UNIT I

MICROSCOPY

Microscopy is the study of devices (microscopes) that are used to view objects or
certain areas that cannot be seen with a naked eye.

Theoretical Principles of Microscopy

 Magnification is the ability of a microscope to produce an image of an

object at a scale larger than its actual size. Magnification serves a useful
purpose only when it is possible to see more details of an object in the
image than when observing the object with the unaided eye.
 Magnification is the ratio of the size of a specific feature of an object or
sample as seen in an image produced by an optical system to the actual size
of the feature on the object itself.
 Thus, lateral magnification, MDIS, can be defined as:

 When observing the image through the eyepieces of a microscope for visual
observation, the total (lateral) magnification is defined as:

MTOT VIS is the total lateral magnification observed through the eyepiece,
MO is the objective lens magnification,
q is the total tube factor (zoom and other tube lenses), and
ME = eyepiece lens magnification.

Resolving Power or Resolution

 Although magnification is important, you must be aware that unlimited
enlargement is not possible by merely increasing the magnifying power of
 the lenses or by using additional lenses, because lenses are limited by a
property called resolving power.
 By definition, resolving power is the ability of a lens to show two adjacent
objects as discrete entities. When a lens cannot discriminate, that is, when
the two objects appear as one, it has lost resolu-tion.
 Increased magnification will not rectify the loss, and will, in fact, blur the
object.
 The resolving power of a lens is dependent on the wave-length of light used
and the numerical aperture, which is a characteristic of each lens and
imprinted on each objective.
 The shorter the wave-length, the greater the resolving power of the lens.
 Resolving power is dependent on another factor, the refractive index. This
is the bending power of light passing through air from the glass slide to the
objective lens. The refractive index of air is lower than that of glass, and as
light rays pass from the glass slide into the air, they are bent or refracted so
that they do not pass into the objective lens. This would cause a loss of
light, which would reduce the numerical aperture and diminish the
resolving power of the objective lens. Loss of refracted light can be
compensated for by interposing mineral oil, which has the same refractive
index as glass, between the slide and the objective lens. In this way,
decreased light refraction occurs and more light rays enter directly into the
objective lens, producing a vivid image with high resolution.
 The numerical aperture is defined as a function of the diameter of the
objective lens in relation to its focal length.
Thus, resolving power is expressed mathematically by Abbe’s Equation, as
follows:

𝟎. 𝟔𝟏 𝛌
𝐋𝐦 =
𝐧𝐬𝐢𝐧𝛂
where,
Lm is the limit of resolution
n is the refractive index
α is the semiangle
 PARTS OF A MICROSCOPE

A simple microscope is a microscope with one lens system.

Real and magnified images of minuscule particles or objects can be achieved using
a combination of lenses. A compound microscope is an intricate gathering of a
combination of lenses that renders a highly maximized and magnified image of
microscopic living entities and other complex details or tissues and cells.

The parts of the compound microscope can be categorized into:

 Mechanical parts
 Optical parts
(A) Mechanical Parts of a Compound Microscope

1. Foot or base
It is a U-shaped structure and supports the entire weight of the compound
microscope.
2. Pillar
It is a vertical projection. This stands by resting on the base and supports the
stage.
3. Arm
The entire microscope is handled by a strong and curved structure known as the
arm.
4. Stage
The flat and rectangular plate that is connected to the arm’s lower end is called
the stage. The specimen is placed on the stage for studying and examining the
various features. The centre of the stage has a hole through which light can pass.
5. Inclination joint
It is a joint, wherein the arm is fastened to the compound microscope’s pillar. The
microscope can be tilted using the inclination joint.
6. Clips
The upper part of the stage is connected to two clips. The slide can be held in its
position with the help of the clips.
7. Diaphragm
The diaphragm is fastened below the stage. It controls and adjusts the intensity of
light that passes into the microscope. The diaphragm can be of two types:
Disc diaphragm
Iris diaphragm
8. Nose piece
The nose piece is circular and a rotating metal part that is connected to the body
tube’s lower end. The nose piece has three holes wherein the objective lenses are
embedded.
9. Body tube
The upper part of the arm of the microscope comprises a hollow and tubular
structure known as the body tube. The body tube can be shifted down and up
using the adjustment knobs.

