Introduction and Methods of Histology PDF

Microanatomy and embryology Lecture modified from Dr. M. Smith RUSVM Sep 2007 Dr. M. Zibrin; Dr. L. Bogdanovic RUSVM 2010, 2011, Dr. C. Fuentealba Sep 2011, Callanan/Bolfa May 2015 Matthew J Valentine BVMS MRCVS PhD Diplomate ACVP ©2021 Ross University School of Veterinary Medicine. All rights reser...

Microanatomy and embryology Lecture modified from Dr. M. Smith RUSVM Sep 2007 Dr. M. Zibrin; Dr. L. Bogdanovic RUSVM 2010, 2011, Dr. C. Fuentealba Sep 2011, Callanan/Bolfa May 2015 Matthew J Valentine BVMS MRCVS PhD Diplomate ACVP ©2021 Ross University School of Veterinary Medicine. All rights reserved. Microanatomy and early embryology MICROANATOMY / MICROSCOPIC ANATOMY = HISTOLOGY The study of the cells and tissues of the body and how these integrate to form organs EMBRYOLOGY / DEVELOPMENTAL BIOLOGY The study of the development of a new individual (development of embryo and fetus) LIVER: GROSS ANATOMY CAN BE SEEN WITH THE NAKED EYE VERSUS MICROANATOMY WHICH REQUIRES A MICROSCOPE MICROANATOMY IN CLINICAL VETERINARY MEDICINE CYTOLOGY - study of the structure and function of cells e.g., vaginal smear for estrus detection in canines CYTOPATHOLOGY - study of diseased cells from fluids or tissues e.g., fine needle aspiration of masses (lumps and bumps) HISTOPATHOLOGY - study of diseased tissues e.g., liver biopsy or intestinal biopsy WHICH IS BEST? LIGHT MICROSCOPY (LM) Light beam is transmitted through a tissue. Examples: – – – – – Bright field microscopy Fluorescence Phase-contrast Polarizing Dissecting Stereomicroscope BRIGHT FIELD MICROSCOPY: review the guides on canvas Requires staining to provide contrast Digital scanners have same objectives to produce a digital image PHASE CONTRAST MICROSCOPY Observation of LIVING non-stained structures e.g. cell and tissue culture, spermatozoa, leukocytes, etc. Dense structures have higher refractive index FLUORESCENCE MICROSCOPY Fluorescent dyes stain specific cell components visualized when viewed under ultraviolet light. Example: Cell culture of kidney cells. Blue fluorescence binding to nuclear DNA. Green fluorescent dye binds to actin filaments POLARIZED MICROSCOPY Adding a polarizing filter to a bright field microscope highlights birefringent materials Bright field microscopy: collagen fibers, thin dark elastic fibers and cell nuclei H&E. e.g. crystalline materials Junqueira, 2010 Polarized light microscopy: collagen fibers are bright red or yellow; elastic fibers and nuclei are not detected DISSECTING STEREOMICROSCOPES  ADVANTAGES: Relatively inexpensive Practical / Versatile Can provide a 3-dimensional image Can be used in microsurgery and with other types of specimens  DISADVANTAGES: Low resolving power Needs to be maintained ADVANTAGE and DISADVANTAGES OF LIGHT MICROSCOPY ADVANTAGES DISADVANTAGES Relatively inexpensive Image is 2-dimensional Provides rapid diagnosis Resolving power is limited by Allows observation of living specimens Resolving power of LM is 0.2μm the wavelength of light Requires maintenance Requires expertise for proper diagnosis (quality analysis and quality control) TRANSMISSION ELECTRON MICROSCOPY (TEM) Based on interaction of electrons and tissue components. The wavelength in the electron beam is shorter than the light beam; resulting in 1,000-fold increase in resolution Sperm tail cross section TEM 50,000x ADVANTAGES versus DISADVANTAGES OF TEM  ADVANTAGES Great resolving power 0.16 – 0.18 nanometres Very useful for rapid diagnosis of viruses and other microscopic organisms & storage diseases  DISADVANTAGES Image is 2-dimensional Image is black & white Cannot be used in living object Very expensive SCAN ELECTRON MICROSCOPY (SEM) Electron beam scans surface (3D effect). Only external structures. Lower resolution than TEM 3D structure of sperm cells and surface of uterine epithelial ciliated and secretory cells ELECTRON MICROSCOPY: TEM versus SEM Hepatocyte: TEM Hepatocytes around central vein: SEM TEM: Transmission Electron Microscopy SEM: Scanning Electron Microscopy RETRIEVING TISSUE FOR MICROSCOPIC EXAMINATION Biopsy: sample of tissue from a living animal Tissue sample/biospecimen: sample of tissue or whole organ from dead animal Images: Carolina Guerrero© Trim to 1 cm3 Place in 10 times volume of 10% formalin fixative OBSERVATION IN MICROSCOPY For light microscopy and electron microscopy the specimen must: be well preserved = retain structure and molecular composition be sufficiently thin to allow light or electron transmission have enough contrast to observe details PRESERVATION OF TISSUE STRUCTURE BY FIXATION AND EMBEDDING 10% buffered formalin (hazardous) coagulates proteins in a life like manner Fixation 10% formalin Ascending % of alcohol Liquid Xylene removes alcohol Paraffin wax Solid A MICROTOME SLICES EMBEDDED TISSUE INTO THIN SECTIONS MICROTOME Slices are cut into 1 to 7 micrometers (µm) thin sections Sections are floated on water for retrieval and staining TISSUE SECTIONS ARE STAINED TO PROVIDE CONTRAST Hematoxylin – Eosin (H&E) are the most common stains used for routine evaluation HAEMATOXYLIN AND EOSIN Lymph node HAEMATOXYLIN =BASIC STAIN -DNA AND RNA BLUE ‘BASOPHILIC’ Skeletal muscle EOSIN =ACIDIC STAIN -PROTEINS PINK ‘EOSINOPHILIC’ SPECIAL STAINS IDENTIFY SPECIFIC TISSUES, SUBSTANCES OR STRUCTURES Masson’s trichrome stains collagen green and smooth muscle grey Mallory’s trichrome stains collagen blue and nuclei red PERIODIC ACID SCHIFF (PAS) IS A SPECIAL STAIN THAT LOCALISES GLYCOGEN, GLYCOPROTEINS AND MUCINS H&E stain: goblet cells are pale PAS stain: goblet cells stain magenta ENZYMES IN CELLS CAN ALSO BE STAINED (ENZYME HISTOCHEMISTRY) Gomori’s method stains alkaline phosphatase in the brush border of proximal convoluted tubules of the kidney black ANTIBODIES LABELLED WITH FLOURESCENT DYES OR ENZYMES BIND TO ANTIGENS IN IMMUNOHISTOCHEMISTRY IMMUNOHISTOCHEMISTRY IS HIGHLY SPECIFIC Glucagon and insulin within the cells of the pancreatic Islets of Langerhans are stained with antibodies labelled with a visible stain Hematoxylin-eosin A cells - Glucagon B cells - Insulin