MICROSCOPY

 Microscopy is the science of investigating tiny objects, such as

cells, tissue, bacteria, viruses, and other small structures using
a microscope.
 The primary function of a microscope is to magnify and
illuminate the objects being viewed
 It allows the observer to see the details that would be invisible
to the naked eye

HISTORY OF MICROSCOPY
 In 1590, Zacharias Jansen and his father, invented the
first compound microscope by placing two lenses in a
tube. This microscope could magnify objects up to 9
times
 In the early 1600s, Galileo Galilei improved on this
design using a convex and a concave lens to create a
more powerful microscope. The microscope could
magnify up to 30 times
 In 1665, Robert Hooke published his book
“Micrographia”, which laid the foundation for the field
of microscopy
 In the 1670s Antonie van Leeuwenhoek, developed a
single-lens microscope that was capable of magnifying
up to 300 times, this microscope was able to observe
bacteria, protozoa, and other microbes
 In the 1800s, the development of achromatic lenses
improved the resolution of microscopes, this allowed
scientists to observe even small structure
 In 1931, Ernst Ruska and Max Knoll developed the first
electron microscope which uses a beam of electrons to
 magnify an object that allowed scientists to observe
structures that were too small to be seen with
traditional optical microscopes
 In 1933, TEMs were built by Ernst Ruska 1933, TEMs use
a beam of electrons to pass through thin samples,
creating images of the sample’s internal structure. This
tech has been critical to advances in fields such as
materials science, molecular biology and nanotech
 In 1938, Manfred von Ardenne built the first SEM
(scanning electron microscope) which uses a focused
beam of electrons to create an image of the surface of a
sample. SEM can produce highly detailed 3d images of
the sample’s surface topography allowing scientists to
study the structure and composition of materials in
greater detail
 Later many microscopes like Confocal Laser Scanning
Microscope (CLSM) which used a laser to scan a
specimen which was developed in the 1980s
 Atomic Force Microscope (AFM) which uses a tiny
probe to scan across the surface of the sample,
detecting small changes in the surface topography this
can be used to study the microbes at the atomic scale
 In the early 2000s several techniques were developed
to surpass the diffraction limit of traditional optical
microscopes

PARTS OF MICROSCOPE
A typical compound light microscope consists of the following main
parts
  Eye Piece: this is the lens through which you view the
specimen
 Objective Lenses: These are the lenses closest to the specimen
and provide the primary magnification of the microscope.
There are several objective lenses (4x, 10x, 40x, 100x)
 Stage: this is the flat platform where the specimen is placed
for observation
 Illumination: The system provides the light necessary to
illuminate the specimen
 Diaphragm: this is a circular disc with several holes of different
sizes used to adjust the amount of light entering the
microscope
 Coarse and Fine Focus Knobs: these knobs are used to adjust
the focus of the microscope
 Arm: the arm supports the upper part of the microscope and
connects it to the base
 Base: The Base supports the microscope and provides stability
during use.
 Condenser: the condenser is a lens system located below the
stage that focuses the light from the illumination source onto
the specimen
 Iris Diaphragm: it is a circular disc with an adjustable opening
that is used to regulate the amount of light that passes
through the specimen

IMPORTANT CONCEPTS OF
MICROSCOPY
 Numerical Aperture: Numerical aperture (NA) is a measure of
the light-gathering ability of an objective lens. It is determined
by the refractive index of the medium (usually air) between
the objective and the specimen and the angle of the cone of
light entering the objective. Higher NA values allow for greater
 light gathering, which in turn increases image resolution and
brightness.

 Magnification: Magnification refers to the degree to which an

object appears larger when viewed through a microscope.
Magnification is determined by the combination of the
objective and eyepiece lenses in a compound microscope. For
example, a 10x objective lens combined with a 10x eyepiece
lens provides a total magnification of 100x.

 Resolving Power: Resolving power, also known as resolution,

is the ability of a microscope to distinguish between two
closely spaced objects as separate entities. It is determined by
the wavelength of light used to illuminate the specimen, as
well as the numerical aperture of the objective lens. Higher
resolving power allows for greater detail and clarity in the
image.

