Introduction to Histology Al-Maarefa College

Objectives Understand what is Histology Realize the size of histological tissues Know basic steps in tissue preparation and staining Understand the importance of different stains Identify different techniques of imaging in histology

What is Histology? The name "Histology" is derived from Greek: "Histos“: tissue "logos" : the study of Histology: the study of tissues

What is Histology? The study of the microscopic anatomy of cells and tissues How? Examining a thin slice (section) of tissue under a light microscope or electron microscope Enhanced by using different histological stains

Levels of body organization Cells: The smallest independently living highly complex structure Tissues: Functional unit: group of cells of similar function and origin (with matrix between cells) Organs: Several tissues grouped together Systems: Several organs working for a common function

Levels of body organization

Tissues Tissues are made of: cells and extracellular matrix Intense interaction between cells and matrix Cells and extracellular matrix form a continuum that functions together and reacts to stimuli and inhibitors together

Tissues Four fundamental tissues are recognized: Epithelial tissue Connective tissue Muscular tissue Nervous tissue

The size spectrum The small size of cells and matrix components makes histology dependent on the use of microscopes

equal to 10−9 m (a billionth of a meter) The size spectrum Electron Microscope 0.1 nm equal to 10−9 m (a billionth of a meter) Light Microscope 300 nm Unaided Eye 300 um 1/1000 of a millimetre, or 0.001mm

Light Microscopy With a maximum magnification power of x 1000 times

Electron Microscopy With a maximum magnification power of x 1000000 times

Electron Microscopy Bronchiole, epithelial cells, X 5000 Surface of epithelial cell, X 100,000 www.visualhistology.com

Basic Techniques Preparation of histological sections Fixation Processing (dehydration and clearing) Embedding Cutting Staining Permanent Mounting

Preparation of histological sections Fixation Fixing tissues by chemicals so they will not change their volume and shape during processing Keeps tissue as close to their living state as possible prevents autolysis and bacterial attack prepares tissues for staining Fixatives: Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde, Methanol and Picric acid.

Preparation of histological sections Dehydration and clearing removes fixative and water from the tissue and replace them with dehydrating fluid Uses hydrophilic or alcohol solutions to extract water Examples of solutions: Ethanol, Methanol, Acetone

Preparation of histological sections Embedding is the process by which tissues are embedded in a medium (agar, gelatin, or wax) which when solidified provides sufficient external support during sectioning

Preparation of histological sections Cutting Small (micro) sections are cut by a Microtome Section thickness 2 to 25 micrometers=um thick for light microscope 60 to 100 nanometers thick for electron microscopy

Preparation of histological sections Staining Various stains are used to see and highlight specific structures and molecules

Preparation of histological sections Staining Various stains are used to see and highlight specific structures and molecules

Hematoxylin and Eosin (H & E) H & E stains are universally used for routine histological examination of tissue sections.

Staining Techniques Hematoxylin and Eosin (H&E) Hematoxylin is a basic dye that stains acidic components of cells a blue (basophilia) Stains the nuclei of cells, and the rER of cytoplasm

Staining Techniques Hematoxylin and Eosin (H&E) Eosin is an acidic dye that stains the basic components of cells reddish-pink (acidophilia) Most of cytoplasm of cells is stained by eosin

Hematoxylin and Eosin (H & E) Nuclei - blue - with some metachromasia Cytoplasm - various shades of pink-identifying different tissue components

Features of H&E Hematoxylin Eosin Blue colored Positively charged (colors negatively charged molecules, such as DNA, rER) The ability of such anionic groups to react with a basic dye - basophilia Eosin Red colored Negatively charged (colors positively charged molecules (amino acids, cytoplasm) The ability of such cationic groups to react with an acidic dye - acidophilia

Staining Techniques Periodic acid-Schiff (PAS) staining mucus, basal lamina, glycogen (carbohydrates) H&E stain does not preserve Mucous in Goblet cells, which Appear empty PAS stain preserves Mucous in Goblet cells, which and stains it with a magenta colour Wheater’s Functional Histology, a text and colour atlas Junqueira and Carneiro, Basic Histology, a text and atlas

Staining Techniques Periodic acid-Schiff (PAS) staining mucus, basal lamina, glycogen (carbohydrates) Glycogen

Staining Techniques Periodic acid-Schiff (PAS) staining mucus, basal lamina, glycogen (carbohydrates) Carbohydrate content in liver tissue stained magenta with PAS stain Clarke F. Millette, Univ. of South Carolina, USA

