HISTOLOGY AND METHODS OF THE STUDY

By Asfaw B.(MSc.)

Hawassa University
2021

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 1

 Outlines

Introduction

Histological techniques

Microscope

Types and parts of the microscope

2
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class
 Introduction

At the end of the class students are expected to effectively:

Describe types and parts of microscope

Explain basic steps in tissue processing

Demonstrate parts of the microscope

Demonstrate steps in tissue processing

3
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class
 Introduction
Histology is the study of the tissues of the body and how these
tissues are arranged to constitute organs.

Histology focus on how cells’ structure and arrangement optimize

functions specific to each organ.

Tissues have two interacting components:

Cells

Extracellular matrix (ECM).

The ECM consists of many kinds of complex macromolecules, such

as collagen fibrils and basement membranes.

4
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class
 Con’t…

ECM supports cells and provide fluid that transports nutrients to

cells and carries away their catabolites and secretory products.

The fundamental tissues of the body are each formed by several

types of cell-specific associations between cells and ECM.

Organs are formed by an orderly combination of several tissues.

The precise combination of these tissues allows the functioning

of each organ.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 5

 Histological techniques

It is the preparation of tissues to be ready for microscopic

examination.

The ideal microscopic preparation is preserved tissue on the

slide has the same structure and molecular composition as it
had in the body.

However, as a practical matter, this is seldom feasible because

of artifacts, distortions, and loss of tissue components during
the preparation process.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 6

 Con’t…

• Basic steps used in tissue preparation for light microscopy are:

1. Fixation 6. Sectioning
2. Dehydration 7. Paraffin removal
3. Clearing 8. Rehydration
4. Impregnation 9. Staining
5. Embedding 10. Dehydration

• Similar steps are used in preparing tissue for TEM, except:

Special fixatives and dehydrating solutions are used
Smaller tissue sample size
Embedding involves epoxy resins which become harder
than paraffin to allow very thin sectioning.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 7

 1. Fixation
Reasons for fixation:
To avoid tissue digestion by enzymes present within the cells
(autolysis)
To protect a sample from bacterial colonization.

To increase tissue’s mechanical strength or stability.

• Fixation involves immersion of tissue in solutions of fixatives.

• Tissues are cut into small fragments before fixation to facilitate

penetration and better ensure tissue preservation.

• No fixative will penetrate a piece of tissue thicker than 1 cm.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 8

 Con’t…

• Intravascular perfusion of fixatives can be used with some

organs or laboratory animals.

• Because the fixative in this case rapidly reaches the tissues

through the blood vessels, fixation is improved.

• One fixative widely used for light microscopy is formalin, a

buffered isotonic solution of 37% formaldehyde.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 9

 Con’t…
• A double-fixation procedure, using a buffered glutaraldehyde
solution followed by immersion in buffered osmium tetroxide,
is a standard method to prepare tissue for studies.

• Osmium tetroxide preserves (and stains) membrane lipids as

well as proteins.

Following factors are important:

Fresh tissue

Proper penetration of tissue by fixatives

Correct choice of fixatives

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 10

 Ideal Fixatives

• Prevents autolysis and bacterial decomposition.

• Preserves tissue in their natural state and fix all components.
• Make the cellular components insoluble to reagent used in
tissue processing.
• Preserves tissue volume.
• Avoid excessive hardness of tissue.
• Allows enhanced staining of tissue.
• Should be non-toxic and non-allergic for user.
• Should not be very expensive.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 11

 Factor affecting fixation

• Size and thickness of piece of tissue.

• Tissue covered by large amount of mucous fix slowly.

• Tissue covered by blood or organ containing very large amount

of blood fix slowly.

• Fatty and lipomatous tissue fix slowly.

• Fixation is accelerated by maintaining temperature around

60oc.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 12

 Con’t…

Tissue fixatives Histochemical fixatives

• Buffered formalin • Formal saline

• Buffered gluteraldehyde • Cold acetone

• Zenker’s formal saline • Absolute alcohol
• Bowen’s fluid

Cytological fixatives

• Ethanol

• Methanol

• Ether 13
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class
 2. Dehydration

• Water is extracted from the fixed tissues by successive transfer

through a graded series of ethanol. e.g. 70% to 100% ethanol.

• The duration for which tissues are kept in each strength of

alcohol depends upon the size & type of tissue of tissue and
fixative used.

• Delicate tissue will get high degree of shrinkage by two great

concentration of alcohol.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 14

 3. Clearing
• Alcohol is replaced with organic solvents in which wax is soluble.
• As the solvent infiltrates the tissues, they become more transparent
(clearing).
• Clearing of tissue is achieved by any of the following reagents:
 Xylene
 Chloroform
 Benzene
 Carbon tetrachloride
 Toluene
Note: Xylene is commonly used. Small piece of tissue are cleaned in
0.5 – 1 hour; whereas larger (5cm or more thick) are cleaned in 2-4
hours.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 15

 4. Impregnation
• The fully cleared tissue is then placed in melted paraffin in an
oven at 52°-60°C.

• At such temperatures the clearing solvent evaporates and the

tissue is filled with liquid paraffin.

