FUNDAMENTAL TISSUES AND MITOSIS
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1 FUNDAMENTAL TISSUES AND MITOSIS Medical School Histology Larry Johnson Texas A&M University Part I Types of microscopy, tissue preparation, and staining
2 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 From: Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual
3 The Microscope Light (bright field) used with stained specimens
4 Kohler illumination 1. Focus specimen 2. Close field diaphragm 3. Focus condenser 4. Center condenser 5. Open field diaphragm
5 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
6 Bright Field Stained dead cells Phase Contrast Live unstained cells Nomarski Differential interference contrast Dark Field Dark, contrasting background
7 Conventional TEM, SEM carbon replica TEM EM 2b EM 4a Types of EM 6 EM 18b Electron EM 7 Micrographs EM 8
8 Conventional TEM, SEM carbon replica TEM EM 2b EM 4a Types of EM 6 EM 18b Electron EM 7 Micrographs EM 8
9 Conventional TEM, SEM carbon replica TEM EM 2b EM 4a Types of EM 6 EM 18b Electron EM 7 Micrographs EM 8
10 Transmission Electron Microscopy (TEM)
11 Scanning Electron Microscopy (SEM)
12 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
13 EM 6a 200,000x EM 2b 60,000x Compare sizes of membranes ribosomes mitochondria EM 7 80,000x
14 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
15 Sample Preparation 1. Fixation 2. Embedding A. Paraffin B. Plastic 3. Sectioning A. 0.5 um for Light Microscopy B nm for Electron Microscopy
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17 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
18 Hematoxylin and Eosin (H&E) Stain Slide 32: Kidney (H&E)
19 Periodic Acid-Schiff (PAS) Used to observe saccharides (hot pink of brush border and basement membrane) Slide 33: Kidney (PAS)
20 Staining 1. Light Microscopy A. Hematoxylin and Eosin (H&E) B. Periodic Acid/Shiff (PAS) C. Toluidine Blue 2. Electron Microscopy (TEM) A. Osmium B. Lead Citrate
21 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
22 Staining 1. Light Microscopy Hematoxylin and Eosin (H&E)) B. Periodic Acid/ (PAS) 2. Electron Microscopy (TEM) Lead A. Osmium citrate B. Lead Citrate
23 Artifacts Artifacts are man-made distortions and aberrations of tissue structure Artifacts are fairly common, beware! Slide 32: Kidney Artifacts
24 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 difiore s Atlas of Histology with Functional Correlations Victor P. Eroschenko
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26 FUNDAMENTAL TISSUES AND MITOSIS Medical School Histology Part II Images of light and electron microscopy
27 Objectives 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) Part II 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 From: Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual
28 Laboratory Experience Primary purpose of this lab is to gain a general impression of similarities and differences in cells and tissues and what can be inferred from that Do not be too concerned about detailed structure, but rather try to gain an overall appreciation for histology
29 Slide 29: Skin Epithelial skin cells Collagen fibers
30 Slide 29: Skin
31 Slide 51: Tongue (cross section) Skeletal muscle Serous glands Adipocytes
32 Slide 51: Tongue (cross section) Serous glands Adipocytes Skeletal muscle
33 Slide 32: Kidney Proximal convoluted tubules with microvilli lining lumen
34 Slide 75: Thyroid Spherical epithelial follicles containing thyroglobulin Simple cuboidal epithelium Extracellular collagen fibrils Blood vessel with red blood cells Simple squamous epithelium lining blood vessel
35 Slide 70: Pancreas Zymogen granules Pancreatic acinus Glandular cell with euchromatic nucleus and basophilic base
36 Endoplasmic Reticulum Granular ER Granular ER Nuclear envelope Granular ER Nuclear pore Nucleolus Nuclear pore Nucleus Nuclear envelope
37 Granular vs. Agranular Endoplasmic Reticulum Granular endoplasmic reticulum Agranular endoplasmic reticulum
38 Plasma Membrane What is the structure of the plasma membrane?
39 Plasma Membrane
40 Plasma Membrane Identify pinocytotic vesicles
41 Mitochondria Identify mitochondria
42 Mitochondria What is the structure and function of mitochondria?
43 Mitochondria & Microbodies Could mitochondria and microbodies be confused morphologically? What is the structure and function of microbodies?
