
• List the different histological microtechniques
• Recognize the advantages and disadvantages of each.
• List the steps of paraffin technique and their importance.
• List the most common histological stains and their uses.
Content
• Light microscope
• Transmission electron microscope
• Scanning electron microscope
• Paraffin technique: advantages, disadvantages, steps.
• Steps of staining.
• Some commonly used stains.
• Freezing technique.
• Celloidin technique.
Light Microscope (LM)
• Light source → visible light
• Condenser lens → focuses the beam.
• Magnifying lenses→ glass lenses:
* Objective lenses (X 4, 10, 20, 40 & 100).
* Eyepiece (X 10, 12, 15)
• The magnified image is reversed from right to left
and is upside down & two-dimensional.
• Resolution = ability to see details = 0.2 μm.
“Minimal distance between 2 points → seen as 2
separate points”
• Magnification power = degree of enlargement
• Magnification power = objective lens X eye piece
Light Microscopes
Electron Microscope (EM)
1- Transmission Electron Microscope (TEM)
- Light source → electron beam
- Lenses → electromagnetic.
* Condenser & magnifying lenses
- Resolution → 0.2 nm.
Transmission Electron Filament
Microscopes (TEM) Site of specimen
Vacuum column
Electromagnetic lenses
Fluorescent screen
Site of camera
Transmission Electron Microscope image
2- Scanning Electron Microscope (SEM)
- Provides a three-dimensional image of the surface of specimen.
- Resolution → 10 nm
Microtechniques
Tissue preparation for microscopic examination
1
1- Paraffin technique
• Most common.
• Gives serial sections
• Easy to stain.
• Xylol & heat destroys fat & enzymes.
1- Fixation:
• Treatment of the tissue with chemical agents.
• Maintains normal architecture (life-like image).
• Retards the alterations of tissue subsequent to death.
• Most common fixatives: buffered formalin & Bouin's.
2- Dehydration:
• Remove water gradually (to prevent shrinkage)
• Using ascending grades of alcohol (50-70-90-100%)
3- Clearing:
• Using Xylol, miscible with paraffin.
• Tissue becomes transparent (clear).
4- Infiltration (impregnation) & Embedding
• In melted paraffin until it is completely infiltrated.
• Harden forming a paraffin block containing the tissue.
5- Sectioning
• Using a microtome with stainless steel blades (5 to 10 μm).
6- Mounting
• On glass slides.
7- Staining
• De-paraffinization using xylene.
• Rehydration (descending grades of alcohol).
• Staining by water-soluble stains.
• Dehydration → then clearing.
• Coverslip fixed using Canada balsam.
Stains
Hematoxylin (basic) → purple → acidic structures of the cell → basophilic
Eosin (acidic) → Pink → alkaline structures of the cell → acidophilic
Some commonly used stains
• Leishman’s stains → blood cells.
• Sudan III, IV & black → lipids.
• Orcein's stain → elastic fibers
• Silver stain → Golgi & reticular fibers
• Metachromatic stains → gives another color than that of the stain → toluidine
blue (blue → purple or red).
2- Freezing technique
• Most rapid.
• Used for rapid diagnosis during surgical operations.
• Most suitable for fat & enzymes examination.
• Gives thick sections (cryostat).
• Difficult to stain.
3- Celloidin technique
• Most perfect.
• Uses celloidin for embedding.
• Very long time.
• Difficult to stain.
• LM uses visible light → resolution 0.2 μm.
• TEM uses electron beam → resolution 0.2 nm.
• SEM → resolution 10 nm.
• Paraffin technique: most common, easy to stain.
• Freezing technique: most rapid, used to diagnose tumors during surgery.
• Celloidin technique: most perfect, but very long time.
• Hematoxylin → basic stain → stains basophilic (acidic) structures.
• Eosin → acidic stain → stains acidophilic (basic) structures.
Test your knowledge
• Calculate the magnification power of a light
microscope if the objective lens is X40 and the
eye piece is X10.
• What is the maximum magnification of LM?
• Using hematoxylin & eosin (H & E) stains,
what should be the colour of nucleus? Why?
References
• Junqueira’s Basic Histology Text & Atlas
• Bancroft's Theory and Practice of Histological Techniques.
