Microscopy

 Microscopes are instruments that are used in science laboratories to visualize

very minute objects, such as cells and microorganisms, giving a contrasting
image that is magnified.
 Discovered by Antonie van Leeuwenhoek
 Microscopes are made up of lenses for magnification, each with its own
magnification powers.
 Depending on the type of lens, it will magnify the specimen according to its focal
strength.
 Their ability to function is because they have been constructed with special
components that enable them to achieve high magnification levels.
 They can view very small specimens and distinguish their structural differences, for
example, the view of animal and plant cells viewing microscopic bacterial cells.
 There are different types of microscopes like light microscope, dark-field
microscope, phase contrast microscope, electron microscope, fluorescent
microscope, etc.
 Microscopes are generally made up of structural parts for holding and supporting the
microscope and its components and the optical parts that are used for magnification
and viewing of the specimen images.

Structural parts of a microscope and their functions

There are three structural parts of the microscope i.e. head, arm, and base.
1. Head – The head is a cylindrical metallic tube that holds the eyepiece lens at one
end and connects to the nose piece at other end. It is also called a body tube or
eyepiece tube. It connects the eyepiece lens to the objective lens. The light coming
from objectives will bend inside this tube. In binocular microscopes, they are
adjustable so that the viewer can adjust the eyepiece for maximum visualization.
2. Arm – This is the part connecting the base to the head and the eyepiece tube to the
base of the microscope. It supports the head of the microscope and is also used
when carrying the microscope. Some high-quality microscopes have an articulated
arm with more than one joint, allowing more movement of the microscopic head
for better viewing.
3. Base – The base is the lowermost part of the microscope that supports the entire
microscope structure. It provides stability for the microscope. Illuminators, light
switches, and electrical wiring systems are fitted in the base.

Optical parts of a microscope and their functions

The optical parts of the microscope are used to view, magnify, and produce an image
from a specimen placed on a slide. These parts include:
1. Eyepiece – The eyepiece (ocular Lens) is closest to the viewer’s eye. They are
located at the top of the microscope. This part is used to look at the
 specimen. These lenses come in different magnification powers from 5X to 30X,
but the most common ocular lenses are of 10X or 15X magnification. They
magnify the image for the second time.
2. Eyepiece tube – It’s the eyepiece holder. It carries the eyepiece just above the
objective lens. In some microscopes, such as the binoculars, the eyepiece tube is
flexible and can be rotated for maximum visualization for variance in distance. For
monocular microscopes, they are none flexible.
3. Nose piece – A nose piece is a movable circular structure that houses all the
objective lenses. It is also called the revolving turret. It is connected to the body
tube and lies just above the stage. It can be rotated clockwise or counterclockwise
to increase or decrease the magnification. The change in magnification results due
to a change in the objective lens.
4. Objective lenses – The objective lens is the lens that is closest to the specimen.
They are fitted on the nosepiece. A standard microscope has 3 to 4 objective lenses
of different magnifying powers: 4X, 10X, 40X, and 100X. The objective lenses
first receive the light transmitted from the specimen and magnify the image for the
first time. Objective lenses are color-coded and are of different sizes. Size and
color depend on the power of the lens. The smallest lens is of the lowest power,
and gradually, the longest will be of the highest power. In most optical
microscopes, objective lenses with 100X or more magnification are of oil
immersion type.
5. The Adjustment knobs – Adjustment Knobs are the control knobs used to focus
the microscope on the specimen. These knobs are of two types;
a. Fine Adjustment Knob: Fine Adjustment Knob is used for fine adjustment. It
is a smaller knob and is used to move the stage up or down very slowly. The stage
covers a very small distance on each rotation of the fine adjustment knob. It is used
to sharpen the image. It is mostly used while viewing under high power.
b. Coarse Adjustment Knob: Coarse Adjustment Knob is used for focusing the
image under low power magnification. It is a larger knob and is used to move the
stage up or down very rapidly. The stage is raised or lowered rapidly with the help
of a coarse adjustment knob.
6. Stage – This is the section in which the specimen is placed for viewing. They have
stage clips that hold the specimen slides in place. The most common stage is the
mechanical stage, which allows the control of the slides by moving the slides using
the mechanical knobs on the stage instead of moving them manually.
7. Stage Control Knobs – Stage Control Knobs are the control knobs used to move
the stage mechanically. There are two knobs; one for moving left and right and the
other for moving forward and backward. This will move the slide in the field of
vision.
8. Aperture – This is a hole in the microscope stage through which the transmitted
light from the source reaches the stage.
9. Microscopic illuminator – A microscopic illuminator is a light source. In some
compound microscopes, a mirror, which reflects the light from an external source
to the sample, is used. In other optical microscopes, different electric bulbs of low
voltages are used as a constant light source. Commonly used illuminators are
tungsten-halogen lamps, 75-150W Xenon lamps, tin-halide lamps, mercury vapor
lamps, etc. The selection of types of bulbs is based on the requirement of intensity
and wavelength for illumination.
[Link] – These are lenses that are used to collect and focus light from the
illuminator into the specimen. They are found under the stage next to the
diaphragm of the microscope. They play a major role in ensuring clear, sharp
images are produced with a high magnification of 400X and above. The higher the
magnification of the condenser, the clearer the image. More sophisticated
microscopes come with an Abbe condenser that has a high magnification of about
1000X.
[Link] – It’s also known as the iris. It is found under the stage of the
microscope, and its primary role is to control the amount of light that reaches the
specimen. It’s an adjustable apparatus, hence controlling the light intensity and the
size of the beam of light that gets to the specimen. For high-quality microscopes,
the diaphragm comes attached with an Abbe condenser, and combined, they are
able to control the light focus and light intensity that reaches the specimen.
[Link] focus knob – This is a knob that moves the condenser up or down,
thus controlling the focus of light on the specimen.
[Link] Switch – Light Switch is an electrical control device. Light switches are
used to on and off the illuminator.
[Link] Adjustment – The brightness adjustment system controls the voltage
supplied to the light bulb, controlling the intensity (brightness) of the light bulb.
Working
In a simple light microscope, a thin specimen containing a slide is placed on the
microscope’s stage.
A beam of light is passed through the condenser to the specimen. The light
transmitted from the specimen enters the objective lens. While passing through the
objectives, the transmitted rays are spread so that they appear to come from the
bigger objects.
The light is then focused on the eyepiece lens. This lens further magnifies the pre-
magnified image coming from the objectives.
Finally, a highly magnified image can be observed.
Applications
 Microscopes are used in examining the ailments by getting a larger view of
the blood sample in detecting the parasites, bacterias attacking the red blood.
 Scientists use a microscope for studying microorganisms, cells, crystalline
structures, and molecular structures.
 Microscopes help doctors diagnose the tissue sample.
 The use of a compound microscope is found in examining the following
samples:
1. Blood cells
2. Cheek cells
3. Parasites
4. Bacteria
5. Algae
6. Tissue
7. Thin sections of organs
8. Detecting the UTI (urinary tract infection) in the urine sample
9. Detecting germs
[Link] crime cases
[Link] in performing research and medical advancements
[Link] the cause of diseases
[Link] aid for prevention of diseases
[Link] electronic devices and circuits
 [Link] of microorganisms
[Link] open branches of sciences
[Link] Forensics