44 Golgi complex Observe the ultrastructure of the Golgi complex
45 Digestion and Storage Vesicles Lipid droplets Secretory granules Lysosomes
46 Digestion and Storage Vesicles Lipofuscin granules Microbodies Glycogen Glycogen
47 Cytoskeleton Microtubules Axonemes encased in membrane Centrioles
48 Cytoskeleton The microvilli in this micrograph contain what type of cytoskeletal element? Hint: What is supporting the microvilli?
49 Clinical Correlation Cell death can occur due to apoptosis or necrosis Apoptosis Programmed cell death Necrosis Accidental cell death resulting from pathological condition Max Planck Institute for Neurological Research
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51 FUNDAMENTAL TISSUES AND MITOSIS Medical School Histology Part III Images of mitosis
52 Objectives 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 Part III Identify stages of mitosis and cellular structures associated with mitosis From: Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual
53 Mitosis Mitosis is cellular multiplication This is the means by which organisms grow Cancer is essentially uncontrolled mitosis We will look at onion root tip and fish egg cells to identify the stages of mitosis
54 Slide 1: Onion root tip Interphase Interphase nucleus
55 Slide 1: Onion root tip Prophase
56 Slide 1: Onion root tip Metaphase Spindle fibers
57 Slide 1: Onion root tip Anaphase Centrosomal area
58 Slide 1: Onion root tip Telophase
59 Slide 2: Fish egg mitosis Interphase Interphase nucleus
60 Slide 2: Fish egg mitosis Prophase
61 Slide 2: Fish egg mitosis Metaphase Spindle fibers
62 Slide 2: Fish egg mitosis Anaphase Centrosomal area
63 Slide 2: Fish egg mitosis Telophase
64 EM 25 & 26 Describe the composition of the nucleolus and the nucleus. What happens to the nucleolus and nuclear membrane (envelope) during mitosis?
65 EM 31 What is the function of centrioles?
66 Clinical Correlation Malignant vs. benign tumors Increased mitotic figures and abnormal mitoses helps distinguish Anti-mitotic drugs are used in cancer chemotherapy Ex: Taxol These drugs also effect normal cells with high proliferation rates causing undesirable consequences
67 Study Questions 1. Does a highly active cell contain a euchromatic or heterochromatic nucleus? 2. Would the cytoplasm of a cell that contains large quantities of protein stain eosinophilic or basophilic? 3. Is a rounded (spherical) nucleus indicative of a squamous, cuboidal or columnar cell? 4. Does hematoxylin preferentially stain chromatin or collagen? (2-3 sentences) 5. Are polysaccharides and mucopolysaccharides best stained with eosin, Sudan black, hematoxylin, or periodic acid Schiff? 6. At which stage of mitosis do sister chromatids separate from each other? 7. During which phase of interphase does the first checkpoint occur? What is the significance of the p53 protein? From: Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual
68 Use your atlas! Use your atlas! pancreas blood Atlas of Normal Histology by DiFiore stomach testis
69 Many illustrations in these VIBS Histology YouTube videos were modified from the following books and sources: Many thanks to original sources! Bruce Alberts, et al Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY. Bruce Alberts, et al Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY. William J. Banks, Applied Veterinary Histology. Williams and Wilkins, Los Angeles, CA. Hans Elias, et al Histology and Human Microanatomy. John Wiley and Sons, New York, NY. Don W. Fawcett Bloom and Fawcett. A textbook of histology. W. B. Saunders Company, Philadelphia, PA. Don W. Fawcett Bloom and Fawcett. A textbook of histology. Chapman and Hall, New York, NY. Arthur W. Ham and David H. Cormack Histology. J. S. Lippincott Company, Philadelphia, PA. Luis C. Junqueira, et al Basic Histology. Lange Medical Publications, Los Altos, CA. L. Carlos Junqueira, et al Basic Histology. Appleton and Lange, Norwalk, CT. L.L. Langley, et al Dynamic Anatomy and Physiology. McGraw-Hill Book Company, New York, NY. W.W. Tuttle and Byron A. Schottelius Textbook of Physiology. The C. V. Mosby Company, St. Louis, MO. Leon Weiss Histology Cell and Tissue Biology. Elsevier Biomedical, New York, NY. Leon Weiss and Roy O. Greep Histology. McGraw-Hill Book Company, New York, NY. Nature ( Vol. 414:88,2001. A.L. Mescher 2013 Junqueira s Basis Histology text and atlas, 13 th ed. McGraw Douglas P. Dohrman and TAMHSC Faculty 2012 Structure and Function of Human Organ Systems, Histology Laboratory Manual - Slide selections were largely based on this manual for first year medical students at TAMHSC
70 The End!
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