Staining Techniques Toluidine blue (Metachromatic stain) Blue stain that stains specific components of tissues a purple color Metachromasia is seen in the matrix of hyaline cartilage, or in the granules of mast cell IJDVL, Omar El Safoury Science Photo Library Blue stain of Hyaline cartilage Metachromasia of Mast cells, skin

Staining Techniques Oil Red O Sudan black Stain lipids red-orange in unfixed frozen sections Sudan black Stains lipids black in unfixed frozen sections

Staining Techniques Impregnation Silver impregnation techniques are also widely used to demonstrate reticular fibers Lymph node, Silver Impregnation Seoul National University

Staining Techniques Reticulin Reticular fibers are stained dark. Used esp. in liver Smooth Muscle, Reticulin stain Clarke F. Millette, Univ. of South Carolina, USA

A liver biopsy stained using the reticulin demonstrating the normal hepatic plate thickness.

Plasmodiun Falciparum (Malaria) Staining Techniques Giemsa stain Blood smears, for disease Wuchereria bancrofti Causing filariasis Plasmodiun Falciparum (Malaria) http://parasitewonders.blogspot.com/ www.sciencebuzz.org

Plasmodiun Falciparum (Malaria) Staining Techniques Giemsa stain Blood smears, for disease Trypanosoma sp. Plasmodiun Falciparum (Malaria) CDC /Dr. Myron G. Schultz 1970 www.sciencebuzz.org

Preparation of histological sections Mounting preserves and supports a stained section mounted on a clear glass slide, and covered with a thin glass cover-slip

Basic Techniques Total preparation time: 16 hours Immediate Frozen section time: 5 minutes For immediate results – (in tumor surgery) Results not as accurate as formalin fixed, wax embedded specimens

Basic Techniques In some cases the tissue to be examined is a very thin membrane (Cell Smears) blood or bone marrow

Basic Techniques In some cases the tissue to be examined is a very thin membrane (Cell Smears) blood or bone marrow http://hepatitiscresearchandnewsupdates.blogspot.com

Basic Techniques In some cases the tissue to be examined is a very thin membrane (Cell Smears) blood or bone marrow epithelial cells (e.g. from the oral cavity, cervix uteri) Crvical carcinoma, PAP stain Univ of Uklahoma Health Science Center NATIONAL CANCER INSTITUTE / SCIENCE PHOTO LIBRARY

Some definitions Histology: Biopsy: Autopsy: microscopic study of tissues Biopsy: removal of tissues for diagnostic purposes Autopsy: examination of organs of a dead body (to find clues to determine cause of death)

Phase-Contrast Microscopy

Phase-Contrast Microscopy An optical microscopy illumination technique A small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. Does not require staining to view the slide Epithelial cell from cheek

Polarizing Light Microscopy Polarization is a property of Electromagnetic waves, such as light that describes the orientation of their oscillations Polarizer is rotated to transmit the reflections as well as possible By rotating the polarizer by 90° , almost all reflected sunlight is blocked 

Polarizing Light Microscopy Polarization is a property of Electromagnetic waves, such as light that describes the orientation of their oscillations

Polarizing Light Microscopy Collagen fibers Yellow birefringence

Fluorescence Microscopy A sample is illuminated with light of a one wavelength which causes fluorescence in the sample. The light emitted by fluorescence, which is at a different, longer, wavelength than the illumination, is then detected through a microscope objective

Fluorescence Microscopy Epifluorescent imaging of the three components in a dividing human cancer cell. DNA is stained blue, a protein called INCENP (Inner centromere protein) is green, and the microtubules are red. Each fluorophore is imaged separately using different filters, and images are captured sequentially using a digital CCD camera( charge coupled device), then overlaid to give a complete image.

Fluorescence Microscopy

Scanning Electron Microscopy Clarke F. Millette, Univ. of South Carolina, USA

Scanning Electron Microscopy Bruce Wetzel/Harry Schaefer, courtesy National Cancer Institute

Scanning Electron Microscopy

Scanning Electron Microscopy

Scanning Electron Microscopy Cut end of human hair Catchrandom.blogspot

Scanning Electron Microscopy Human skin

Summary What is histology The size spectrum and organizational structure Light microscope / Electron microscope

Summary Preparation of tissue Different stains and special techniques fixation processing embedding cutting staining mounting Different stains and special techniques

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