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

The size spectrum

Unaided Eye300 um

The size spectrum

Unaided Eye300 um

Light Microscope300 nm

The size spectrum

Unaided Eye300 um

Light Microscope300 nm

Electron Microscope0.1 nm

Light MicroscopyWith a maximum magnification power of x 1000 times

Electron MicroscopyWith a maximum magnification power of x 1000000 times

Electron Microscopy

Surface of epithelial cell, X 100,000Bronchiole, epithelial cells, X 5000

www.visualhistology.com

Basic Techniques• Preparation of histological sections

1. Fixation2. Processing (dehydration and clearing)3. Embedding4. Cutting5. Staining6. 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– 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 preserveMucous in Goblet cells, which

Appear empty

PAS stain preserves Mucous in Goblet cells, which and stains it

with a magenta colourWheater’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)

Clarke F. Millette, Univ. of South Carolina, USA

Carbohydrate content in liver tissue stained magenta with PAS stain

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

Metachromasia of Mast cells, skinScience Photo Library

Blue stain of Hyaline cartilage

Staining Techniques • Oil Red O

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

Seoul National UniversityLymph node, Silver Impregnation

Staining Techniques • Reticulin

Reticular fibers are stained dark. Used esp. in liver

Clarke F. Millette, Univ. of South Carolina, USASmooth Muscle, Reticulin stain

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

Staining Techniques • Giemsa stain– Blood smears, for disease

www.sciencebuzz.org

Plasmodiun Falciparum (Malaria)Trypanosoma sp.

CDC /Dr. Myron G. Schultz 1970

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

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)

Univ of Uklahoma Health Science Center NATIONAL CANCER INSTITUTE / SCIENCE PHOTO LIBRARYCrvical carcinoma, PAP stain

Some definitions• Histology:– 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

Catchrandom.blogspot

Cut end of human hair

Scanning Electron Microscopy

Human skin

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

Summary• Preparation of tissue– fixation– processing– embedding– cutting– staining– mounting

• Different stains and special techniques

Thank you