In forensics, a microscope is used to study general criminal science, forensic

epidemiology, forensic anthropology, and forensic pathology.

Magnification
 It is the ratio of the size of an object seen under microscope to the actual size
observed with unaided eye.
 The total magnification of microscope is calculated by multiplying the magnifying
power of the objective lens by that of eye piece.
 Magnification refers to the process of enlarging the apparent size of an object,
allowing for the observation of details that are too small to be seen with the naked
eye.
 It's quantified by a size ratio, or the ratio of the image size to the object's actual size.
 Microscopes utilize lenses to achieve this, with the total magnification being the
product of the objective lens magnification and the eyepiece magnification

Resolving power
 It is the ability to differentiate two close points as separate.
 The resolving power of human eye is 0.25 mm
 The light microscope can separate dots that are 0.25µm apart.
 The electron microscope can separate dots that are 0.5nm apart.
 It depends on Numerical Aperture (NA) of objective and wavelength of light.
 Resolving power = λ / 2*n sinθ
= λ / 2*NA , where NA = n sinθ
 Working distance
 It is the distance between the objective and the object slide.
 Working distance decreases with increase in the magnification.
 Working distance in microscopy is the space, or linear distance, between the front
lens of a microscope objective and the point where the specimen is in sharp focus,
typically the surface of the specimen or the coverslip.

Numerical aperture
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. Image-forming light
waves pass through the specimen and enter the objective in an inverted cone as
illustrated in Figure 1. A longitudinal slice of this cone of light shows the angular
aperture, a value that is determined by the focal length of the objective.
 θ

The angle µ is one-half the angular aperture (A) and is related to the numerical aperture through the
following equation:

Numerical Aperture (NA) = n(sin θ)

