Principle and Application of Microscopy

• Microscopy is a technique used for making very tiny things visible to the
naked eyes and the instrument used to make things visible to the unaided
or naked eye is known as Microscope.

• The magnification provides by the microscope enables us to see

microorganisms and their structures otherwise invisible to the naked eye.

• Microscope is an instrument used in the field of basic and applied sciences

to observe microscopic cells.

• The naked or unaided human eye can see the objects, which are more than
0.1 mm in size.

• The function of the microscope is to enlarge, magnify and capture the

images of the microscopic cells.
• The efficiency of microscopes is indicated by its resolution

• Resolution is the ability of microscope to distinguish two closely

related points

• In simple microscope, single lens is used for magnification

whereas in compound microscope, multiple lenses are used.

• Monocular microscope has one eyepiece and binocular has two

eyepieces

• As research has advanced, we now have different types of

microscopes that are commercially available
• In 1609 Galileo Galilei perfected the first device known as a
microscope

• Galileo's microscope used a bi-concave eyepiece and bi-convex objective

lens to provide up to 30 times magnification

• A convex lens or converging lens focuses the light rays to a specific

point, whereas a concave lens or diverging lens diverges the light rays.

• Galileo contributed to the field of microscopy by popularizing lenses

capable of perceiving much smaller images than were previously
available.
• Antony Van Leeuwenhoek invented the first simple microscope
in 1683. Leeuwenhoek was the first to observe bacteria and
microscopic animals
• Leeuwenhoek is considered the founder of the study of microscopy and
played a vital role in the development of cell theory

• Van Leeuwenhoek was the first to observe and to experiment with 

microbes, which he originally referred to for "small animals“

• He was the first to relatively determine their size. Most of the

"animalcules" are now referred to as unicellular organisms

• Zaccharias Janssen and Hans Lipperhey are noted as the first men to
develop the concept of the compound microscope
• Single-lensed simple microscopes can magnify up to 300× and
are capable of revealing bacteria

• while compound microscopes can magnify up to 2,000×

• A simple microscope can resolve below 1 micrometre (μm; one

millionth of a metre). A compound microscope can resolve down
to about 0.2 μm

•  Electron microscope, which uses a beam of electrons in its

image formation.
• Scanning electron microscope (SEM) has magnifying powers
upto 250000
• The transmission electron microscope (TEM) has magnifying
powers of more than 1,000,000×. 
• Robert Hooke around mid 1600 improved the design of the
existing compound microscope  which illuminated and
enlarged specimens.

• Robert Hook published a volume called Micrographia which

introduced a wide range of microscopic views of familiar objects
(fleas, lice etc).

• Leeuwenhoek effectively launched microbiology in 1674, and

single-lensed microscopes remained popular until the 1850s.

• In 1827 they were used by Scottish botanist Robert Brown to

demonstrate the ubiquity of the cell nucleus, a term he coined in
1831.
• lenses that collect, reflect, and focus light into the specimen

• lenses promotes magnification without altering the quality of

the image produced

• Objec&ve lenses are responsible for collec%ng and

concentra%ng light into the specimen

• An instrument called an eyepiece magnifies the object by

changing the wavelength of light
• Eyepiece: It is present at the top of microscope. It is the part
through which we observe the sample/object. The magnifying
power of the eyepiece lens is generally 10X.

• Nosepiece: This part connects the eyepiece tube to objective

lenses. The flexibility of nosepiece allows switching the objective
lenses.

• Objective lens: In compound microscope, in general 3 objective

lenses are placed. Their magnifying power is 10X, 40X and
100X respectively. The total magnification of respective lenses
would be 10X x 10X = 100X, 40X x 10X = 400X and 100X x
10X = 1000X (the magnifying power of eyepiece lens is 10X).
 Types of Microscopes
• Several types of light microscopes are used in microbiology: bright-field, phase-
contrast, dark-field and fluorescence.

• Brightfield Microscope

• Brightfield Microscope is also known as the Compound  light microscope.

• It is an optical microscope that uses light rays to produce a bright

background.

• It is the standard microscope that is used in Biology, Cellular Biology, and

Microbiological Laboratory studies.

• This microscope is used to view fixed and live specimens, that have been
stained with basic stains which gives a contrast between the image and the
image background.
• It is specially designed with magnifying glasses known as lenses that modify
the specimen to produce an image seen through the eyepiece.

