Part I Types of Microscopy, Tissue Preparation, and Staining

FUNDAMENTAL TISSUES AND MITOSIS Part I Types of microscopy, tissue preparation, and staining Objectives Part I • Operate the “virtual microscope” • Describe steps for tissue processing and discuss different types of microscopy • Interpret cytological significance of differential staining by hematoxylin and eosin (H&E) • Recognize and identify cells in microscopic tissue preparations • Understand that morphology reflects the function of cells • Identify stages of mitosis and cellular structures associated with mitosis The Microscope Light (bright field) used with stained specimens Kohler illumination 1. Focus specimen 2. Close field diaphragm 3. Focus condenser 4. Center condenser 5. Open field diaphragm Types of Microscopy • Light (bright field) • Most common, used to observe stained sections. • Phase Contrast • Used to observe living cells, cells appear as black and white image • Nomarski (differential interference) • Similar to phase contrast, but greater resolution of 3D appearance and optical sectioning • Dark Field • Illuminates cells and tissues against dark background to allow for contrast • Transmission Electron Microscopy (TEM) • Magnifies image to allow visualization of intracellular structures • Scanning Electron Microscopy (SEM) • Scans cell surfaces at great magnifications providing highly resolved surface details • Bright Field • Stained dead cells • Phase Contrast • Live unstained cells • Nomarski • Differential interference contrast • Dark Field • Dark, contrasting background Conventional TEM, SEM carbon replica TEM EM 2b EM 4a EM 18b EM 6 Types of Electron Micrographs EM 7 EM 8 Conventional TEM, SEM carbon replica TEM EM 2b EM 4a EM 18b EM 6 Types of Electron Micrographs EM 7 EM 8 Conventional TEM, SEM carbon replica TEM EM 2b EM 4a EM 18b EM 6 Types of Electron Micrographs EM 7 EM 8 Transmission Electron Microscopy (TEM) Scanning Electron Microscopy (SEM) Transmission Electron Microscopy (TEM) Comparing Magnifications EM 8f 9,000x EM 12a 13,200x Compare the magnification by comparing sizes of • membranes • ribosomes • mitochondria EM 4c 60,000x EM 7 80,000x EM 6a 200,000x EM 2b 60,000x Compare sizes of • membranes • ribosomes • mitochondria EM 7 80,000x Tissue Preparation 1. Fixation – Chemically prevents autolysis of cells and preserves morphology 2. Dehydration – organic solvents 3. Embedding –paraffin or plastic used to stiffen tissue 4. Sectioning – cutting thin sections (1-10 microns thick) 5. Staining – different stains react with different biochemical components of cells 6. Mounting – preserve section 7. Histological viewing – using microscope Sample Preparation 1. 2. Fixation Embedding A. B. 3. Paraffin Plastic Sectioning A. 0.5 um for Light Microscopy B. 60-80 nm for Electron Microscopy Differential Staining Properties Hematoxylin and Eosin (H&E) are the most widely used stains • Hematoxylin • Blue-black dye with basic pH, preferentially binds acid molecules • Nuclear DNA and cytoplasmic ribosomes darkly stain with hematoxylin based on density of material Dark blue nuclei of lymphocytes • Eosin • Red dye with acidic pH, preferentially binds basic molecules • Proteins (amines) stain with eosin based on density of material Red cytoplasm of smooth muscle cells Hematoxylin and Eosin (H&E) Stain • Kidney (H&E) Periodic Acid-Schiff (PAS) • Used to observe saccharides (hot pink of brush border and basement membrane) • Kidney (PAS) Staining 1. Light Microscopy A. B. C. 2. Hematoxylin and Eosin (H&E) Periodic Acid/Shiff (PAS) Toluidine Blue Electron Microscopy (TEM) A. B. Osmium Lead Citrate Staining 1. Light Microscopy A. Hematoxylin and Eosin (H&E) B. Periodic Acid/Shiff (PAS) Color provides clues C. Toluidine Blue Shape Size Intensity of staining Staining 1. Light Microscopy Hematoxylin and Eosin (H&E)) B. Periodic Acid/ (PAS) 2. Electron Microscopy (TEM) Lead citrate A. Osmium B. Lead Citrate Artifacts • Artifacts are man-made distortions and aberrations of tissue structure • Artifacts are fairly common, beware! • Kidney Artifacts Identifying Cells • Cells < Tissues < Organ < Organ System < Human • Somatic vs. Germ Cells • Morphology accomplishes specific function • Distinct features of cell • Nucleus: shape is indicative of cell morphology • Cytoplasm: Contains various organelles such as RER, Golgi, and mitochondria