where n is the refractive index of the imaging medium between the front lens of the
objective and the specimen cover glass and θ is ½ angle of oblique ray that enters the
lens of object.
 LIGHT MICROSCOPY
A light microscope is a biology laboratory instrument or tool, that uses visible
light to detect and magnify very small objects and enlarge them.
 They use lenses to focus light on the specimen, magnifying it thus producing an
image. The specimen is normally placed close to the microscopic lens.
 Microscopic magnification varies greatly depending on the types and number of
lenses that make up the microscope. Depending on the number of lenses, there are
two types of microscopes i. e Simple light microscope (it has low magnification
because it uses a single lens) and the Compound light microscope (it has a higher
magnification compared to the simple microscope because it uses at least two sets
of lenses, an objective lens, and an eyepiece). The lenses are aligned in that, they
can be able to bend light for efficient magnification of the image.
 The functioning of the light microscope is based on its ability to focus a beam of
light through a specimen, which is very small and transparent, to produce an image.
The image is then passed through one or two lenses for magnification for viewing.
The transparency of the specimen allows easy and quick penetration of light.
Specimens can vary from bacterial to cells and other microbial particles.
PRINCIPLE
As mentioned earlier, light microscopes visualize an image by using a glass lens, and
magnification is determined by, the lens’s ability to bend light and focus it on the
specimen, which forms an image. When a ray of light passes through one medium into
another, the ray bends at the interface causing refraction. The bending of light is
determined by the refractive index, which is a measure of how great a substance slows
the speed of light. The direction and magnitude of the bending of the light are
determined by the refractive indexes of the two mediums that form the interface.
 PARTS
 Eyepiece (Ocular lens) – it has two eyepiece lenses at the top of the microscope
which focuses the image from the objective lenses. this is where you see the formed
image from, with your eyes.
 The objective lenses which are made up of six or more glass lenses, which make
a clear image clear from the specimen or the object that is being focused.
 Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob,
found on the microscopes’ arm, which can move the stage or the nosepiece to focus
on the image. Their function is to ensure the production of a sharp image with
clarity.
 The stage is found just below the objectives and this is where the specimen is
placed, allowing movement of the specimen around for better viewing with the
flexible knobs and it is where the light is focused on.
 The condenser: It is mounted below the stage which focuses a beam of light onto
the specimen. It can be fixed or movable, to adjust the quality of light, but this
entirely depends on the microscope.
 The arm: This is a sturdy metallic backbone of the microscope, used to carry and
move the microscope from one place to another. They also hold the
microscope base which is the stand of the microscope. The arm and the base hold
all the microscopic parts.
 It has a light illuminator or a mirror found at the base or on the microscope’s
nosepiece.
 The nosepiece has about two to five objective lenses with different magnifying
power. It can move round to any position depending on the objective lens to focus
on the image.
 An aperture diaphragm (contrast): It controls the diameter of the beam of light
that passes through the condenser. When the condenser is almost closed, the light
comes through to the center of the condenser creating high contrast and when the
condenser is widely open, the image is very bright with very low contrast.
 APPLICATIONS
Brightfield Microscope is used in several fields, from basic biology to understanding
cell structures in cell Biology, Microbiology, Bacteriology to visualizing parasitic
organisms in Parasitology. Most of the specimens to be viewed are stained using
special staining to enable visualization. Some of the staining techniques used include
Negative staining and Gram staining.
Some of its applications include:
1. Used to visualize and study the animal and plant cells
2. Used to visualize and study the morphologies of bacterial cells
3. Used to identify parasitic protozoans such as Paramecium.
Advantages of Brightfield Microscope
1. It is simple to use with few adjustments involved while viewing the image.
2. It can be used to view both stained and unstained.
3. The optics of the microscope do not alter the color of the specimen.
4. The microscope can be adjusted and modified for better viewing such as installing
a camera, to form a digital microscope or in the way image illumination is done
such as by use of fluorochromes on the specimen and viewing under a dark
environment, forming a darkfield microscope.
Disadvantages of Brightfield microscope
1. It can not be used to view live specimens such as bacterial cells. Only fixed
specimens can be viewed under the brightfield microscope.
2. The maximum magnification of the brightfield microscope is 100x but
modification can readjust the magnification to 1000x which is the optimum
magnification of bacterial cells.
3. It has low contrast hence most specimens must be stained for them to be visualized.
4. The use of oil immersion may distort the image
5. The use of a coverslip may damage the specimen
6. Staining may introduce extraneous unwanted details into the specimen or
contaminate the specimen.
7. It is tedious to stain the specimen before visualizing it under the brightfield
microscope.
8. The microscope needs a strong light source for magnification and sometimes the
light source may produce a lot of heat which may damage or kill the specimen.
 DARK FIELD MICROSCOPY
 This is similar to the ordinary light microscope; however, the condenser system is
modified so that the specimen is not illuminated directly.
 The condenser directs the light obliquely so that the light is deflected or scattered
from the specimen, which then appears bright against a dark background.
 Living specimens may be observed more readily with darkfield than with
brightfield microscopy.
Principle
 A dark field microscope is arranged so that the light source is blocked off,
causing light to scatter as it hits the specimen.
 This is ideal for making objects with refractive values similar to the background
appear bright against a dark background.
 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.
 Working
1. The dark-ground microscopy makes use of the dark-ground microscope, a special
type of compound light microscope.
2. The dark-field condenser with a central circular stop, which illuminates the object
with a cone of light, is the most essential part of the dark-ground microscope.
3. This microscope uses reflected light instead of transmitted light used in the
ordinary light microscope.
4. It prevents light from falling directly on the objective lens.
5. Light rays falling on the object are reflected or scattered onto the objective lens
with the result that the microorganisms appear brightly stained against a dark
background.

Applications
 It is useful for the demonstration of very thin bacteria not visible under
ordinary illumination since the reflection of the light makes them appear
larger.
 This is a frequently used method for rapid demonstration of Treponema
pallidum in clinical specimens.
 It is also useful for the demonstration of the motility of flagellated bacteria
and protozoa.
 Darkfield is used to study marine organisms such as algae, plankton, diatoms,
insects, fibers, hairs, yeast and protozoa as well as some minerals and
crystals, thin polymers and some ceramics.
 Darkfield is used to study mounted cells and tissues.
 It is more useful in examining external details, such as outlines, edges, grain
boundaries and surface defects than internal structure.

Advantages of Darkfield Microscope

 Dark-field microscopy is a very simple yet effective technique.
 It is well suited for uses involving live and unstained biological samples, such as a
smear from a tissue culture or individual, water-borne, single-celled organisms.
 Considering the simplicity of the setup, the quality of images obtained from this
technique is impressive.
 Dark-field microscopy techniques are almost entirely free of artifacts, due to the
nature of the process.
 A researcher can achieve a dark field by making modifications to his/her
microscope.