• Principle of Brightfield Microscope

• For a specimen to be the focus and produce an image under the Brightfield
Microscope, the specimen must pass through a uniform beam of the
illuminating light.

• Through differential absorption and differential refraction, the microscope

will produce a contrasting image.

• The specimens used are prepared initially by staining to introduce color for
easy contracting characterization.

• The colored specimens will have a refractive index that will differentiate it
from the surrounding, presenting a combination of absorption and refractive
contrast.
• The functioning of the microscope is based on its ability to produce a high-
resolution image from an adequately provided light source, focused on the
image, producing a high-quality image.

• The specimen which is placed on a microscopic slide is viewed under oil

immersion or/and covered with a coverslip.

• Light path
• The light path consists of:
• A transillumination light source, commonly a halogen lamp in the microscope
stand;

• a condenser lens, which focuses light from the light source onto the sample;
• an objective lens, which collects light from the sample and magnifies
the image;

• oculars and/or a camera to view the sample image.

• Performance

• Bright-field microscopy typically has low contrast with most biological

samples, as few absorb light to a great extent. Staining is often required
to increase contrast

• Bright-field illumination is useful for samples that have an intrinsic

color, for example chloroplasts in plant cells.
• What is Phase-contrast microscopy?

• Unstained living cells absorb practically no light. Poor light absorption

results in extremely small differences in the intensity distribution in the
image. 

• This makes the cells barely, or not at all, visible in a brightfield

microscope. It was first described in 1934 by Dutch physicist Frits
Zernike.

• Principle of Phase contrast Microscopy

• When light passes through cells, small phase shifts occur, which are
invisible to the human eye.
• In a phase-contrast microscope, these phase shifts are converted into
changes in amplitude (vibration), which can be observed as differences
in image contrast.

• Light waves change phase by 180° when they reflect from the surface of
a medium with higher refractive index than that of the medium in which
they are travelling.

• A light wave travelling in air that is reflected by a glass barrier will

undergo a 180° phase change, while light travelling in glass will
not undergo a phase change if it is reflected by a boundary with air. 

• Phase-contrast microscopy is particularly important in biology.

• It reveals many cellular structures that are invisible with a 

bright-field microscope. These structures were made visible by staining,
but this required additional preparation and death of the cells.
• Dark-field microscope

• In dark filed microscope background is dark thus picture appears bright

• In the dark-field microscope, light reaches the specimen from the sides only.
•
• Obstruction (Annular filter) is between light source to condenser lens.

• Light reaches to condenser lens through the sides of the obstruction.

• It contains special type of condenser that scatters light

• Bright field microscopy shows clear magnification while dark field image
shows minute details

• Light from condenser lens falls on the sample, sample scatters this light.
Scattered light then reaches to objective lens, objective lens magnifies this
light
• There is no need to stain the specimen

• Study external features

• The only light that reaches the lens is that scattered by the specimen,
and thus the specimen appears light on a dark background.

• Resolution by dark-field microscopy is often better than by light

microscopy, and some objects can be resolved by dark-field that cannot
be resolved by bright-field or even by phase-contrast microscopes.
• Dark-field microscopy is a particularly good way to observe microbial
motility, as bundles of flagella (the structures responsible for swimming
motility) are often resolvable with this technique.

• Principle of the Darkfield Microscope

• A dark field microscope is arranged so that the light source is blocked off,
causing light to scatter as it hits the specimen.

• When light hits an object, rays are scattered in all azimuths or directions.

• The design of the dark field microscope is such that it removes the dispersed
light, or zeroth order, so that only the scattered beams hit the sample.
• The introduction of a condenser and/or stop below the stage ensures that
these light rays will hit the specimen at different angles, rather than as a
direct light source above/below the object.

• The result is a “cone of light” where rays are diffracted, reflected and/or
refracted off the object, ultimately, allowing the individual to view a
specimen in dark field.
 Principles of Microscope

• The microscope works on three principles of physics:

• Magnification
• Resolving power
• Numerical aperture

Magnification: It is the ability of lenses to enlarge an object visually.

If the magnifying power of lens is 10X it means that the given lens can enlarge
the object up to 10 times.

In compound microscope, the magnification is the product of magnifying power

of the lenses. The magnifying power of lens depends on focal length. Lower
the focal length, higher is the magnifying power of lens.

