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 microscopesPhase 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 microscope2- Microscope used in the field of tissue culturePhase 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 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 MicroscopeTRANSMISSION ELECTRON MICROSCOPE (TEM) specimen’s internal featureSCANNING ELECTRON MICROSCOPE (SEM):Scanning electron microscopy views only the surface as 3 D image
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 fluidsFunction: To inhibit action of autolysis
13 2- Celloidin techniqueThe tissue is embedded in celloidin instead of paraffin and cut into sections using sliding microtome
14 3- Freezing techniqueIn 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 FIXATIONDEHYDRATIONCLEARINGEMBEDDINGSECTIONINGSTAININGMOUNTING
16 BOUIN’s FLUID for (liver) FIXATIONIS THE TREATMENT OF THE TISSUE WITH CHEMICAL OR PHISICAL AGENTSAVOID TISSUE AUTOLYSIS – DIGESTION BY ENZYMES PRESENT WITHIN THE CELLSALLOW TO PRESERVE THE STRUCTURE AND MOLECULAR COMPOSITION OF THE TISSUE, MAINTAINING NORMAL ARCHITECTURE OF TISSUETherefore pieces of organ removed from body should be as soon as possible treated by specific fixativesSIMPLE FIXATIVES:ALDEHYDEneutral 4% solution of formaldehyde, formalinCOMPOSITE FIXATIVES:BOUIN’s FLUID for (liver)(picric acide + formalin),
18 CLEARING is the treatment with xylene to make tissue transparent. Xylene is totally miscible with both the dehydrating fluid and embedding mediumxyleneCLEARING 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 slaidsFor light microscopy, the thickness of each section is 3-5 μm
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 STAININGthe paraffin must be removed from the section using xylene
24 STAINING Coverslipping PERMIT THE EXAMINATION OF THE TISSUES BY LIGHT MICROSCOPEMOUNTINGCoverslippingThe 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 matrixSpecific dyes stains the fibrous components of the extracellular matrixMetallic salts penetrate into the tissues, forming metal deposits within the tissue
26 LIGHT MICROSCOPE BASIC DYES: ACID DYES Hematoxylin Toluidine blue Metylene blueBasic fuchsinACID DYESEosinOrange GAcid fuchsinBASIC DYES HAVE AFFINITY TO ACIDIC (BASOPHILIC) COMPONENTS OF CELL AND TISSUEACID 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 matrixHEMATOXYLIN 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 matrixHEMATOXYLIN AND EOSIN (H-E)
29 HISTOLOGIC STAINS Reagent Result Hematoxylin Blue: nucleus; acidic regions of the cytoplasm; cartilage matrixEosinPink: basic regions of the cytoplasm; collagen fibersOrcein's elastic stainBrown: elastic fibersSilver stainBlack: reticular fibers, collagen fiber with blackIron hematoxylinBlack: striations of muscle, nuclei, erythrocytesPeriodic acid-SchiffMagenta: glycogen and carbohydrate-rich molecules