Limitations of Darkfield Microscope

 The main limitation of dark-field microscopy is the low light levels seen in the
final image.
 The sample must be very strongly illuminated, which can cause damage to the
sample.
 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.
 Phase-contrast microscopy is an optical microscopy technique that converts phase
shifts in the light passing through a transparent specimen to brightness changes in
the image.
 It was first described in 1934 by Dutch physicist Frits Zernike.

Principle
 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, which can be observed as differences in image contrast.
Parts
Phase-contrast microscopy is basically a specially designed light microscope with
all the basic parts in addition to which an annular phase plate and annular diaphragm
are fitted.
 The annular diaphragm
 It is situated below the condenser.
 It is made up of a circular disc having a circular annular groove.
 The light rays are allowed to pass through the annular groove.
 Through the annular groove of the annular diaphragm, the light rays fall on the
specimen or object to be studied.
 At the back focal plane of the objective develops an image.
 The annular phase plate is placed at this back focal plane.
The phase plate
 It is either a negative phase plate having a thick circular area or a positive phase
plate having a thin circular groove.
 This thick or thin area in the phase plate is called the conjugate area.
 The phase plate is a transparent disc.
 With the help of the annular diaphragm and the phase plate, the phase contrast is
obtained in this microscope.
 This is obtained by separating the direct rays from the diffracted rays.
 The direct light rays pass through the annular groove whereas the diffracted light
rays pass through the region outside the groove.
 Depending upon the different refractive indices of different cell components, the
object to be studied shows a different degree of contrast in this microscope.
Working
1. Partially coherent illumination produced by the tungsten-halogen lamp is directed
through a collector lens and focused on a specialized annulus (labeled condenser
annulus) positioned in the substage condenser front focal plane.
2. Wavefronts passing through the annulus illuminate the specimen and either pass
through undeviated or are diffracted and retarded in phase by structures and phase
gradients present in the specimen.
3. Undeviated and diffracted light collected by the objective is segregated at the rear
focal plane by a phase plate and focused at the intermediate image plane to form
the final phase-contrast image observed in the eyepieces.
 Applications
To produce high-contrast images of transparent specimens, such as
1. living cells (usually in culture),
2. microorganisms,
3. thin tissue slices,
4. lithographic patterns,
5. fibers,
6. latex dispersions,
7. glass fragments, and
8. subcellular particles (including nuclei and other organelles).
Advantages of Phase contrast Microscopy
 Living cells can be observed in their natural state without previous fixation or
labeling.
 It makes a highly transparent object more visible.
 No special preparation of fixation or staining etc. is needed to study an object under
a phase-contrast microscope which saves a lot of time.
 Examining intracellular components of living cells at relatively high resolution.
eg: The dynamic motility of mitochondria, mitotic chromosomes & vacuoles.
 It made it possible for biologists to study living cells and how they proliferate
through cell division.
 Phase-contrast optical components can be added to virtually any brightfield
microscope, provided the specialized phase objectives conform to the tube length
parameters, and the condenser will accept an annular phase ring of the correct size.
Limitations of Phase contrast Microscopy
 Phase-contrast condensers and objective lenses add considerable cost to a
microscope, and so phase contrast is often not used in teaching labs except perhaps
in classes in the health professions.
 To use phase-contrast the light path must be aligned.
 Generally, more light is needed for phase contrast than for corresponding bright-
field viewing, since the technique is based on the diminishment of the brightness
of most objects.
 FLUORESCENCE MICROSCOPE

 A fluorescence microscope is an optical microscope that uses fluorescence and

phosphorescence instead of, or in addition to, reflection and absorption to study the
properties of organic or inorganic substances.
 Fluorescence is the emission of light by a substance that has absorbed light or other
electromagnetic radiation while phosphorescence is a specific type of
photoluminescence related to fluorescence.
 The fluorescence microscope was devised in the early part of the twentieth century
by August Köhler, Carl Reichert, and Heinrich Lehmann, among others.
 Light microscopes that use fluorescence microscopy have the ability to excite
fluorophores and then detect the luminescence signal.
 Photoluminescence is a phenomenon that occurs when a molecule is excited by
ultraviolet or visible light photons to produce luminescence.
 Depending on the electronic configuration of the excited state and the emission
pathway, photoluminescence is divided into two categories: fluorescence and
phosphorescence.
 When light excites or transfers an electron to a higher energy state, fluorescence is
produced, which results in the generation of light after a brief time period (less
than a microseconds) with a longer wavelength, lower energy, and a different hue
from the first absorbed light. This time period is known as the fluorescence
lifetime.
 Similar to fluorescence, phosphorescence happens in a similar way but has a
significantly longer excited state lifespan.
Principle
 The initial step in the observation of the sample through a fluorescence microscope
includes labelling the sample with fluorescent dyes.
 Then, light source which emits white light is allowed to fall onto the excitation
filter.
 This filter selects the light of a specific wavelength that can excite the fluorescent
molecules tagged in the specimen and this excitation light incidents onto the
dichroic mirror.
 The light after reflection from the dichroic mirror passes onto the specimen after
emerging from the objective lens.
 This small wavelength light falls into the specimen stained with a fluorescent dye
that results in emission of high wavelength light which passes again through the
condenser lens and dichroic mirror.
 This allows green light in maximum along with some blue light to pass towards
the emission filter.
 However, this filter only permits the longer wavelength green light to pass into the
eyepiece and detector while at the same time rejecting the blue light completely.
 The detector detects the green light and permits it to fall back onto the specimen
thereby, forming fluorescent green specimens against a dark background.