Focal length is a measure of how strongly the system converges or diverges
light
• Resolution: The resolving power is the ability to distinguish two closely
placed points.

• The resolution power of lens allows us to observe the details of an object.

The resolution of microscope can find out by using Abbe’s equation.
• d=0.5λ
• n sinθ
• Where,
• d – distance between two closely distant points
• λ lambda– wavelength of light
• n sin θ – numerical aperture

• The microscope with higher magnification has small d value.

• λ is the wavelength of light, shorter is the wavelength; higher is the

resolution.
• The wavelength of visible light is from 300 to 700 nm.
• The best resolution for light microscope is obtained in the range of 450 to 500
nm. ‘n’ is the refractive index of medium.

• Refractive index is the ability of the medium to bend the light.

• The angle of cone of light is affected by the refractive index of medium.

• The refractive index of air is 1. ‘θ’ is the half of the angle of the cone of light
that enters the microscope.

• The value of ‘Sin θ’ cannot be more than 1 because angle of entering cone of
light cannot be more than 90° and value of sin 90 is 1.
• d= 0.5 x 450 nm
•               1
• d= 225 nm 0r 0.2 μm
• Hence, the resolution limit of light microscope is 0.2 μm.
• The numerical aperture of a microscope objective is a measure of its ability
to gather light and resolve fine specimen detail at a fixed object distance.

• Application of Microscopy

• Biological sampling

• Microscope can visualize the structure of tissues and biomaterial of

organisms and humans.

• Microscopes are used in examining the ailments by getting a larger view of the
blood sample in detecting the parasites, bacteria attacking the red blood [E.
coli produce toxins (Shiga toxin)that damage RBC membranes].
• Microscope is used to study microorganisms, cells, crystalline structures, and
molecular structures.

• It is used to study plant-microbe interaction: symbiosis, mycorehizae, animal-

microbe interactions

• Cellular and tissue structure

• Blood Microscopy
• Microscopy is used to observe live blood cells especially red blood cells
(RBCs), white blood cells (WBCs), plasma [liquid portion containing
coagulants mainly fibrinogen and plasma proteins (albumin and globulin)].

• Within the plasma it can observe undigested food particles, fungus, crystals,
bacteria, viruses.

• It can also observe absorption of proteins, nutrients, lipids, immune system

disorders (anemia)in the plasma. It can observe erythrocytes (red blood cells)
and leukocytes in the blood.
• Sputum Microscopy
• It can observe bacteria (Staphylococcus auerus (abscess), Klabsella,
Streptococcus), virus (Rhinovirus (causes common cold), Influenza) and fungi
(Aspergillosis, a respiratory disease) in the sputum especially the causative
agent of tuberculosis.

• Immunohistochemistry
• It can study, identify, and diagnose a variety of antigens and antibodies
(IgG)/IgM produced that are present in diseased tissue.  

• It can be used in diagnosing different types of cancer, determining if a tumor is

benign (not cancerous) or malignant (cancerous) and identifying its stage,
grade and place of origin

• Urine Analysis
• It is used for urine analysis which can detect various types of disorders which
includes kidney disease, urithritis (inflammation of urethra), urinary tract
infection. It will involve examination of appearance, concentration as well as
content (creatine, urea, uric acid and some elements) of the urine sample.
• Crystals can be found in the urine they include triple phosphate crystals,
calcium oxalate and amorphous phosphates. Casts (cylindrical structures
produced in the kidney).

• Nanotechnology

• In the field of nanotechnology, microscopy can be used to study nanostructure

properties at dimensions between 1 and 100 nanometers.

• Electron microscopes such as transmission electron microscopes (TEM) and 

scanning electron microscopes (SEM) provide topographical, morphological
and compositional data.

• Biotechnology
• It is used to study cells, genes, biofilm formation
• Forensic
• In forensics, a microscope is used to study general criminal science,
forensic epidemiology (cause of disease or death in case of forensics)
and forensic pathology.

• Microscope in Forensic science helps doctors examine organs, bones, and

other parts of the body to know the cause of the death.
 Scanning electron microscopy (SEM)

• The first Scanning electron microscope was initially made by Mafred von

Ardenne in 1937.

• High-resolution power is used to scan specimen using beam of electrons.

• He also aimed at reducing the problems of chromatic aberrations (failure of

lens to focus all colors to the same point) images produced by the
Transmission electron Microscopes. 

