Lecture 1- Micro techniques & microscopes.pdf

BMS111 Lecture 1 histology Microtechnique Dr Manal Shaaban Hafez Professor of histology and cell biology in Galala University Faculty of Medicine, Fall 2024-2025 Galala University...

BMS111 Lecture 1 histology Microtechnique Dr Manal Shaaban Hafez Professor of histology and cell biology in Galala University Faculty of Medicine, Fall 2024-2025 Galala University gu.edu.eg NORMAL Prof. dr. Manal shaaban Hafez STRUCTURE OF Professors of Histology and Cell Biology HUMAN BODY Presented By MODULE Prof. dr. Noha Abdel Latif Professors of Histology and Cell Biology Course Code (BMS 111 )  Assessment of the course (BMS 111 Assessment Measures (Methods) Weight ) Course Activities (Assignments, group 20% dynamic, Quizzes, Presentations, Portfolio) Midterm Exam 10% Practical Exam 30% Final Exam 40% Total 100% Lecture 1 MICROTECHNIQUES & MICROSCOPES Reference Junqueira's Basic Histology, Text and Atlas. Mescher AL, 15th Edition, 2018. Chapter 1: Pages: Pages: 1-4 & 9-11  Learning Objectives By the end of this Lecture, you should be able to: Define histology & name the basic types of tissues. Enumerate & describe the micro-techniques used in LM. Identify & compare between advantages & disadvantages of different techniques. Identify the most commonly used stains used in histological slides. Define the magnification & resolution power of different microscopes. Describe structure & characters of light and electron microscopes. What is the meaning of histology ? (Histo = tissue, Logia = branch of learning) Histology means study of microscopic structure of the cells, tissues and organs 1. Definite tissue diagnosis Uses of histology 2. Tissue culture 3. Stem cell researches  Organization of the human body 4 3 1 Human 2 body  How can we prepare & examine tissues and organs? Microtechnique Microscopes s MICRO-TECHNIQUES Cell structure is studied in slices of the tissue, called sections. Micro-techniques are the methods by which histological sections are prepared. The most common method is the paraffin technique.  Steps of Paraffin technique 1 2 4 5 Tissue Sampling 3 6 Paraffin block Fixation Dehydration Clearing Impregnation Embedding 7 Sectioning 8 9 Section mounting Section Staining 1-Tissue sampling Small pieces of tissue (1cm). Cut with sharp instrument. It is taken immediately: After death, After operation (biopsy),or From experimental animal.  2-Fixation What is the fixative? Chemical agent to preserve tissues as life-like state. What are the most common fixatives? Formol saline (10%) Bouin’s What are the functions of fixatives? Prevent putrefaction: stop autolysis (post-mortem changes) & kill bacteria. Preserve tissue molecular and morphological structure. Harden the tissue: by coagulation of its proteins. 3-Dehydration 4-Clearing Removal of water (as water is Removal of Alcohol. immiscible with paraffin). Using organic solvent as Xylol It should be gradual (to prevent (Miscible with both alcohol & shrinkage of tissue) paraffin). Using ascending grades of alcohol Tissue becomes transparent (50% , 70%, 90% then absolute (clear). 100%). Before After  5-Impregnation & 6-Embedding In molten soft paraffin (50°C) to In molten hard paraffin wax infiltrate the tissue completely & (55°C) in another oven. to replace xylol. Carried in an oven for 15 min. Placed in a small mould → cooled down to harden forming paraffin block containing the tissue. 7- Sectioning 8-Mounting Using a rotatory microtome with stainless On glass slides smeared with steel blades. Thin section (4 to 10 µm). egg albumin. Sections adhere to one another forming a Now ready to be stained. ribbon.  9. Staining Methods I. Routine stains: Hematoxylin & Eosin (H & E) II. Special stains e.g. Silver, PAS, Sudan III, toluidine blue ……are used to stain different cells and tissue Hematoxylin (H) Eosin (E) (Basophilic) (Acidophilic) Basic stain Acidic stain Blue in color Red to pink in color Binds to acidic Binds to basic components components e.g. Nuclei, e.g. Collagen, proteins Ribosomes and RER Mitochondria , SER  Paraffin technique Advantag Disadvantages es Short time (1-3 days). Xylol dissolve fat content. Thin section, easy to be stained. Heat damages enzymes. Serial sections for research. Not suitable for demonstration of chemical components of cells. Q: MATCHING 1. Fixatio a. Sections on slide smeared with egg albumin. n 2. Clearing b. The tissue is transparent. 3. Embedding c. Preservation of tissue. 4. Mounting d. Hard paraffin. 5. Impregnation e. Removal of water to be replaced by alcohol 6. Dehydration f. Soft paraffin.  Freezing technique (most rapid) Cryostat Tissue sample immediately frozen in liquid nitrogen (temperature -15 to -30) Thick sections cut by cryostat and stained within few minutes. Advantage Disadvantages s of Rapid diagnosis: tumors Thick & fragile sections; not during surgical operation. easily stained. Preserve fat & enzymes. No serial sections. Question No 1 As regarding paraffin micro-technique, which of the following procedures involving immersion of fixed tissues in increasing concentrations of alcohol ? a. Dehydration. b. Staining. c. Impregnantion. d. Mounting. e. Embedding.  Question No 2 Frozen technique may be required to preserve which substances when preparing tissue for paraffin technique? a. Nucleic acids. b. Basic protein. c. Carbohydrates. d. Enzymes MICROSCOPES Micro = very small) & scope = something for looking with Microscope is optical instrument for examination of histological sections Common Types of Microscopes: 1. Light microscope 2. Electron microscope 3. Phase-contrast microscope  MICROSCOPES Some important terms: The magnification power: The degree of enlargement. The resolution power: The least distance between two point that can be seen as two and not as one. Resolution Resolution (R): the smallest distance between two points that could be demonstrated as separate. The maximum average resolution power: By naked eye it is 0.2 mm By light microscope it is up to 0.2 µm By electron microscope it is up to 0.2 nm.  Light microscope Light source: Mirror to reflect day light or electrical lamp. Lenses: 3 Condenser lens gathers & focuses the beam. Magnifying lenses glass lenses: *Objective lenses: enlarging & projecting image (4, 10, 20, 40 & 100) *Eyepiece: further magnifying & projecting to viewer’s eye (5, 10, 15) Light Microscope The magnification power = eye piece power X objective lens power. * The magnification power = 1500 times. * The resolution power = 0.2 µm  Electron Microscope Transmission electron Scanning electron microscope microscope (SEM) (TEM) Transmission electron microscope Electrons (TEM) Light source: → electron beam. Condenser & magnifying lenses → electromagnetic. Ultrathin tissue sections (50 - 100 nm) stained with osmic acid The magnification power = up to 400,000 The resolution power = 0.2 nm Two Dimention images (2D images)  Scanning electron Electrons microscope (SEM) Beam of electrons scans only the surfaces of different body structures as RBCs. Three dimension images (3D images) The magnification power = up to 1000,000 The resolution power= 10 nm Cell & tissue culture Living cells and tissues can be maintained and studied outside the body in culture. Tissue can be examined in the living state without stain by Phase-contrast light microscopy. Uses: Study cell growth (proliferation) & differentiation. Study effect of new drugs on cells. Cultivation of virus & vaccine production.  Any questions? Thank You gu.edu.eg