• The duration of impregnation depends on size and types of

tissues and the clearing agents employed.

• Longer periods are required for larger pieces and harder tissue
like bones & skin as compared to liver kidney, spleen, lung etc.

• Total duration of 4 hours is sufficient for routine impregnation.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 16

 5. Embedding

• Tissues are embedded in a solid medium to facilitate sectioning

• Impregnated tissues are placed into a fresh melted paraffin wax

and allowed to settle, solidify and form blocks.

• These blocks are ready for microtomes.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 17

 6. Sectioning

• A hardened block containing tissue and paraffin is placed in an

instrument called microtome- slices into extremely thin sections

• Paraffin sections are generally cut at 1-10 μm thickness, while

the glass or diamond knives of ultramicrotomes produce
sections of less than 1 μm for electron microscopy.

• The very thin sections are then placed on glass slides and
stained for examination.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 18

 7. Staining

• Most cells and ECM are completely colorless.

• To be studied microscopically sections must typically be

stained (dyed).

• Dyes stain tissue components more or less selectively.

• Anionic(acidic) components of cells such as nucleic acids

stain with basic dyes and are termed basophilic.

• Cationic(basic) components, such as proteins, have affinity for

acidic dyes and are termed acidophilic.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 19

 Con’t…

• Examples of basic dyes: • Examples of acid dyes:

toluidine blue Eosin
alcian blue orange G
methylene blue acid fuchsin
Hematoxylin • They stain an acidophilic
• They stain basophilic components of tissues such
compnents such as DNA, as mitochondria, secretory
RNA & glycosaminoglycans. granules, and collagen.

 Most commonly the combination of hematoxylin and eosin is used

 Hematoxylin produces a dark blue or purple color
 Eosin stains produces pink color

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 20

Stained tissue

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 21

 Con’t…
• Other dyes, such as the trichromes (eg, Mallory stain, Masson
stain), are used in more complex histologic procedures.

• The trichromes, besides showing the nuclei and cytoplasm

very well, help to distinguish extracellular tissue components
better than H&E.

• The whole procedure takes from 12 hours to 2 days

• It depends on the size of the tissue, the fixative, embedding

medium, and method of staining.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 22

 Microscope

• A microscope is an instrument used to see objects that

are too small to be seen by the naked eye.

• Microscopy is the science of investigating small objects

and structures using such an instrument.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 23

 Types of microscope

Based on what interacts with the sample to generate the

image:
1. Light Microscope
based on the interaction of light with tissue components:
Bright-field microscope
Fluorescence microscope
Phase-contrasting microscope
Confocal microscope
2. Electron Microscope:
Scanning electron microscope
Transmission electron microscope

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 24

 Simple light microscope

• Similar to a magnifying
glass and has only one lens

eg-hand lens

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 25

 Compound light microscope

Lets light pass through an

object and then through two
or more lenses.

It has two parts:

I. The Optical System

II. The Mechanical System

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 26

 Con’t…

The Optical System

a. Illuminator(7): artificial light,

usually supplied by a light
bulb, to illuminate the
specimen.

b. Condenser(8): collects and

focuses a cone of light that
illuminates the tissue slide on
the stage.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 27

 Con’t…
c. Objective(3) Lens: the lens
closest to the specimen;
enlarges and projects the image
of the object in the direction of
the eyepiece.

d. Eyepiece(1): the lens closest

to the eye: oculars magnify this
image another X10 and project
it to the observer’s retina.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 28

 Con’t…
Mechanical parts:

Body tube: Connects the eyepiece to the

objective lenses. Holds the objective
lenses and the ocular lens at the proper
distance

Nose piece(2): it holds the objective

lenses and can be turned to change the
magnification

Coarse adjustments(4): Moves the stage

up and down (quickly) for focusing your
image.
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 29
 Con’t…

Fine adjustments(5): used to fine-tune

the focus of your specimen after using
the coarse adjustment knob. This knob
moves the stage SLIGHTLY to sharpen
the image.

Stage(6): is where the sample, slide, or

specimen is placed for examination

Stage clips: two 2 clips hold the

slide/specimen in place on the stage
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 30
 Con’t…

• Iris diaphragm: allows you to control the

amount of light on the specimen that
comes through the aperture.

• Aperture: is the hole in the stage that

allows light to pass through.

• Arm: Used to support the microscope

when carried. Holds the body tube, nose
piece and objective lenses

• Metal base: Supports and carry the

microscope.
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 31
Parts of the microscope

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 32

 Function of the microscopy

A. Magnification: apparent enlargement of an object.

Eyepiece Lens X Objective Lens = Total Magnification

B. Resolution= clarity, sharpness & ability of a microscope to
show two very close points separately.

It is the power of the microscope to distinguish fine details

The power of resolution therefore increases as the limit of
resolution decreases.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 33

 Con’t…

Numerical Aperture (NA) of a lens:

It is a measure of the amount of light entering the lens.

