Download Introduction to Histology

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 Eye
300 um
The size spectrum
Light Microscope
300 nm
Unaided Eye
300 um
The size spectrum
Electron Microscope
0.1 nm
Light Microscope
300 nm
Unaided Eye
300 um
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
www.visualhistology.com
Surface of epithelial cell, X 100,000
Basic Techniques
• Preparation of histological sections
1.
2.
3.
4.
5.
6.
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
– 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
Wheater’s Functional Histology, a text and colour atlas
PAS stain preserves Mucous in
Goblet cells, which and stains it
with a magenta colour
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
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.
Staining Techniques
• Giemsa stain
– Blood smears, for disease
Trypanosoma sp.
CDC /Dr. Myron G. Schultz 1970
Plasmodiun Falciparum (Malaria)
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
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:
– 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
– fixation
– processing
– embedding
– cutting
– staining
– mounting
• Different stains and special techniques
Thank you