 Oil Immersion Objective: An oil immersion objective is a

specialized objective lens designed to work with immersion oil,
a high-refractive index fluid with a refractive index similar to
that of glass. The oil is placed between the specimen and the
objective lens, which allows for a higher numerical aperture
and better-resolving power. Oil immersion objectives are
typically used for high-magnification imaging of small
structures, such as bacteria or small organelles within cells.

WORKING PRINCIPLE:
Microscopes work by using lenses to magnify small objects or
structures that are too small to be seen with the naked eye. Light
microscopes, the most commonly used type of microscope, use
visible light to illuminate the specimen placed on a glass slide. The
light passes through the objective lens, which magnifies the image
of the specimen. The magnified image then passes through the
eyepiece lens, which further magnifies the image for the observer.

PROCEDURE OF USE:
1. Turn on the microscope and adjust the light source to the
appropriate intensity.
2. Place the specimen on a glass slide and cover it with a
coverslip.
3. Place the slide on the microscope stage and adjust the position
of the slide using the mechanical stage knobs.
4. Use the coarse adjustment knob to focus the image, moving
the objective lens closer to or farther away from the slide as
needed.
5. Use the fine adjustment knob to refine the focus and obtain a
clear image.
6. Adjust the magnification by changing the objective lens or
eyepiece lens as needed.
7. Use the microscope to observe and study the specimen.

HANDLING:
1. Always handle the microscope with care to avoid damage to
the lenses and other delicate parts.
2. Use lens paper or a soft cloth to clean the lenses and other
surfaces of the microscope, being careful not to scratch or
damage the glass.
3. Do not touch the lenses with your fingers, as this can leave
fingerprints and oils that can damage the lenses.
 4. When changing lenses or adjusting the focus, be gentle and
take care not to force any of the moving parts.

MAINTENANCE:
1. Regularly clean the lenses and other surfaces of the
microscope to ensure that they remain free of dust and other
debris.
2. Store the microscope in a clean, dry place when not in use, and
cover it with a protective cover or case.
3. Have the microscope serviced by a qualified technician
regularly to ensure that it is functioning properly and that any
necessary repairs or adjustments are made promptly.
4. Replace any worn or damaged parts, such as lenses or light
bulbs, as needed to maintain the microscope's performance
and functionality.