10. Fine adjustment knob

It is the smaller knob, which is used for sharp and fine focusing of the object. For
accurate and sharp focusing, this knob can be used.
11. Coarse adjustment knob
It is a large knob that is used for moving the body tube down and up for bringing
the object to be examined under exact focus.

(B) Optical Parts of Compound Microscope

1. Eyepiece lens or Ocular

At the top of the body tube, a lens is planted which is known as the eyepiece. On
the rim of the eyepiece, there are certain markings such as 5X, 10X, 15X, etc.
These indicate the magnification power. The object’s magnified image can be
observed with the help of an eyepiece. Sometimes two ocular lenses are present
in a microscope (Binocular microscopes).
2. Mirror
A mirror is found attached wither to the pillar or the lower end of the arm. It
consists of a concave mirror on one side and a plain mirror on the other side. It
can be used for reflection of light rays into the microscope.
3. Objective lenses
At the bottom of the body tube, there are two objective lenses, which are
connected to the revolving nose piece. The three objective lenses are as follows:
Oil immersion objective – 100X
High power objective – 45X
Low power objective – 10X
 TYPES OF MICROSCOPE
There are mainly two microscopy techniques:
1. Light Microscopy
2. Electron Microscopy
LIGHT MICROSCOPY
Light microscopy is used to make small structures and samples visible by
providing a magnified image of how they interact with visible light, e.g., their
absorption, reflection and scattering. This is useful to understand what the
sample looks like and what it is made of, but also allows us to see processes of the
microscopic world, such as how substances diffuse across a cell membrane.

Image formation in a compound microscope

Light microscopy can be of following types:

a) Brightfield Microscopy
b) Darkfield Microscopy
c) Phase Contrast Microscopy
d) Fluorescence Microscopy
e) Confocal Microscopy
 BRIGHTFIELD MICROSCOPY

Brightfield Microscope is also known as the Compound Light Microscope. It is an

optical microscope that uses light rays to produce a darker image against a highly
lit 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 give 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
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.
Advantages
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

Disadvantages
1. It cannot 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
3. It has low contrast hence most specimens must be stained for them to be
visualized.
4. It is tedious to stain the specimen before visualizing it under the brightfield
microscope.
5. Low resolution
6. Staining may introduce extraneously unwanted details into the specimen or
contaminate the specimen.
7. 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.

Applications
1. Used to visualize and study the animal cells
2. Used to visualize and study plant cells.
3. Used to visualize and study the morphologies of bacterial cells
4. Used to identify parasitic protozoans such as Paramecium.

DARKFIELD 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.

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. This is ideal for making objects with refractive values similar to the
background appear bright against a dark background. 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.
The dark-ground microscopy makes use of the dark-ground microscope, a special
type of compound light microscope. 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.This microscope uses reflected light
instead of transmitted light used in the ordinary light microscope.It prevents light
from falling directly on the objective lens.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.

Advantages
1. Dark-field microscopy is a very simple yet effective technique.
2. 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.
3. Considering the simplicity of the setup, the quality of images obtained from
this technique is impressive.
4. Dark-field microscopy techniques are almost entirely free of artifacts, due to
the nature of the process.
5. A researcher can achieve a dark field by making modifications to his/her
microscope.

Disadvantages
1. Sensitive to dust so a clean environment is needed
2. Sensitive to water bubbles
3. A dark room is always required for better visualisation

Applications
1. 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.
2. This is a frequently used method for rapid demonstration of Treponema
pallidum in clinical specimens.
3. It is also useful for the demonstration of the motility of flagellated bacteria and
protozoa.
4. 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.
5. Darkfield is used to study mounted cells and tissues.
6. It is more useful in examining external details, such as outlines, edges, grain
boundaries and surface defects than internal structure.

