Types of microscopes & Microtechniques

1. Types of microscopes &
Microtechniques

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

3. 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. 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.

7. 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. Paraffin technique Freezing technique
Celloidin technique

12. 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. The tissue is embedded in celloidin
instead of paraffin and cut into
sections using sliding microtome

14. 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
1. FIXATION
2. DEHYDRATION
3. CLEARING
4. EMBEDDING
5. SECTIONING
6. STAINING
1. MOUNTING

16. 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%
Alcohole
70%
Alcohole
90%
Alcohole
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 xylene xylene xylene
CLEARING is replacing the dehydrating fluid with the clearing fliud -
xylene

19. ◦
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. 
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

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

23. 
the paraffin must be removed from the section using xylene

24. 
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. 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. 
BASIC 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. 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.
Dyes stain acidic and basic components of the cell and extracellular m

28. Dyes stain acidic and basic components of the cell and extracellular m

29. 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