It depends on sine the angle (B) which is half the angle of a
cone of light gathered by the objective lens and on the
refractive index of the medium b/n the specimen and objective.

This medium is air in the case of low and high powers and oil
in the case of oil immersion objective.

NA = Sine (B) X refractive index

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 34

 Factors affecting power of resolution

Numerical aperture (N.A.)

Objective magnification

Wave length of the light used λ

The quality of the image

The maximal resolving power of the L.M. is around 0.1 um.

This permits good images magnified 1000-1500 times.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 35

Factors affecting limit of resolution

L.r is directly proportional to wave length of the light used (λ) and

Indirectly proportional to the numerical aperture (NA) of the

objective lens.

• L.r.= K X λ ⁄ NA K= Constant=0.61

The light microscope can not distinguish two close details unless
they are at least 0.24 µm apart or

can not distinguish any structure less than 0.24 µm in diameter.

-i.e. L.r. of light microscope is 0.24 µm

NB. The limit of resolution of the human eye is 0.1mm (100 um).
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 36
 Bright-Field Microscopy
• Widely used by students, stained preparations are examined
by means of ordinary light that passes through the specimen.
• The microscope includes an optical system and mechanisms to
move and focus the specimen.

Advantages:
 simplicity Enhancements:
Limitation  Reducing or increasing the
 Very low contrast of most amount of the light source
biological samples. by the iris diaphragm
 Low apparent optical resolution  Oil immersion can be
 Colorless and transparent added on glass cover to
samples cannot be seen well. improve resolution

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 37

 Phase-Contrast Microscope
• Used to study unstained cells and tissue sections, which are
usually transparent and colorless
• It uses a lens system that produces visible images from
transparent specimens; e.g living cells, cultures cells.
• It is based on the principle that light changes its speed when
passing through cellular and extracellular structures with
different refractive indices.
• These changes are used by the phase-contrast system to
cause the structures to appear lighter or darker in relation to
each other.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 38

 Con’t…

Limitations
Applications  Costly
1) living cells (in culture),  To use phase-contrast the
• ..
2) microorganisms, light path must be aligned.
3) thin tissue slices,  more light is needed for
4) fibers, phase contrast than for
5) subcellular particles corresponding bright-field

Advantages
 Living cells can be observed in their natural state
 It makes a highly transparent object more visible
 No special preparation of fixation or staining, etc. is needed
 High resolution
 Allows studying living cells how they proliferate
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 39
 Fluorescence Microscope

• Uses fluorescence to generate an image.

• A specimen that is to be studied must be coated with special

compounds that possess the quality of fluorescence.

• Such compounds are called fluorochromes.

• These are Auramine O, acridine orange, and fluorescein are

well-known fluorochromes.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 40

 Confocal Microscope

• It concentrates light and sharp focus by using:

(1) a small point of high-intensity light, often from a laser, and
(2) plate with a pinhole aperture in front of the image detector.
• The light source, focal point of the lens, and detector’s pinhole
aperture are all optically aligned to each other in the focal
plane (confocal), and unfocused light does not pass through the
pinhole.
• This greatly improves resolution of the object in focus and
allows the localization of specimen components with much
greater precision than with the bright-field microscope.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 41

 Electron microscope
• They are based on the interaction of electrons and tissue
components
• Uses a beam of accelerated electrons as a source of
illumination.
• Uses a magnetic field to bend beams of electrons; instead of
using lenses to bend beams of light.
• Electron microscopes are used to investigate the ultrastructure
of a wide range of biological and inorganic specimens
including microorganisms, cells, large
molecules, biopsy samples, metals, and crystals
• There are two types:
transmission E.M.
Scanning E.M.

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 42

 Electron microscope Light microscope
High resolving power Low resolving power
Minimal useful magnification Maximum magnification=1500
400,000
Very thin sections (50nm) Thin sections (5-8um)
Glass or diamond knives Microtome metal knives
Embedding medium: Hard epoxy Embedding Medium: Paraffin
plastic wax
Short wave length stream of electrons Photons

Electro-Magnetic lenses Glass lenses

Heavy metal salts Staining: Any dye
Osmic acid Any type of fixative
On metal grids Mounting on glass slide
5nm (Scanning E.M.), 0.24 nm Resolution limit 0.2-0.4 um
03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 43
Microscope care

• Always carry with 2 hands

• Only use lens paper for cleaning
• Do not force knobs
• Always store covered
• Keep objects clear of cords
• Clean only with a soft cloth/tissue
• Make sure it’s on a flat surface

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 44

 References

• Gerard G. Tortora, principles of Human Anatomy, 12th edition

• Junquiera’s Basic Histology Text and Atlas 13th Edition

03/22/2024 04:51 PM Asfaw B.(MSc.) Medical student class 45