1. Types of microscopes &
Microtechniques

2. Some type of microscope
* Light microscope . * Electron microscope . A- Transmission electron microscope . B- Scanning electron microscope . * Darkground microscope . * Phase-contrast microscope . *Interference microscope . * Fluorescence microscope . * Confocal microscope .

3. Classification of the microscopes
I- Microscopes using visible beams:
1-Optical light microscope.
2-Modified microscopes
Phase contrast microscope.
Interference microscope.
Polarizing microscope.
Dark field microscope.

4. II- Microscopes using invisible beam:
1- Ultraviolet microscope.
2- X-ray microscope.
3- Electron microscope.

5. Other classification:
1- High resolution microscope.
Electron microscope
2- Microscope used in the field of tissue culture
Phase contrast microscope.
3-Microscope used in the field of surgery.
Dissecting microscope (Stereomicroscope).
4- Image analyzer, which is special programmed system provided with a microscope, vidio camera,
and software system for quantitative microscopic measurements.

6. Light Microscope

7. Fluorescent Microscope
FLUORESCENCE MICROSCOPE :
are extremely important in immunohistological staining.dectation of anti- bodies

8. TRANSMISSION ELECTRON MICROSCOPE (TEM) specimen’s internal feature
SCANNING ELECTRON MICROSCOPE (SEM):
Electron Microscope
TRANSMISSION ELECTRON MICROSCOPE (TEM) specimen’s internal feature
SCANNING ELECTRON MICROSCOPE (SEM):Scanning electron microscopy views only the surface as 3 D image

9. Microtome

10. The MICROTOME -
Cryostat

11. Microtechnique Paraffin technique Freezing technique
Celloidin technique

12. 1- Paraffin technique Tissue sampling :
A small piece of tissue is obtained by biopsy under anaesthesia or taken immediately after death .
Sample should be very small in thickness 0.5 cm 
why to be small ?? -=> to allow entrance of fluids
Function: To inhibit action of autolysis

13. 2- Celloidin technique
The tissue is embedded in celloidin instead of paraffin and cut into sections using sliding microtome

14. 3- Freezing technique
In this technique the tissue is frozen using liquid nitrogen .
sections are cut inside cold cabinet using microtome .
this machine is called cryostat , sections are then stained and examined .

15. STEPS IN PREPARATING SECTIONS FOR LIGHT MICROSCOPE
FIXATION
DEHYDRATION
CLEARING
EMBEDDING
SECTIONING
STAINING
MOUNTING

16. BOUIN’s FLUID for (liver)
FIXATION
IS THE TREATMENT OF THE TISSUE WITH CHEMICAL OR PHISICAL AGENTS
AVOID TISSUE AUTOLYSIS – DIGESTION BY ENZYMES PRESENT WITHIN THE CELLS
ALLOW TO PRESERVE THE STRUCTURE AND MOLECULAR COMPOSITION OF THE TISSUE, MAINTAINING NORMAL ARCHITECTURE OF TISSUE
Therefore pieces of organ removed from body should be as soon as possible treated by specific fixatives
SIMPLE FIXATIVES:
ALDEHYDE
neutral 4% solution of formaldehyde, formalin
COMPOSITE FIXATIVES:
BOUIN’s FLUID for (liver)
(picric acide + formalin) ,

17. DEHYDRATION
To remove water
Alcohole
50%
70%
90%
100%

18. CLEARING is the treatment with xylene to make tissue transparent.
Xylene is totally miscible with both the dehydrating fluid and embedding medium
xylene
CLEARING is replacing the dehydrating fluid with the clearing fliud - xylene

19. EMBEDDING Paraffin-infiltrated tissue is placed into a small mould,
covered with melted paraffin,
and allowed to cooled and harden, forming a paraffin block containing the tissue.

20. SECTIONING BY MICROTOME
Paraffin block is mounted in a microtome.
The microtome is the machine equipped in a sharp steel blade, that undercontrol of crank cuts thin slices of paraffin block containing tissue.
slices are placed onto well-adhered glass slaids
For light microscopy, the thickness of each section is 3-5 μm

21. SECTIONING

22. UNSTAINED PARAFFIN SECTION
Many tissue elements have approximately the same optical densities, therefore for light microscopy they have to be stained with water- soluble stains.

23. BEFORE THE STAINING
the paraffin must be removed from the section using xylene

24. STAINING Coverslipping
PERMIT THE EXAMINATION OF THE TISSUES BY LIGHT MICROSCOPE
MOUNTING
Coverslipping
The stained section on the slide must be covered with a thin piece plastic or glass to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope with . Canda balsam or DPX (mixture of distyrene, a plasticizer, and xylene )

25. HISTOLOGIC STAINS Classes of histological stains:
Dyes stain acidic and basic components of the cell and extracellular matrix
Specific dyes stains the fibrous components of the extracellular matrix
Metallic salts penetrate into the tissues, forming metal deposits within the tissue

26. LIGHT MICROSCOPE BASIC DYES: ACID DYES Hematoxylin Toluidine blue
Metylene blue
Basic fuchsin
ACID DYES
Eosin
Orange G
Acid fuchsin
BASIC DYES HAVE AFFINITY TO ACIDIC (BASOPHILIC) COMPONENTS OF CELL AND TISSUE
ACID DYES HAVE AFINITY TO BASIC (ACIDOPHILIC) COMPONENTS OF CELL AND TISSUE

27. HEMATOXYLIN AND EOSIN (H-E)
Dyes stain acidic and basic components of the cell and extracellular matrix
HEMATOXYLIN AND EOSIN (H-E)
The most commonly use stains in histology:
Hematoxylin is a base that colors the acidic components of the cell a bluish tint.
The organella - nucleus (DNA, RNA), and regions of the cytoplasm rich in ribosomes or another acidic components are stain dark blue;
The components stain with hematoxylin are referred to as basophilic.
Eosin is an acid that stains the basic components of the cell a pinkish color.
Most of cytoplasmic components have a basic pH and stain pink;
The cytoplasmic elements stain with eosin are said to be acidophilic.

28. HEMATOXYLIN AND EOSIN (H-E)
Dyes stain acidic and basic components of the cell and extracellular matrix
HEMATOXYLIN AND EOSIN (H-E)

29. HISTOLOGIC STAINS Reagent Result Hematoxylin
Blue: nucleus; acidic regions of the cytoplasm; cartilage matrix
Eosin
Pink: basic regions of the cytoplasm; collagen fibers
Orcein's elastic stain
Brown: elastic fibers
Silver stain
Black: reticular fibers, collagen fiber with black
Iron hematoxylin
Black: striations of muscle, nuclei, erythrocytes
Periodic acid-Schiff
Magenta: glycogen and carbohydrate-rich molecules

30. LONGITUDINAL, CROSS & SLANTWISE SECTION

31. LONGITUDINAL & CROSS SECTION