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Histology Intro

• Histology comes from Greek words: "Histos" meaning web/tissue, and "Logos" meaning the study of.
• Histology is used not only for the study of tissue alone, but also to observe cells and fine structures of organs at the microscopic level
• Histology is also known as Microscopic Anatomy
• Histology provides a structural basis for functional correlation of organs or tissues
• It serves as a prerequisite for studying pathology (abnormal tissue).

General Architecture of the Body

• The body is organized in a hierarchical manner:
  • Cells -> Tissues -> Organs -> Systems -> Body

Tissue Basics

• Tissues are composed of specialized cells and intercellular matrix
• There are four basic types of tissues in the body, each with specific functions:
  • Epithelial tissue: protection
  • Connective tissue: support
  • Muscular tissue: contraction
  • Nervous tissue: conduction

Measurement Units in Histology

• For light microscopy, the term micrometer (µm) is now used instead of micron (μ).
  • 1 micrometer/micron = 0.001 mm or 10^-6 m
• For electron microscopy, the term nanometer (nm) is now used instead of angstrom (A°).
  • 1 nanometer = 0.001 µm or 10^-9 m
  • 1 angstrom = 0.1 nm or 10^-10 m

Section Interpretation

• Microscopic study usually involves observing 2D sections of 3D tissue structures
• Serial sectioning prepares tissue in a sequential order to get information about 3D architecture

Processing Tissues for Light Microscopy (Paraffin Wax Embedding)

• Tissues are processed to obtain thin sections for microscope examination via transillumination
• The procedure involves:
  • Fixation, Dehydration, Clearing, Embedding, Section Cutting (Microtomy)

Fixation and Fixatives

• Substances like formalin, mercuric chloride, acetic acid, picric acid, and glutaraldehyde act as fixatives to preserve tissues
• All fixatives have both desirable and undesirable effects
• A combination of fixatives is often prepared to maximize desirable effects
• Commonly used fixative combinations include:
  • Bouin's fluid (formalin, acetic acid, and picric acid)
  • Formal sublimate (formalin and mercuric chloride)
  • Helly's fluid (formalin, mercuric chloride, and potassium dichromate)
  • Zenker's fluid (acetic acid, mercuric chloride, and potassium dichromate)
• Small pieces of fresh tissues are placed in common fixatives like 10% neutral formal saline for 24 hours
• The purpose of fixation is to:
  • Preserve morphology and chemical composition, preventing autolysis and putrefaction
  • Harden the tissue for easy manipulation
  • Solidify colloidal material
  • Influence Staining

Decalcification

• Hard tissues (bone, tooth) containing calcium salts require decalcification after fixation before dehydration
• Decalcification makes hard tissues soft for microtome cutting
• Decalcifying agents include:
  • 10% nitric acid
  • 5% trichloroacetic acid
  • Ethylene diamine tetra acetic acid (EDTA).

Dehydration

• Water is gradually removed from tissues by immersion in ascending grades of alcohol (50%, 70%, 90%, absolute alcohol)
• This prepares the tissue for paraffin wax embedding, as paraffin wax is not miscible in water
• Tissue remains in each grade for 30-60 minutes

Clearing

• After dehydration, tissues are treated with paraffin solvents (clearing agents) like xylene or toluene for 2-3 hours
• These agents penetrate and replace the alcohol, making the tissue translucent

Embedding

• Tissues must be infiltrated to create a rigid consistency and prepare for thin sections with a microtome
• Various embedding media exist, including paraffin wax, celloidin, gelatin, and plastic resins (for EM)
• Paraffin is the embedding medium routinely used medium for for light microscopy
• Embedding involves two steps:
  • Impregnation
  • Casting or block making

Impregnation

• Following clearing, tissue is impregnated with molten paraffin wax (58°-60°C) in a hot air oven for 2 hours with 3 changes
• The melting point of paraffin wax is 56°C

Casting or Block Making

• Impregnated tissue is placed in 'L' moulds containing molten paraffin
• Molten wax cube cools with the tissue so that the paraffin block can then be removed from the mould

Section Cutting (Microtomy)

• 5-7 µm-thick sections are cut with a rotary microtome
• Cut paraffin sections are affixed to albuminised glass microslides after flattening on warm water
• Microslides with sections are either air-dried or incubated overnight at 37°C and then stored at room temperature

