Microtechniques & Microscopy PDF

‫كلية طب وجراحة الفم واألسنان‬ Microtechniques & Microscopy By Assmaa Othman Professor of Histology and Cell Biology  Microtechniques Definition: preparación of the tissue for microscopic study. Main goal: preservation of tissue structrues &chemical compositi...

‫كلية طب وجراحة الفم واألسنان‬ Microtechniques & Microscopy By Assmaa Othman Professor of Histology and Cell Biology  Microtechniques Definition: preparación of the tissue for microscopic study. Main goal: preservation of tissue structrues &chemical composition as it have in the living body. The most common is paraffin technique Steps of paraffin technique  1. Tissue sampling From anaesthized animals or immediately after death Sharp instrument Small speciment for proper fixation  2. Fixation The aim of fixation: 1- Maintains normal structure of tissues 2- Prevents autolysis and putrefaction (bacterial attack). 3- Hardens the tissues to be easily cut into thin sections Common fixatives: buffered formalin & Bouinꞌs fluid  3. Dehydration - Aim of dehydration:  Remove water from the tissue and replace it with paraffin (water not miscible ‫امتزاج‬with paraffin).  Gradual dehydration (avoid shrinkage)  Ascending grades of alcohol (50%- 70%- 95%-100%).   4. Clearing ‫وضوح‬ - Aim of clearing:  To remove alcohol to render tissue more transparent  Xylene is totally miscible with paraffin &alcohol. Clearing agent: Xylene  5. Embedding in soft paraffin Aim of embedding: -- penetrate tissue deeply& replace xylol -- Allow tissues to be bulky and harder to be easily sectioned -- Using melted paraffin wax 6. Embedding in hard paraffin Molds  7. Sectioning & Mounting ‫تركيب‬ Using the microtome: sections are 5-10 µm thick Using albumin Sections are put on glass es. Microtome  7. Mounting & Staining The most common stains of LM: Hematoxylin & eosin (H&E) Hematoxylin: basic dye that stains the acidic component= blue colour (basophilic) as Nucleus (DNA & RNA) Eosin: acidic dye that stains the basic componen t= red colour (acidophilic) as filaments, muscle or Cytoplasm Cover slip Choose the correct answer: 1- Which of the following is used for dehydration of tissue sample in microtechniques? a) Buin`s fixative b) Ethyl alcohol c) Paraffin wax d) Xylene 2- Which of the following prevent autolysis of tissue sample? a) Xylene c) Ethyl alcohol b) Buin`s fluid d) paraffin wax  Microscopes - The importance of a microscope: its capacity to magnify small objects (magnification) and to reveal their fine details (resolution). The most important types are: 1- Light microscope (LM) 2- Transmission Electron microscopes (TEM) The Ordinary Light Microscope - Magnification of eye piece=10X - Mag. of objective lens = 4X, 10X, 40X or100X Total Magnification of a histological section = The power of ocular lens X the power of objective lens e.g. 10 X 40 = 400 Resolution power is the least distance between two particles that can be seen as separate objects. Transmission Electron Microscope Transmission Electron Microscope  Transmission Electron Microscope  Used to study ultrastructure of the cell (very high magnification power)  Illuminated by beam of electrons  Electromagnetic lenses  Fluorescent screen  No slides but electron micrographs  Staining: (salts of heavy metals)  So, Structures either appear dark (electron dense) pale (electron lucent)  TEM LM Beam of electron Day light or electric Source of lamb illumination Electromagnetic lenses Glasses lenses lenses 0.2 nm 0.2 micron Resolution power Very high Limited Magnification Not colored Colored Images Cells: - The structural units of all living organisms Cells Tissue Organ System - Cells vary in size, shape and function - Each cell consists of nucleus and cytoplasm  The cell ‫غشاء‬Cell membrane ‫عضيات‬Organelles ‫هيكل‬Cytoskeleton ‫شوائب‬Inclusions