Introduction to the Study of Histology & its Methods of Study

Lecture one

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 Objectives of study of Histology
• To explain the methods of tissue preparation and its microscopic method of study

• To know the basic components of cell

• To understand that cells can have a different shape and size depending on their
functional roles

• To know the microscopic three dimensional tissue structures

• T0 know the relationship between tissue structure & function

• Gives the basis for histopathology

• Gives the basis for treating diseased & injured tissues

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Levels of structural organization of the human body

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Units of measure in histological study
• Millimetre (mm) = 10-3 (one thousands) of a metre
• Micrometre (µm) = 10-6 (one millionth) of a metre
• Nanometre (nm) = 10-9 (one billionth) of a metre

• Thickness of tissue for:

• Light microscope is 5-10 µm
• Electron Microscope is 40-70 nm

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Tissue processing for Microscopy
• To get thin sections the tissue should be:
• Prevented from decomposing➔ 1. Fixation: by formalin/glutaraldehyde
• Optimally soft to be cut but also rigid to be sliced
➔ Several steps that replace water in the tissue by embedding agent

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To make tissue soft to be cut but also rigid to be sliced
–By replacing water in the tissue by embedding agent though:
2. Dehydration - by alcohol, e.g. ethanol
3. Clearing – by xylene
4. Infiltration (Impregnating) – by paraffin wax
5. Embedding – by paraffin wax or resins are used for EM

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 Embedding
• Treatment with agents that solidify within tissue through change in temperature or
chemical composition
• Gives support & rigidity, but soft enough to be cut
• Paraffin Wax (with a melting point of 50 - 60oC) is the most popular for routine
sectioning for LM
• Resins are used for EM

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 Microtomy
• Sectioning of the tissue blocks➔ steel knife (LM) & diamond knife (EM)

At 5-10 µm thick for LM

At 40-70 nm thick for EM

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Appearances of Tissue and Organ sections under the LM

• Depends on the plane of sectioning

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Appearances of Tissue and Organ sections under the LM

• Depends on the plane of sectioning

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Appearances of Tissue and Organ sections under the LM
• A section through a single coiled tube may appear as
sections of many separate tubes

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Most histological tissue sections are translucent

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 Histological Tissue Staining
• Generally, staining are classified by the following chemical basis:
1. Basic dyes
2. Acidic dyes

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Basic dyes
• React with the acidic components of the tissues, which are basophilic
in nature, and contain:
• Phosphate groups of nucleic acids in nuclei and cytoplasmic RNA
• Sulfate groups of glycosaminoglycans
• Carboxyl groups of proteins

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 Acidic dyes
• React with the basic components of the tissue, which are acidophilic in
nature that contain ionized amino groups, and include:
• Most cytoplasmic filaments, especially of muscle cells
• Most intracellular membranous components and unspecialized
cytoplasm
• Most extracellular fibers

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 General tissue stains
• Uses one or two dyes to differentiate the nucleus from the cytoplasm
• Widely used are the haematoxylin and eosin
• Haematoxylin (acts like a basic dye) - stains the basophilic components of
the tissue blue black
• Eosin (is an acidic dye) - stains the acidophilic components of the tissue
various shades of pink, orange & red

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 Special Stains
• Used to demonstrate specific tissue components
Mallory azan Azocarmine and aniline blue Nuclei: red
Cytoplasm: light pink
Collagen, mucigen: light-blue

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 Metachromasia
• Basophilic tissue components that show color change from blue to purple-red
• By polymerization of the dye by tissue macromolecules with electronegative
radicals (polyanions) present in the tissue

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 Microscopy

• Use

1. Magnification

2. Resolution

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Types the microscope

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Which one is LM and EM view?

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 Light microscopy
• Basic requirements
1. Light source
2. Different lenses

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Microscopic lenses
• Condenser lens

• Objective lenses

• Ocular lens(es)

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 Condenser lens
• Located between light source & specimen
• Focuses the light upon the specimen

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 Objective lenses
• Several with different magnifications on a rotating turret located between
specimen and ocular lens
• Form a magnified real image of the object within the microscope

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 Ocular lens (Eyepiece)
• One on monocular, but two on binocular microscopes

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 Ocular lens(es)
• Use as object the image formed by the objective lens to form a magnified
virtual image in the plane of the object being observed and project it onto
retina of the eye, screen or photographic emulsion, etc

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Total Magnification
• Is the product magnification factors of:
1. Of the objective lens
2. Of the eyepiece
3. In the body tube, when present

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 Resolution
• Ability to show two closely located points appear separate
• Depends on the size and the distance they are separated
• Determines the clarity & richness of details of an image
• Is property of objective lens

1) ..
2) .. ..

3) .. . .

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 Resolution

• Quality of a lens depends on the closeness of its resolving power to the theoretical limit
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Resolution

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Magnification & Resolution

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Magnification & Resolution

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