Histology: An Introduction
Diane Hickson-Bick, PhD
Learning Objectives:
 Understand the basic concepts of microscopy
 Understand the techniques used for histological sample preparation.
Key Words: Microscopy, Resolution, H & E
HISTOLOGY: Histos = tissue; ology = knowledge. Histology is the study of cells and tissues and their
arrangement in the constitution of organs.
Tissue: Composed of cells + extracellular matrix.
4 fundamental tissues:
 Epithelia
 Connective Tissue
 Muscle tissue
 Nervous tissue
Microscopy: Small size(s) makes histology dependent on the use of microscopy.
Units:
 1μm=0.001mm
 1nm=0.001μm (10-3μm)
 Angstrom (Å = 1 x 10-7mm)
Resolution: Smallest distance between two particles when the particles can be discerned as separate
entities.
 d =0.612λ/NA
 d = resolution
 λ = wavelength of illuminating light source
 NA = numerical aperture of the lens
Size
100μm
8.0μm
0.2μm
Resolution
Eye
light microscope
light microscope



Human Ovum
Red blood cell
Bacteria

Light microscopy: The most commonly used microscope. A light beam is transmitted through a
specimen; samples must be thin and translucent. Samples from a tissue are cut precisely using a
microtome. Tissue structure and molecular composition should be maintained in its native state.
 Glass lenses and an illuminating source in the visible spectrum.
 Condenser collects and focuses light on the specimen
 Objective lens enlarges and projects image towards eyepiece
 Eyepiece further magnifies and projects image towards the detector (retina)
 Total magnification is the magnifying power of the objective lens multiplied by the
magnifying power of the ocular (eyepiece) lens.
Figure 1. Schematic
drawing of a light
microscope showing its
main components and
the pathway of light
from the substage lamp
to the eye of the
observer. (Courtesy of
Carl Zeiss Co.)

Phase Contrast Microscopy: Used for viewing unstained cells and tissues. Light changes speed
when passing through tissue components with different refractive indices. These components then
appear lighter or darker relative to each other.

Polarizing Microscopy: Light passes through a polarizing filter and exits vibrating in only one
direction. A second filter is placed perpendicular to the first filter i.e. no light will pass through.
However, if a sample containing oriented molecules like collagen or microtubules their ordered
structure rotate the axis of the light and appear as bright structures on a dark background.

Confocal Microscopy: Allows precise focusing of light source (laser) on a very thin plane of cell
or tissue. Only light from the focused plane reaches the detector, all others are blocked.
Figure 4. Principle of confocal
microscopy. While a very small
spot of light originating from
one plane of the section crosses
the pinhole and reaches the
detector, rays originating from
other planes are blocked by the
blind. Thus, only one very thin
plane of the specimen is
focused at a time.

Fluorescence Microscopy:
Some substances which when illuminated at a particular
wavelength emit light at a longer wavelength i.e. fluoresce. Cells and tissues are usually
illuminated with UV light with emission monitored in the visible spectrum.
 Some fluorescent molecules have an affinity for certain macromolecules e.g. acridine orange
binds DNA and RNA


Fluorescent compounds can be coupled to marker molecules that specifically bind to different
tissue or cell components.
Electron Microscopy: Microscopes use electrons as illuminating source. These are passed
through electromagnetic lenses and a small aperture. These permit high resolution of images
(0.1nm).
 Transmission or scanning EM. One fixed, in the latter the electron beam is moved across the
specimen.
 Electrons interact with a thin metal coat applied to the specimen.
Tissue Preparation for Light Microscopic Analysis
 Fixation: Should be carried out as soon as possible to minimize autolysis and/or bacterial
contamination. Fixatives are stabilizing or cross-linking reagents. Chemical fixation most
common especially formaldehyde and glutaraldehyde.
 Embedding: Tissues embedded in solid medium to facilitate sectioning. Paraffin and plastic
resins often used. Resins can be used for both light and electron microscopy. Prior to embedding
tissue is dehydrated by successive alcohol washes. A solvent miscible with the embedding
material, usually xylent for paraffin, replaces the alcohol. Once impregnated with solvent the
tissue is placed in melted resin or paraffin, hardened by cooling or cross-linking, then sectioned.
 Staining: Tissues and cells must usually be stained for visualization. Most stains are acidic or
basic compounds. Tissue components with an affinity for acidic dyes like mitochondria and
collagen are acidophilic. Tissue components with an affinity for basic dyes (nucleic acids and
glycosaminoglycans) are basophilic.
 Basic Dyes: Hematoxylin, toluidine blue, methylene blue
 Acidic Dyes: Eosin, Orange G, acid fuchsin.
 Hematoxylin and Eosin (H & E) is the most common dye combination used.
Histochemistry and Cytochemistry: Methods for detecting different substances in tissue sections. Most
based on a specific chemical reaction or high-affinity interactions between molecules. Usually produce a
colored or electron-dense compound. Examples:
 Phosphatases, peroxidases and dehydrogenases used to react with specific chemical bonds.
 Lipid soluble dyes like Oil red O and Sudan black used to detect lipids
 Periodic-Schiff (PAS) reaction transforms glycol groups into aldehydes for detection of
polysaccharides.
Immunocytochemisty; Depends on a specific interaction between an antigen and its antibody. Labeled
antibodies can identify and localize specific proteins.