• More studies followed by scientists and research institutions such as

Cambridge Scientific Instrument Company who eventually developed a fully
constructed Scanning electron Microscope, in 1965 and named it a Stereoscan. 

• The price of the Scanning Electron Microscope (SEM) is approximately $1

million.
• Scanning electron microscopy (SEM) has been widely used in
environmental microbiology to characterize the surface structure of
biomaterials and to measure cell attachment and changes in
morphology of bacteria.
•  surfaces of solid objects

• Moreover, SEM is useful for defining the number and distribution of

microorganisms that adhere to surfaces.

• Traditionally, inability to provide phylogenetic or genetic information

about microorganisms has been one limitation of SEM in environmental
microbiology.
• The Scanning electron microscope works on the principle of applying
accelerated electrons carrying sufficient kinetic energy to produce signals
on the interaction of the electrons.

• These are secondary electrons are used to produce an image

• The secondary electrons are emitted from the specimen play the primary
role of detecting the morphology and topography of the specimen.

• These are also used to view crystallized elements and photons

 
• SEM provides magnifications up to ×300,000 and has a high spatial
resolution of less than 2 nm (as measured by gold particles on carbon).

• No elaborate specimen-preparation techniques are required for

examination in the SEM, and large and bulky specimens may be
accommodated.

• It is desirable that the specimen be rendered electrically conducting;

otherwise, a sharp picture will not be obtained.
• Conductivity is usually achieved by evaporating a film of metal, such as 
gold, 50–100 angstroms thick onto the specimen (such a thickness does not
materially affect the resolution of the surface details).

• How does the Scanning Electron Microscope (SEM) work?

• The source of the electrons are from tungsten filament lamps that are placed
at the top of the column.

• The electrons are emitted after thermal energy is applied to the electron
source and allowed to move in a fast motion to the anode, which has a
positive charge and resulted in the formation of beam of electrons.
• The beam of electrons interacts with the specimen to produce signals that
give information about the surface topography and composition of the
specimen.

• However, microbial specimens need fixation, dehydration, and drying

in order to maintain the structural features of the cells and to prevent
collapsing of the cells when exposed to the high vacuum of the
microscope.
• Primary fixation with aldehydes (lipids), secondary fixation with osmium
tetroxide (lipids), dehydration with solvents such as ethanol or acetone,
drying, mounting on stub, then to make specimen conductive

• The samples are mounted and coated with thin layer of heavy metal
elements to allow spatial scattering of electric charges on the surface of
the specimen allowing better image production, with high clarity.
• When the electrons reach the specimen, the surface releases a tiny staw of
electrons known as secondary electrons which are then trapped by a special
detector apparatus.

• When the secondary electrons reach and enter the detector, they strike a
scintillator (a luminescence material that fluoresces when struck by a charged
particle).

• This emits flashes of light which gets converted into an electric current by a
photomultiplier, sending a signal to the cathode ray tube.

• This produces an image that looks like a television picture that can be viewed
and photographed.
• The quantity of secondary electrons that enter the detector is highly
defined by the nature of the specimen i.e raised surfaces to receive high
quantities of electrons, entering the detector while depressed surfaces
have fewer electrons reaching the surface and hence fewer electrons
enter the detector.

• Therefore raised surfaces will appear brighter on the screen while

depressed surfaces appear darker.
 Parts of a Scanning Electron Microscope (SEM)

• The major components of the Scanning Electron Microscope include;

• Electron Source – This is where electrons are produced under thermal heat at
a voltage of 1-40kV. the electrons condense into a beam that is used for the
creation of an image and analysis.

• There are three types of electron sources that can be used i. e Tungsten
filament, Lanthanum hexaboride and Field emission gun (FEG)

• Lenses – it has several condenser lenses that focus the beam of electrons from
the source through the column forming a narrow beam of electrons that form
a spot called a spot size.

• Scanning Coil – they are used to deflect the beam over the specimen surface.
• Detector – It’s made up of several detectors that are able to differentiate
the secondary electrons.

• The functioning of the detectors highly depends on the voltage speed, the
density of the specimen.

• The display device (data output devices)

• Power supply

• Vacuum system
SEM image of Tradescantia pollen and stamens
 Applications of the Scanning Electron Microscope (SEM)

• It is used in a variety of fields including Industrial uses, nanoscience studies,

Biomedical studies, Microbiology

• Used in the analysis of cosmetic components which are very tiny in size.