Parts
 Fluorophore: These are reactive fluorescent dyes which form a fluorescent image
by generating highly contrasted visible green light after being activated by highly
illuminating UV radiation.
 Light Source: Major light sources include xenon arc lamps, mercury-vapour
lamps, lasers and high-power LEDs. A simple epifluorescent microscope makes
use of a light source made up of xenon lamps, mercury lamps, and LEDs. On the
other hand, laser light is mostly used by the advanced confocal fluorescence
microscope.
 Excitation filter: The excitation filter is a bandpass filter that functions to narrow
the wavelength of the light that illuminates the sample by blocking other sources
of exciting light. Such light of shorter wavelength can be easily absorbed by the
fluorescent dye.
 Dichroic Mirror: It is also known as dichromatic mirror or beam-splitter. It
selectively reflects or transmits light with specific wavelengths.
 Objective/ Condenser lens: The light is then directed through a system of optics
that serves as both a condenser, gathering the light into a narrow beam on the
sample, and a focusing objective for the light emitted back by the specimen.
 Sample stage: The sample stage holds the specimen and contains x, y, and z-axis
movements that can be controlled manually or by a computer.
 Emission filter: It is a bandpass filter that functions similarly to an excitation
filter. The light emitted from the specimen includes light reflected from the sample,
which will be the excitation wavelength as well as light emitted from the
fluorescent components of the specimen. Since, certain samples may include
different fluorescent material, light of different wavelengths may be emitted. It
allows fluorophore light radiations to pass while blocking excitation light.
 Eyepieces and detector: The light after passing through an emission filter is
directed toward a set of eyepieces to facilitate the user to see or to a camera. The
fluorescence images can then be quantitatively analysed using a combination of
digital imaging and image processing.
Working
 The specimen to be observed are stained or labeled with a fluorescent dye and then
illuminated with high intensity ultra violet light from mercury arc lamp.
 The light passes through the exciter filter that allows only blue light to pass
through.
 Then the blue light reaches dichroic mirror and reflected downward to the
specimen.
 The specimen labeled with fluorescent dye absorbs blue light (shorter wavelength)
and emits green light.
 The emitted green light goes upward and passes through dichroic mirror, reflects
back blue light and allows only green light to pass the objective lens, then it reaches
barrier filter which allows only green light.
 The filtered fluorescent emission is sent to a detector where the image can be
digitized.

Applications
 Neurotransmitters such as dopamine, serotonin, norepinephrine, and epinephrine
can be detected using fluorescence microscopes even though they are much too
small to be seen by a conventional light microscope.
 These are used by the food chemists in quality control by accessing the structural
organisation and chemical processes of the food components and ensuring the
safety of the consumers.
 It enables researchers to understand the cycles of protein synthesis and degradation
as well as how proteins travel. The location of the mineral’s origin can also be
determined more easily.
 It is employed in textile industries for the analysis of fibres and obtaining clear 3D
images of fibres and yarns.
 It is used to examine the porosity of ceramics, enabling researchers to determine
an object’s density, strength, and durability.
Advantages of Fluorescence Microscope
 Adaptable: Many conventional bright-field microscopy techniques can be
replaced and enhanced by fluorescence microscopy techniques. If the instruments
are outfitted with a few fluorescence attachments, the majority of compound
microscopes can be utilized for fluorescence microscopy.
 Easy: Examining fluorescent specimens is easy due to the great contrast.
 Rapid: Specimens can be studied directly on a slide, frequently without any
preliminary purification or concentration
 Reliable: The great sensitivity and specificity of fluorescence microscopy
techniques are responsible for the good reliability and accuracy of microscopic
analysis. Even when performed by technicians with less training, the likelihood of
incorrect observations or discrepancies in test interpretation is decreased.
 Sensitive: Small things are simple to see because of the comparatively bright
fluorescent picture on a dark background.
 Specific: High specificity for details is achieved by using modern fluorochromes
and interference light filters.
 Universal: Numerous biomedical fields, including bacteriology, mycology,
virology, parasitology, serology, immunology, autoimmunology, cytology, cell
biology, and histochemistry, can benefit from the use of fluorescence microscopy.
Limitations of Fluorescence Microscope
 The addition of probes and dyes to a membrane system has the ability to alter the
characteristics of the liposomal delivery system. However, it has been
demonstrated that the physical characteristics of the membrane are only slightly
affected by the application of low dye concentrations (1 mol%).
 The selection of the fluorescent dye is an important step since some dyes might
produce significant changes in the host membrane and/or experimental errors,
leading to incorrect data interpretation.
 Bleaching and a reduction in fluorescence intensity can also happen after
prolonged exposure to fluorescent light.
 CONFOCAL MICROSCOPY
 Confocal microscopy is also known as confocal laser scanning microscopy (CLSM)
or laser confocal scanning microscopy (LCSM).
 Confocal microscope is an advanced form of flurosence microscope.
 It is an optical imaging technique for increasing the optical contrast and resolution
of a microscope sample using a spatial pinhole to remove the out-of-focus light in
forming the image.
 Fluorescently stained specimens are usually examined.
 A focused laser beam strikes a point in the specimen .
 Uses pin hole to form high resolution images.
PRINCIPLE
 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 are started 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.
WORKING
 Confocal microscope incorporate 2 ideas :
 Pin – point illumination of the specimen.
 Rejection of out of focus of light.
 Light source of very high intensity is used – Zirconium arc lamp in Minsky’s design
and laser light source in morden design.
A). Laser provide intense blue excitation light
B). The light reflects off a dichoric mirror, which directs it to an assembly of
vertically and horizontally scanning mirrors.
C). These mirrors scan the laser beam across the specimen
D). The specimen is scanned by moving the stage back and forth in the vertical &
horizontal directions and optics are kept stationary.
 Dye in the specimen is excited by the laser light & fluroescens.
 The fluroscent ( green )light is descanned by the same mirrors that are used to scan
the excitation ( blue) light from the laser beam.
 Then it passes through the dichoric mirror.
 Then it is focussed on to pinhole
 The light passing through the pinhole is measured by the detector such as
photomultiplier tube
 For visualisation, detector is attached to the computer, which builds up the image
at the rate of 0.1 – 1 sec for single image