LIGHT MICROSCOPY
1. Bright field microscope: Working principle: In a bright field
microscope, a light source illuminates the specimen from
below, and the light passes through the specimen and into the
objective lens. The objective lens then magnifies the image,
which is further magnified by the eyepiece. The specimen
appears dark against a bright background.
Procedure: First, prepare a thin, transparent specimen on a
microscope slide using a bright field microscope. Place the slide on
the stage of the microscope and adjust the focus using the coarse
and fine focus knobs. Use the diaphragm to adjust the light amount
and the objective lens to achieve the desired magnification.
Application: Bright field microscopes are commonly used in biology
and medical research to observe stained tissue samples, blood
smears, and microorganisms.
2. Dark field microscope: Working principle: In a dark field
microscope, a special condenser illuminates the specimen
from the side, causing the light to scatter and creating a bright
image against a dark background. The scattered light does not
enter the objective lens, so only the scattered light is visible.
Procedure: To use a dark field microscope, first prepare a thin,
transparent specimen on a microscope slide. Place the slide on the
stage of the microscope and adjust the condenser to create a dark
field of view. Use the coarse and fine focus knobs to adjust the focus
and achieve the desired magnification.
Application: Dark field microscopes are commonly used to observe
live, unstained specimens such as bacteria, protists, and small
aquatic organisms.
3. Phase contrast microscope: Working principle: In a phase
contrast microscope, a special condenser and objective lens
create phase shifts in the light passing through the specimen,
producing a high-contrast image of transparent specimens.
The different refractive indexes of different parts of the
specimen cause a phase shift in the light, which is detected by
the objective lens.
Procedure: To use a phase contrast microscope, first prepare a thin,
transparent specimen on a microscope slide. Place the slide on the
stage of the microscope and adjust the phase ring to create the
desired phase contrast. Use the coarse and fine focus knobs to
adjust the focus and achieve the desired magnification.
Application: Phase contrast microscopes are commonly used to
observe living cells and tissues, particularly in medical research.
 4. Fluorescent microscope: Working principle: In a fluorescent
microscope, a light source excites fluorescent molecules in the
specimen, causing them to emit light of a different colour. The
emitted light is then detected by the objective lens and
eyepiece, producing a fluorescent image.
Procedure: To use a fluorescent microscope, first prepare a
specimen that has been labelled with fluorescent dyes or antibodies.
Place the slide on the stage of the microscope and adjust the focus
using the coarse and fine focus knobs. Use the filter wheel to select
the appropriate excitation wavelength and emission filter, and
adjust the objective lens to achieve the desired magnification.
Application: Fluorescent microscopes are commonly used in biology
and medical research to observe specific structures or molecules
within cells and tissues.
5. Confocal microscope: Working principle: In a confocal
microscope, a laser beam is focused on a small area of the
specimen, producing a fluorescent image. The laser is scanned
across the specimen, producing a series of images that can be
combined into a 3D image of the specimen.
Procedure: To use a confocal microscope, first prepare a specimen
that has been labelled with fluorescent dyes or antibodies. Place the
slide on the stage of the microscope and adjust the focus using the
coarse and fine focus knobs. Use the software to adjust the laser
and scanning parameters and adjust the objective lens to achieve
the desired magnification.
Application: Confocal microscopes are commonly used in biology
and medical research to observe the structure and function of cells
and tissues
ELECTRON MICROSCOPY
1. Transmission electron microscope (TEM): Working principle: A
beam of electrons is transmitted through an ultra-thin
specimen in a transmission electron microscope. The electrons
are focused by magnetic lenses and detected by a fluorescent
screen or a digital detector, producing a high-resolution image
of the internal structure of the specimen.
Procedure: To use a TEM, first prepare a thin specimen that is less
than 100 nm thick. The specimen is placed on a copper grid and
inserted into the vacuum chamber of the microscope. The electron
beam is then directed at the specimen, and the image is recorded
on a detector.
Application: TEMs are commonly used in materials science, biology,
and medical research to observe the internal structure of cells,
tissues, and materials at the nanoscale.
2. Scanning electron microscope (SEM): Working principle: In a
scanning electron microscope, a beam of electrons is scanned
across the surface of a specimen, and the emitted electrons
are detected by a detector. The electron signal is processed by
a computer to produce a high-resolution 3D image of the
surface of the specimen.
Procedure: To use an SEM, first prepare a specimen that is flat and
conductive, such as a metal or carbon-coated sample. The specimen
is placed on a stage in the vacuum chamber of the microscope. The
electron beam is then directed at the specimen, and the emitted
electrons are detected by a detector. The signal is processed by a
computer to produce an image.
Application: SEMs are commonly used in materials science, geology,
and biology to observe the surface morphology of specimens, such
as cells, tissues, and materials. They can also be used to analyse the
chemical composition of the specimen by detecting backscattered or
secondary electrons.