• Used to study the filament structures of microorganisms.

• Used to study the topography of elements used in industries.

• Limitations

• They are very expensive to purchase

• They are bulky to carry

• They must be used in rooms that are free of vibrations and free of
electromagnetic elements

• They must be maintained with a consistent voltage

• They should be maintained with access to cooling systems

 Transmission Electron Microscopy
• Transmission electron microscopy uses high energy electrons (up to 300 kV
accelerating voltage) which are accelerated to nearly the speed of light.

• The electron beam behaves like a wavefront with wavelength about a

million times shorter than light waves.

• Working Principle of TEM

• The TEM operates on the same basic principles as the light microscope but
uses electrons instead of light.

• When an electron beam passes through a thin-section of specimen of a

material, electrons are scattered.

• A sophisticated system of electromagnetic lenses focuses the scattered

electrons into an image. 
• In a Transmission electron microscope, the electron beam is transmitted
through a very thin specimen or object and forms a highly magnified and
detailed image of the sample.

• This microscope uses electron beams instead of light.

• The specimen used in Transmission Electron Microscope, should be very thin,

less than 100 nm thick.

• The electron beam behaves like a wavefront with wavelength about a million
times shorter than light waves

• TEM can tell us the structure, crystallization, morphology, and stress of the
specimen in a better way as compared to a simple microscope.

• The formed image is then magnified and visualized on a  fluorescent screen

(layer of photographic film)
 Working principle of Transmission Electron Microscope

• Electron Microscope follows the same principle as a light microscope

follow.

• The major difference is, the light microscope uses artificial light or
natural light to create an image of the specimen, whereas an electron
microscope uses electron beams.

• In EM when electron beams cross through a specimen, the electron

particles starts to scatter.

• The electromagnetic lens on EM focusses the scattered electron on a

screen and creates an image of the specimen.
 Mechanism of Transmission Electron Microscope
• First of all, a tungsten filament is heated, which is also called an electron
gun.

• The heated tungsten filament or electron gun will start to release electron
beams.

• An electromagnetic coil and high voltage(up to several million volts) applied

to these electron beams to accelerate their speed (extremely high speeds ). 

• A condenser lens with a high aperture eliminates all the high angle electrons
and focused all the electron beams into a thin, small beam.

• The high-speed electron beams are now transmitted through the specimen.

• The transmitted electron beams are focused into an image with the help of an
objective lens.
• The electron beams are projected on to a phosphorescent screen, which creates
an image of the specimen, also called a micrograph.

• All the images are captured by a charge-coupled device (CCD) camera, which
is located underneath the screen.
• Parts of A Transmission Electron Microscope
• 1. Electron Gun

• Electron guns consist of four important parts, the filament, a biasing circuit, a
Wehnelt cap, and an extraction anode.

• When an electron gun is connected with a power supply it starts to generate

electron beams.

• These electron beams are now moved towards the anode plate and the TEM
column.
• Functions:
• It generates electron beams.

• 2. Vacuum system

• It creates a vacuum to prevent the interaction between air particles and

electrons. So that electron will not be scattered.

• 3. Specimen stage

• It has an  airlocks system to insert the specimen inside the vacuum with
minimal loss of vacuum in other areas of the microscope. 

• 4. Electron lens

• These act as an optical lens by focusing parallel electrons at some

constant focal distance.
• 5. Apertures

• Apertures are annular metallic plates, which consist of a small metallic

disc.

• This disc permits the axial electrons to pass through it and exclude those
electrons that are at a distance from the optic axis.

• By permitting the  central electrons, apertures decrease the intensity of

electron beams in TEM, which is good for the beam sensitive samples.

• By this way it also removes those electrons are scattered to high angles.
 Applications of the Transmission Electron Microscope (TEM)

• It can achieve a resolution of ~0.1 nm, thousand times better resolution,

cannot be reached by the light microscope.

• The beam of electrons passes through the specimen and analyzes the internal
structure of the specimen in the form of images.

• TEMs provide information on element and compound structure

• Images are high-quality and detailed

• TEMs offer the most powerful magnification, potentially over one million times
or more
 Disadvantages

• TEMs are large and very expensive

• Laborious sample preparation

• Operation and analysis requires special training

• TEMs require special housing and maintenance

• A Transmission Electron Microscope requires constant upkeep including

maintaining voltage, currents to the electromagnetic coils and cooling
water.
 Fluorescence Microscope
• Fluorescence microscopy is a light microscope that works on the principle of
fluorescence.