APPLICATIONS
 Used to describe physiological processes in vivo in the cornea, kidney and liver.
 It has also been shown to be able to illustrate the healing of wounds in four
dimensions(x, y, z, t) at a cellular level.
 Used for qualitative analysis, and quantification of endothelial cells of the cornea.
 Used for localizing of filamentary fungal elements in the corneal stroma incases of
keratomycosis.
 Used to create 3d image of samples.

TYPES
1. Laser scanning confocal microscope
 It uses laser and illumination pinhole to get point like light source llumination
on specimen.
 PMTs are used as detecting device.
 Main disadvantage is slow frame rate, photo bleaching, phototoxicity.

2. Multiphoton laser confocal microscope

 This type of confocal uses two lasers of longer wavelength.
 Advantage of MP is its low phototoxicity , can work with even thicker specimen

3 . Disk scanning confocal microscope

 In this a spinning disk with multiple small holes between light source and
specimen is used.
 CCD camera is used as detector.
 The main advantage is its high scan speed
 ADVANTAGES
 3d image can be generated
 Depth of field can be controlled
 Used for both in-vitro and in-vivo imaging
 Higher resolution
 Imaging of thick specimen
 Photobleaching, phototoxicity.
 They have a limited number of excitation wavelengths, with very narrow bands.
 They are expensive to produce the ultraviolet rays used by the confocal microscopes.
 They are also expensive to manufacture and to purchase.
DISADVANTAGES
 Photobleaching, phototoxicity.
 They have a limited number of excitation wavelengths, with very narrow bands.
 They are expensive to produce the ultraviolet rays used by the confocal microscopes.
 They are also expensive to manufacture and to purchase.
 ELECTRON MICROSCOPY
 An electron microscope is a microscope that uses a beam of accelerated electrons
as a source of illumination.
 It is a special type of microscope having a high resolution of images, able to
magnify objects in nanometres, which are formed by controlled use of electrons
in a vacuum captured on a phosphorescent screen.
 Ernst Ruska (1906-1988), a German engineer and academic professor, built the
first Electron Microscope in 1931, and the same principles behind his prototype
still govern modern EMs.
Working Principle of Electron Microscope
 Electron microscopes use signals arising from the interaction of an electron beam
with the sample to obtain information about structure, morphology, and
composition.
1. The electron gun generates electrons.
2. Two sets of condenser lenses focus the electron beam on the specimen and then
into a thin tight beam.
3. To move electrons down the column, an accelerating voltage (mostly between 100
kV-1000 kV) is applied between the tungsten filament and anode.
4. The specimen to be examined is made extremely thin, at least 200 times thinner
than those used in the optical microscope. Ultra-thin sections of 20-100 nm are cut
which is already placed on the specimen holder.
5. The electronic beam passes through the specimen and electrons are scattered
depending upon the thickness or refractive index of different parts of the specimen.
6. The denser regions in the specimen scatter more electrons and therefore appear
darker in the image since fewer electrons strike that area of the screen. In contrast,
transparent regions are brighter.
7. The electron beam coming out of the specimen passes to the objective lens, which
has high power and forms the intermediate magnified image.
8. The ocular lenses then produce the final further magnified image.
 Parts of Electron Microscope
Electron Microscope is in the form of a tall vacuum column that is vertically
mounted. It has the following components:
1. Electron gun
 The electron gun is a heated tungsten filament, which generates electrons.
2. Electromagnetic lenses
 The condenser lens focuses the electron beam on the specimen. A second
condenser lens forms the electrons into a thin tight beam.
 The electron beam coming out of the specimen passes down the second of
magnetic coils called the objective lens, which has high power and forms the
intermediate magnified image.
 The third set of magnetic lenses called projector (ocular) lenses produce the final
further magnified image.
 Each of these lenses acts as an image magnifier all the while maintaining an
incredible level of detail and resolution.
3. Specimen Holder
 The specimen holder is an extremely thin film of carbon or collodion held by a
metal grid.
4. Image viewing and Recording System
 The final image is projected on a fluorescent screen.
 Below the fluorescent screen is a camera for recording the image.