Staining Procedure

• Done with basic and acidic dyes to stain tissue components selectively

Basophilic Components:

• Tissue components stain more readily with basic dyes and are blue

Acidophilic Components:

• Tissue components have an affinity for acid dyes and are pink/orange

Stain Examples

• Hematoxylin & Eosin (H&E) is the most used stain
• Periodic Acid Schiff (PAS) reagent
• Osmic acid
• Mallory and Masson's
• Trichrome stain

Staining Procedure Steps

• Deparaffinization, Hydration, Staining, Dehydration, Clearing and Mounting

Deparaffinization

• Xylol is used to remove the paraffin from the section, using three changes for 3-5 minutes each

Hydration

• Slides are passed through the following series to hydrate the sections:
  • Absolute alcohol – 5 min (2 changes), 90% alchohol (3 min), 70% alchohol (3 min), 50% alchohol (3 min), Distilled water (3 min)

Staining

• The technique used is differential staining
• This involves: A staining with haematoxylin for 5-7 minutes, washing with tap water, differentiation with acid/alcohol for 5 seconds, repeated washing, and using 1% eosin for a minute

Dehydration

• Stained sections are dehydrated in the following series:
  • 50% alcohol – 10 sec
  • 70% alcohol – 10 sec
  • 90% alcohol – 30 sec
  • Absolute alcohol – 5 min (2 changes)

Clearing and Mounting

• Sections are cleared in xylene and mounted in DPX

Examples of Dyes

Basic Dyes

• Toluidine Blue
• Alcian Blue
• Methylene Blue
• Hematoxylin dyes are known for staining tissue components with net negative or acidic structures (ex: DNA, RNA, Glycosaminoglycans)

Acidic Dyes

• Eosin
• Orange G
• Acid Fuchsin
• Acidic dyes stain the structures with a positive charge or basic components in the tissue (ex: mitochondria, secretory granules, and collagen)

Special Stains and What They Stain

• Feulgen stain highlights DNA (Red against a Green background),
• Periodic Acid Schiff stain highlights Glycogen (Purple)
• Sudan Black highlights Lipids, which turn from Blue to Black
• Gram Stain is used to differentiate Gram- with Pink and Gram+ bacteria which appear purple/blue

Microscopy Basics

• Once stained with haematoxylin and eosin (H&E) or special stains, sections are viewed with a light microscope

Dark Ground Microscope

• A modified light microscope for examining objects using dark ground illumination
• A small circle of black paper is inserted in the center of the filter carrier of the condenser for illumination
• Used to examine minute particles (colloid suspension) or large transparent objects (living protozoa, crystals)

Phase-Contrast Microscope

• Relies on the refractive index
• Phase differences are transformed into differences of light intensity to see structures within the cells
• Used to view transparent living biological specimens without staining.

Polarizing Microscope

• Modified LM uses 2 filters: Polarizer and Analyzer
• Polarizer and analyzers are kept at right angles, which gives a darkfield effect because no light can pass
• Structures with a linear (bones, muscle, collagen, nerve fibres) or radial fashion (lipid droplets, starch granules), appear as bright structures because they rotate light's polarized vibration
• Birefringency is the capacity to rotate the direction of polarized light vibration and is typical for crystal substances

Electron Microscopes

• These use shorter electron wavelengths instead of light rays
• Can achieve a very high resolution, as low as 3Å, to view cells and organelles in details
• Electrons are deflected/scattered by electromagnetic lenses, similar to light deflection by glass lens of optical microscope

TEM (Transmission Electron Microscope) vs SEM (Scanning Electron Microscope)

Transmission Electron Microscope (TEM)

• Utilizes the transmitted electrons that penetrate the specimen
• These electrons are focused by an objective coil or lens, then enlarged by one or two projector coils/lenses and are projected on a screen/photographic film to produce an electron micrograph

Scanning Electron Microscope (SEM)

• The electrons cannot pass through the specimen because it is thick and coated with heavy metals (e.g. gold)
• Utilizes only what's reflected/backscattered, the secondary electrons that are deflected because of the coated surface by the electrons
• Collected by special detectors to send electrical signals to a television tube which gives a 3D image of the specimen
• This is used when the surface topography of a sample is needed
• This has less resolution than TEM