• A substance is said to be fluorescent when it absorbs the energy of invisible

shorter wavelength radiation (such as UV light) and emits longer
wavelength radiation of visible light (such as green or red light).

• This phenomenon, also called fluorescence, is widely used in clinical and

diagnostic settings to detect microorganisms, antibodies, and many other
substances rapidly.

• Some cells fluoresce naturally under ultraviolet light because they contain
fluorescent substances such as chlorophyll.

• Pyoveridin in Pseudomonas florescence

•
• If the specimen to be viewed does not naturally fluoresce, it can be
stained with fluorescent dyes called fluorochromes.

• Commonly used fluorescent dyes are; DAPI (49,6-diamidino-2-

phenylindole), acridine orange, auramine-rhodamine, Alexa Fluors,
or DyLight 488.

• When fluorescence microscopy is used for the detection of antigen-

antibody, it is known as immunofluorescence.
• Working Principle
• Most cellular components are colorless and cannot be clearly distinguished
under a microscope.

• The basic premise of fluorescence microscopy is to stain the components

with dyes.
 
• To observe the sample through a fluorescence microscope, it should first be
labeled with fluorescent dyes/substances known as fluorophores.

• Fluorescent dyes, also known as fluorophores or fluorochromes, are

molecules that absorb excitation light at a given wavelength (generally UV),
and after a short delay emit light at a longer wavelength.

• Higher energy shorter wavelength lights (UV rays or blue light) generated
from mercury vapor arc lamp pass through the excitation filter.
• The excitation filter allows only the short wavelength of light to pass
through and removes all other non-specific wavelengths of light.

• The filtered light is reflected by the dichroic filter and falls on the
fluorophore-labeled sample. The fluorochrome absorbs shorter wavelength
rays and emits rays of longer wavelength (lower energy) that pass through
the emission filter. 

• The emission filter blocks (suppresses) any residual excitation light and
passes the desired longer emission wavelengths to the detector. 

• Thus the microscope forms glowing images of the fluorochrome-labeled

microorganisms against a dark background.

• To the observer, the background is dark, as there is no visible light and only the
labeled specimen (cells, microorganisms, etc.) appear bright (fluoresce).
• Light Path In Fluorescence Microscopy/Parts of Florescence Microscopy

• Fluorescence microscope uses a high-intensity Mercury lamp as a Source of

light.  This lamp emits blue light.

• The exciter filter transmits only blue lights to the specimen and blocks out all
other colors.

• The blue light is reflected downward to the specimen by a dichroic mirror,

which reflects the lights of certain colors but transmits light of other colors.

• The specimen is stained with a fluorescent dye certain portions of the

specimen retains the dye others do not

• The stained portion absorbs blue light and emits green light,  which passes
upward penetrates the dichroic mirror and reaches the barrier filter.
• This filter allows the green light to pass to the eye; however, it blocks out
any residual blue lights from the specimen which may not have been
completely deflected by the dichroic mirror.

• Thus the eye perceives the stained portion of the specimen as glowing green
against a jet black background whereas the unstained portion of the specimen
is invisible.
• Parts of Fluorescence Microscope

• Light Source
• Most of the Fluorescence Microscope Uses Mercury light lam as a primary
source of light. This lam emits blue lights.
• Fluorescent dyes or Fluorochromes

• Fluorescent dyes are a type of chemical compound that emits light rays when
they are excited with UV, blue rays.

• Excitation filter
• It is a type of bandpass filter, which passes the wavelengths absorbed by the
fluorophore
• Emission filter

• It is also a type of bandpass filter.

• It only passes only those wavelengths that are emitted from a

fluorophore.

• It blocks all unwanted wavelengths outside this band – especially the

excitation light. As a result of this action, it creates a dark background.

• Dichroic mirror

• It is a type of accurate color filter.

• It only passes small range of colors and reflects other colors.

• Application of Fluorescence Microscope

• The Fluorescence microscope has become an essential tool in medical

microbiology and microbial ecology.

• Bacterial pathogens can be identified after staining them with fluorescent or

specifically labeling them with fluorescent antibodies using
immunofluorescence produce.