Applications of Electron microscope

 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.
 Industrially, electron microscopes are often used for quality control and failure
analysis.
 Modern electron microscopes produce electron micrographs using specialized
digital cameras and frame grabbers to capture the images.
 The science of microbiology owes its development to the electron microscope. The
study of microorganisms like bacteria, virus, and other pathogens have made the
treatment of diseases very effective.
Advantages of Electron microscope
 Very high magnification
 Incredibly high resolution
 Material rarely distorted by preparation
 It is possible to investigate a greater depth of field
 Diverse applications

Limitations of Electron microscope

 The live specimen cannot be observed.
 As the penetration power of the electron beam is very low, the object should be
ultra-thin. For this, the specimen is dried and cut into ultra-thin sections before
observation.
 As the EM works in a vacuum, the specimen should be completely dry.
 Expensive to build and maintain
 Requiring researcher training
 Image artifacts resulting from specimen preparation.
 This type of microscope is large, cumbersome extremely sensitive to vibration and
external magnetic fields.
Types of Electron microscope
There are two types of electron microscopes, with different operating styles:
1. Transmission Electron Microscope (TEM)
 The transmission electron microscope is used to view thin specimens through
which electrons can pass generating a projection image.
 The TEM is analogous in many ways to the conventional (compound) light
microscope.
 TEM is used, among other things, to image the interior of cells (in thin sections),
the structure of protein molecules (contrasted by metal shadowing), the
organization of molecules in viruses and cytoskeletal filaments (prepared by the
negative staining technique), and the arrangement of protein molecules in cell
membranes (by freeze-fracture).
Principle of Transmission Electron Microscope (TEM)
 The working principle of the Transmission Electron Microscope (TEM) is similar
to the light microscope.
 The major difference is that light microscopes use light rays to focus and produce
an image while the TEM uses a beam of electrons to focus on the specimen, to
produce an image.
 Electrons have a shorter wavelength in comparison to light which has a long
wavelength.
 The mechanism of a light microscope is that an increase in resolution power
decreases the wavelength of the light, but in the TEM, when the electron illuminates
the specimen, the resolution power increases increasing the wavelength of the
electron transmission.
 The wavelength of the electrons is about 0.005nm which is 100,000X shorter than
that of light, hence TEM has better resolution than that of the light microscope, of
about 1000times.
 This can accurately be stated that the TEM can be used to detail the internal
structures of the smallest particles like a virion particle.
Parts of Transmission Electron Microscope (TEM)
Their working mechanism is enabled by the high-resolution power they produce which
allows it to be used in a wide variety of fields. It has three working parts which include:
1. Electron gun
2. Image producing system
3. Image recording system
 Electron gun
 This is the part of the Transmission Electron Microscope responsible for producing
electron beams.
 Electrons are produced by a cathode that is a tungsten filament that is V-shaped
and it is normally heated. The tungsten filament is covered by a control grid known
as a Wehnelt cylinder made up of a central hole which lies columnar to the tube.
The cathode lies on top of or below the cylindrical column hole. The cathode and
the control grid are negatively charged with an end of the anode which is disk-
shaped that also has an axial hole.
 When electrons are transmitted from the cathode, they pass through the columnar
aperture (hole) to the anode at high voltage with constant energy, which is efficient
for focusing the specimen to produce an accurately defined image.
 It also has the condenser lens system which works to focus the electron beam on
the specimen by controlling the energy intensity and the column hole of the
electron gun. The TEM uses two condenser lenses to converge the beam of
electrons to the specimen. The two condenser lens each function to produce an
image i.e the first lens which has strong magnification, produces a smaller image
of the specimen, to the second condenser lens, directing the image to the objectives.
Image- Producing system
 Its made up of the objective lens, a movable stage or holding the specimen,
intermediate and projector lenses. They function by focusing the passing electrons
through the specimen forming a highly magnified image.
 The objective has a short focal length of about 1-5mm and it produces an
intermediate image from the condenser which are transmitted to the projector
lenses for magnification.
 The projector lenses are of two types, i.e the intermediate lens which allows great
magnification of the image and the projector lens which gives a generally greater
magnification over the intermediate lens.
 To produce efficient high standard images, the objectives and the projector lenses
need high power supplies with high stability for the highest standard of resolution.
Image-Recording System
 Its made up of the fluorescent screen used to view and to focus on the image. They
also have a digital camera that permanently records the images captured after
viewing.
 They have a vacuum system that prevents the bombardment or collision of
electrons with air molecules disrupting their movement and ability to focus. A
vacuumed system facilitates the straight movement of electrons to the image.
 The vacuumed system is made up of a pump, gauge, valves and a power supply.
 The image that is formed is called a monochromatic image, which is greyish or
black and white. The image must be visible to the human eye, and therefore, the
electrons are allowed to pass through a fluorescent screen fixed at the base of the
microscope.
 The image can also be captured digitally and displayed on a computer and stored
in a JPEG or TIFF format. During the storage, the image can be manipulated from
its monochromatic state to a colored image depending on the recording apparatus
eg use of pixel cameras can store the image in color.
 The presence of colored images allows easy visualization, identification, and
characterization of the images.
Working
From the instrumentation described, the working mechanism is a sequential process
of the parts of the TEM mentioned above. To mean:
 A heated tungsten filament in the electron gun produces electrons that get focus on
the specimen by the condenser lenses.
 Magnetic lenses are used to focus the beam of electrons of the specimen. By the
assistance offered by the column tube of the condenser lens into the vacuum
creating a clear image, the vacuum allows electrons to produce a clear image
without collision with any air molecules which may deflect them.
 On reaching the specimen, the specimen scatters the electrons focusing them on
the magnetic lenses forming a large clear image, and if it passes through a
fluorescent screen it forms a polychromatic image.
 The denser the specimen, the more the electrons are scattered forming a darker
image because fewer electron reaches the screen for visualization while thinner,
more transparent specimens appear brighter.

