Types of microscopes & Microtechniques

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 .

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.

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

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.

Light Microscope

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

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

Microtome

The MICROTOME - Cryostat

Microtechnique Paraffin technique Freezing technique Celloidin technique

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

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

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 .

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

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) ,

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

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

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.

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

SECTIONING

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.

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

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 )

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

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

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.

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

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

LONGITUDINAL, CROSS & SLANTWISE SECTION

LONGITUDINAL & CROSS SECTION