LIMITATIONS OF ELECTRON
MICROSCOPY
Despite their numerous advantages, electron microscopy
techniques also have some limitations. Some of the major
limitations are:
 High cost: Electron microscopes are expensive and require
specialized equipment and skilled personnel to operate and
maintain.
 Sample preparation: Electron microscopy techniques require
the sample to be very thin and dry, which can be a time-
consuming and delicate process. Some samples may not be
amenable to electron microscopy due to their size, shape, or
composition.
 Vacuum requirement: Electron microscopes operate in a high
vacuum environment, which can limit the types of samples
that can be analysed. Biological samples, for example, need to
be carefully dehydrated and fixed to withstand the vacuum,
which can alter their structure.
 Radiation damage: Electron microscopy techniques expose the
sample to high-energy electrons, which can cause radiation
damage, especially in biological samples. This can lead to
artefacts and distortions in the image.
 Limited depth of field: Electron microscopy has a limited depth
of field, meaning that only a thin slice of the sample can be
imaged at one time. This can make it challenging to create 3D
images of complex structures.
 Limited resolution: Despite their high resolution, electron
microscopes have a limit to their resolution, which is
determined by the wavelength of the electrons used. This can
limit the ability to observe structures at the atomic level.
SPECIMEN PREPARATION-LIGHT
MICROSCOPY
Specimen preparation is a crucial step in light microscopy to obtain
clear and accurate images of biological specimens. Different types of
techniques can be used for specimen preparation, including simple
staining, differential staining, and wet mounting. Here are the
details of each technique:
1. Simple staining: Principle: Simple staining is a technique that
uses a single dye to colour the entire specimen. The stain is
usually a basic dye that carries a positive charge and binds to
negatively charged components of the cell, such as the cell
wall, cytoplasm, or nucleus.
Procedure: To perform a simple stain, a drop of the specimen is
placed on a clean slide and allowed to air dry. Then, a small amount
of the stain is added to the specimen and allowed to sit for a few
minutes. Excess stain is rinsed off with water, and the slide is air-
dried and viewed under the microscope.
Advantages: Simple staining is a quick and easy method that can
reveal the morphology and arrangement of cells. It can also help
identify the presence or absence of particular structures, such as
endospores, flagella, or capsules.
2. Differential staining (Gram staining): Principle: Differential
staining is a technique that uses multiple dyes to distinguish
different types of microorganisms based on their cell wall
composition. Gram staining is the most common type of
differential staining and is based on the difference in the
thickness and composition of the cell wall of bacteria.
Procedure: To perform Gram staining, a heat-fixed smear of the
specimen is first flooded with crystal violet stain for a minute,
followed by iodine solution for another minute. Then, the smear is
washed with alcohol or acetone to remove the stain from some
types of bacteria, called Gram-negative bacteria, but not from
others, called Gram-positive bacteria. The smear is then
counterstained with a second dye, usually safranin, for a few
seconds. The slide is rinsed with water, air-dried, and viewed under
the microscope.
Advantages: Gram staining is a valuable technique for identifying
and classifying bacteria based on their cell wall structure. It can
distinguish between Gram-positive and Gram-negative bacteria and
provide information about their shape, arrangement, and other
features.
3. Wet mounting:
Principle: Wet mounting is a technique that involves placing a
drop of the specimen in a liquid medium on a clean slide and
covering it with a coverslip. The liquid medium provides a
suitable environment for the specimen to remain alive and active
during observation.
Procedure: To perform a wet mount, a small drop of the specimen is
placed on the centre of a clean slide, and a coverslip is placed on top
of the specimen at an angle to allow the air bubbles to escape. The
coverslip is gently pressed down to spread the specimen and
eliminate excess liquid. The slide is then viewed under the
microscope.
Advantages: Wet mounting is a simple and quick technique that can
be used to observe living specimens in their natural state, including
movement, growth, and reproduction. It can also be used to
observe small or transparent specimens that are difficult to see with
other techniques.
ELECTRON MICROSCOPY
SPECIMEN PROCESSING
Electron microscopy requires special specimen processing to
prepare the samples for imaging. The following steps are generally
involved in the specimen processing for electron microscopy:
1) Fixation: The first step in specimen processing for electron
microscopy is fixation. The sample is treated with a chemical
fixative to preserve its structure and prevent any changes due to
processing. Common fixatives include glutaraldehyde and
formaldehyde.
2) Dehydration: The next step is dehydration, where the sample is
gradually dehydrated using a series of alcohol solutions. This is
done to remove water from the sample, which can cause
artefacts during imaging.
3) Embedding: Once the sample is dehydrated, it is embedded in a
resin to provide support for the sample during imaging. The most
common resin used is epoxy resin.
4) Sectioning: The embedded sample is then sliced into thin sections
using a microtome. The sections are typically around 50-100
nanometres thick for transmission electron microscopy (TEM)
and several micrometres thick for scanning electron microscopy
(SEM).
normal sectioning and ultra-thin sectioning are two different
techniques used to prepare samples for imaging. The main
difference between these techniques lies in the thickness of the
sections produced.
 a) Normal sectioning involves slicing the sample into several
micrometres thick sections, typically between 1-5
micrometres. These sections are thicker than those produced
by ultra-thin sectioning and are commonly used in scanning
electron microscopy (SEM) to study the surface features of a
sample.

b) Ultra-thin sectioning, on the other hand, involves slicing the

sample into much thinner sections, typically between 50-100
nanometres thick. These sections are used in transmission
electron microscopy (TEM) to study the internal structure of
cells and tissues at high resolution
In summary, the main difference between normal sectioning and
ultra-thin sectioning is the thickness of the sections produced,
with normal sectioning producing thicker sections used for SEM,
and ultra-thin sectioning producing thinner sections used for
TEM.