Applications of Transmission Electron Microscope (TEM)

TEM is used in a wide variety of fields From Biology, Microbiology,
Nanotechnology, forensic studies, etc. Some of these applications include:
1. To visualize and study cell structures of bacteria, viruses, and fungi
2. To view bacteria flagella and plasmids
3. To view the shapes and sizes of microbial cell organelles
4. To study and differentiate between plant and animal cells.
5. Its also used in nanotechnology to study nanoparticles such as ZnO nanoparticles
6. It is used to detect and identify fractures, damaged microparticles which further
enable repair mechanisms of the particles.
Advantages of Transmission Electron Microscope (TEM)
1. It has a very powerful magnification of about 2 million times that of the Light
microscope.
2. It can be used for a variety of applications ranging from basic Biology to
Nanotechnology, to education and industrial uses.
3. It can be used to acquire vast information on compounds and their structures.
4. It produces very efficient, high-quality images with high clarity.
5. It can produce permanent images.
6. It is easy to train and use the Transmission Electron Microscope
Limitations of Transmission Electron Microscope (TEM)
1. Generally, the TEMs are very expensive to purchase
2. They are very big to handle.
3. The preparation of specimens to be viewed under the TEM is very tedious.
4. The use of chemical fixations, dehydrators, and embedments can cause the dangers
of artifacts.
5. They are laborious to maintain.
6. It requires a constant inflow of voltage to operate.
7. They are extremely sensitive to vibrations and electro-magnetic movements hence
they are used in isolated areas, where they are not exposed.
8. It produces monochromatic images, unless they use a fluorescent screen at the end
of visualization.
 2. Scanning Electron Microscope (SEM)

 Conventional scanning electron microscopy depends on the emission of secondary

electrons from the surface of a specimen.
 Because of its great depth of focus, a scanning electron microscope is the EM
analog of a stereo light microscope.
 It provides detailed images of the surfaces of cells and whole organisms that are
not possible by TEM. It can also be used for particle counting and size
determination, and for process control.
 It is termed a scanning electron microscope because the image is formed by
scanning a focused electron beam onto the surface of the specimen in a raster
pattern.
 Scanning Electron Microscope (SEM) is a type of electron microscope that scans
surfaces of microorganisms that uses a beam of electrons moving at low energy to
focus and scan specimens.
 The development of electron microscopes was due to the inefficiency of the
wavelength of light microscopes.
 Electron microscopes have very short wavelengths in comparison to the light
microscope which enables better resolution power.
Principle of Scanning Electron Microscope (SEM)
 Unlike the Transmission Electron Microscope which uses transmitted electrons, the
scanning electron Microscope uses emitted electrons.
 The Scanning electron microscope works on the principle of applying kinetic
energy to produce signals on the interaction of the electrons.
 These electrons are secondary electrons, backscattered electrons, and diffracted
backscattered electrons which are used to view crystallized elements and photons.
 Secondary and backscattered 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 while the backscattered
electrons show contrast in the composition of the elements of the specimen.
Working
 The source of the electrons and the electromagnetic lenses are
from tungsten filament lamps that are placed at the top of the column and it is
similar to those of the transmission electron Microscope.
 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.
 The beam of electrons activates the emission of primary scattered (Primary)
electrons at high energy levels and secondary electrons at low-energy levels from
the specimen surface.
 The beam of electrons interacts with the specimen to produce signals that give
information about the surface topography and composition of the specimen.
 The specimen does not need special treatment for visualization under the SEM,
even air-dried samples can be examined directly.
 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.
 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.
 Scanning by this microscope is attained by tapering a beam of electrons back and
forth over a thin section of the microscope.
 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 or high-
energy photon).
 This emits flashes of light which get 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
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, backscattered electrons, and diffracted backscattered
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
 Like the transmission electron Microscope, the Scanning electron microscope should
be free from vibrations and any electromagnetic elements.
Applications
It is used in a variety of fields including Industrial uses, nanoscience studies,
Biomedical studies, Microbiology
1. Used for spot chemical analysis in energy-Dispersive X-ray Spectroscopy.
2. Used in the analysis of cosmetic components which are very tiny in size.
3. Used to study the filament structures of microorganisms.
4. Used to study the topography of elements used in industries.
Advantages of the Scanning Electron Microscope (SEM)
 They are easy to operate and have user-friendly interfaces.
 They are used in a variety of industrial applications to analyze surfaces of solid
objects.
 Some modern SEMs are able to generate digital data that can be portable.
 It is easy to acquire data from the SEM, within a short period of time of about 5
minutes.
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