• In ecological studies, the Fluorescence microscope is used to observe

microorganisms stained with Fluorochrome-label probes or
Fluorochromes that bind specific cell constituents.

• Another Important use of the fluorescence microscope is the localization of

specific proteins within the cell.
 Confocal Microscope

• It is also called confocal laser scanning microscopy (CLSM) or laser

confocal scanning microscopy (LCSM)

• It is an optical (vision) imaging technique for increasing optical resolution

 and contrast of a micrograph by means of using a spatial pinhole to block
out-of-focus light in image formation.

• In a conventional (i.e., wide-field) fluorescence microscope, the entire 

specimen is flooded evenly in light from a light source. All parts of the
sample can be excited at the same time and the resulting fluorescence is
detected by the microscope's photodetector or camera including a large
unfocused background part.

• In contrast, a confocal microscope uses point illumination

• The concept of confocal microscopy was initially developed by Marvin
Minsky in the 1950s, at Harvard University with an aim of viewing the
neural network without staining the tissues.

• It did not bear fruit due to lack of enough light source and a
computerized system to store the large data.

• The work was later adapted by David Egger M. and Mojmir Petran,
forming a multiple-beam confocal microscope in the late 1960s.

• The technique was later modified and published by Egger forming a

mechanical scanned confocal laser microscope, that was able to
visualize images of cells.
• The first commercial confocal microscope was developed in 1987 with high
scanning efficiency.

• To date, these microscopes have been used to investigate molecules,

microbial cells and tissues.

• Principle of Confocal Microscope

• Confocal Microscope uses fluorescence lights to create micrographs of

specimens

• In a confocal microscope, the laser light is focused onto a defined spot at a

specific depth within the sample.

• As a result, fluorescent lights start to emit from the exact point.

• A pinhole located inside the optical pathway, it only allows the fluorescence
signals from the illuminated spot to enter the light detector and cuts off
signals that are out of focus.

• It scans the specimen in a raster pattern and creates a 3D picture of the

specimen.

• How Confocal Microscope Works

 
• A confocal microscope uses laser beams instead of lights. The laser beams
are released from their source and then focused onto a fluorescent stained
sample.

• Neutral density filters and a set of scanning mirrors control the intensity of
the laser light by moving them very precisely and quickly.
• One mirror tilts the beam within the X route, the opposite within the Y
route. Together, they tilt the beam in a raster style (rectangular, scan from
top to bottom).

• Then an objective lens focuses it onto the sample.

• The fluorochrome stained sample will be excited and then it will emit
fluorescent lights.

• These fluorescent lights will travel back into the objective lens through the
same path that the laser travels.

• The main effects of these scanning mirrors are on this light is to generate a
spot of light which is not scanning, but standing still.
• Then a semi-transparent mirror (dichronic) reflects this fluorescent
light away from the laser and toward the detection system.

• Before entering into the detection system, it passes through a pinhole.

This pinhole allows only a small central portion of the light through to
the light detectors.

• Confocal microscope produces a very low-intensity light, so the light is

amplified by a photomultiplier tube (PMT).

• Photomultipliers have the ability to amplify a faint signal around one

million times without introducing a single noise.

• After that, the PMT releases an electrical signal, which is then converted
into an image by using a computer.
• Advantages of Confocal Microscopy

• It creates a high-resolution image.

• It creates 3D image of the specimen.

• Living and fixed cells can be used.

• Confocal Microscope illuminates uniformly across the focus points.

• Disadvantages of Confocal Microscopy

• Confocal Microscopes are very expensive.

• It contains a limited number of excitation wavelengths, with very narrow

bands.
 Applications of the Confocal Microscope

• The Confocal Microscope is used in a wide range of fields including

Biomedical sciences, Cells Biology, genetics, Microbiology, Developmental
Biology, Spectroscopy, Nanoscience (nanoimaging), and Quantum Optics.

• 1. In Biomedical sciences, it is used in the analysis of eye corneal infections,

by quantifying and qualitatively analyzing the endothelial cells of the
cornea.

• 2. Used to identify the presence of fungal elements in the corneal stroma,

during keratomycosis infection, or rapid diagnosis and quick therapeutic
response. It is used in pharmaceutical industries, to ensure the maintenance
of thin-film pharmaceuticals, allowing control of the quality and
uniformity of drug distribution.
• It is used in Stem cell research, DNA hybridization
• Photobleaching studies, Bioluminescent proteins