5) Staining: The sections are stained with heavy metals, such as

uranyl acetate or lead citrate, to improve contrast and enhance
the visibility of the structures in the sample.
6) Mounting: The sections are mounted on a copper grid for TEM or
an SEM stub for SEM. The grid or stub is coated with a conductive
material, such as carbon or gold, to prevent charging during
imaging.
7) Imaging: Finally, the sample is imaged using an electron
microscope. In TEM, the sample is illuminated with a beam of
electrons that pass through the sample to produce an image. In
SEM, a beam of electrons scans the surface of the sample to
produce an image. The resulting images are typically black and
white but can be colourized for visualization purposes.
Some of the techniques used to prepare samples for imaging are
a) Shadowing: Shadowing is a technique used to visualize the
three-dimensional structure of macromolecules and viruses.
The sample is coated with a thin layer of metal, such as
platinum or gold, at an angle so that one side of the sample is
in shadow and the other side is illuminated. This creates a
contrasting image that highlights the surface features of the
sample.
b) Freeze-fracturing and etching: Freeze-fracturing and etching is
a technique used to study the internal structure of biological
membranes. The sample is rapidly frozen and then fractured to
expose the internal structure. The fractured surface is then
etched with a chemical solution, such as sodium meta
periodate, to remove the organic material and reveal the
underlying structure.
c) Sputter coating: Sputter coating is a technique used to coat the
sample with a thin layer of metal, typically gold or platinum, to
improve conductivity and reduce charging during imaging. The
sample is placed in a vacuum chamber and bombarded with
high-energy ions, which cause atoms from the metal target to
be ejected and deposited onto the sample.
d) Surface replica: A surface replica is a technique used to create
a physical replica of the sample surface. The sample is coated
with a thin layer of plastic, such as epoxy or polycarbonate,
and then the plastic film is peeled off to create a replica of the
surface. The replica can then be imaged using SEM to study
the surface features of the sample.
In summary, shadowing, freeze-fracturing and etching, sputter
coating, and surface replica are techniques used in electron
microscopy specimen processing to prepare samples for
imaging at high resolution. These techniques are often used in
conjunction with other sample preparation methods to
provide a complete picture of the sample structure and
morphology.
APPLICATION OF MICROTOMES IN
SAMPLE PREPARATION
Microtomes are instruments used in sample preparation to produce
thin sections of biological specimens, such as tissues and cells, for
microscopic examination. Microtomes are commonly used in
histology, pathology, and other biological sciences for a variety of
applications.
Some common applications of microtomes in sample preparation
include:
1. Histology: Microtomes are used extensively in histology to
prepare thin sections of tissues for examination under the
microscope. Thin sections of tissues are cut using a microtome,
mounted on a slide, and stained to visualize the different
structures of the tissue.
2. Immunohistochemistry: Microtomes are also used in
immunohistochemistry to prepare thin sections of tissues for
staining with antibodies. Thin sections of tissues are cut using a
microtome, mounted on a slide, and stained with antibodies to
visualize specific proteins or other molecules in the tissue.
3. Electron microscopy: Microtomes are used in electron
microscopy to prepare ultra-thin sections of tissues for
examination under the electron microscope. Ultra-thin
sections of tissues are cut using a microtome, mounted on a
grid, and stained with heavy metals to visualize the different
structures of the tissue at high resolution.
4. Pathology: Microtomes are used in pathology to prepare thin
sections of tissues for examination by pathologists. Thin
sections of tissues are cut using a microtome, mounted on a
slide, and stained to diagnose diseases and other pathological
conditions.
In summary, microtomes are versatile instruments used in sample
preparation for a variety of applications in biological sciences,
including histology, immunohistochemistry, electron microscopy,
and pathology.