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.
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.

Principle

Small phase changes in light rays induced by differences in the thickness and
refractive index of different parts of an object can be transformed into differences
in brightness or light intensity.

Working of Phase Contrast Microscopy

 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.
 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.
 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.
Parts of a Phase Contrast Microscope:
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 micro-scope.

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
1. Living cells can be observed in their natural state without previous fixation
or labeling.
2. It makes a highly transparent object more visible.
3. 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.
4. Examining intracellular components of living cells at relatively high
resolution. eg: The dynamic motility of mitochondria, mitotic chromosomes
& vacuoles.
5. It made it possible for biologists to study living cells and how they
proliferate through cell division.
6. 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.

Disadvantages
1. 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.
2. To use phase-contrast the light path must be aligned.
3. 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.

FLUOROSCENCE MICROSCOPY

Principle
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. 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
reaction, it is known as immunofluorescence.

Components of Fluorescence Microscope

The fluorescence microscope has a
Light source: Xenon arc lamps or mercury-vapor lamps are a common source of
ultraviolet light; power LED and lasers are used in more advanced forms.
A set of optical filters: Optical filters include a set of a compatible excitation filter,
emission filter, and dichroic beam splitter;
An excitation filter selects the wavelengths to excite a particular dye within the
specimen.
A dichroic beam splitter/ dichroic mirror reflects light in the excitation band and
transmits light in the emission band, enabling the classic epifluorescence incident
light illumination.
An emission filter provides quality control by letting only the wavelengths of
interest emitted by the fluorophore pass through.
Darkfield condenser: It provides a black background against which the
fluorescent objects glow.
The filters are often plugged together in a filter cube (compound microscopes) or
a flat holder (mainly stereo microscopes).

Working of Fluorescence Microscope

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

Applications

1. Acid-fast bacilli (AFB) in sputum or CSF are detected when stained

with auramine fluorescent dye.
2. Detection of Trichomonas vaginalis, intracellular gonococci, and other
parasites when stained by acridine orange.
3. In immunodiagnosis of infectious diseases, using both direct and indirect
antibody techniques. Immunofluorescence is especially useful in
diagnosing syphilis and rabies.
4. Tracking down proteins
5. Tracking metabolic pathways within cell.
6. Bacterial infection studies can be performed

Advantages

1. The high degree of specificity possible thanks to modern fluorophore

probes allows specific proteins or other biological structures to be
emphasized
2. The sensitivity of these probes
3. Great contrast and magnification

Disdvantages

1. Fluorophores gradually lose their ability to fluoresce as they are illuminated in

photobleaching. Photobleaching can severely limit the time a sample can be
observed by fluorescence microscopy. However, several techniques exist to
reduce photobleaching, such as using more robust fluorophores, minimizing
illumination, or using photoreactive scavenger chemicals.
2. Fluorescence microscopy has enabled the analysis of live cells, but fluorescent
molecules generate reactive chemical species under illumination that
enhances the phototoxic effect, to which live cells are susceptible.
3. Fluorescence microscopy only allows observation of the specific structures
labeled for fluorescence. For example, observing a tissue sample prepared
with a fluorescent DNA stain by fluorescence microscopy only reveals the
organization of the DNA within the cells and reveals nothing else about the cell
morphologies.

CONFOCAL MICROSCOPY

In standard microscopy, unless the specimen is very thin then areas of the
specimen above and below the focal plane still contribute to the image as "out of
focus blur".

In confocal microscopy, a pinhole between specimen and detector is used to

select information from a single focal plane, producing a sharply focussed optical
slice through the specimen. Taking a series of optical slices from different focus
levels in the specimen generates a 3D data set.

Principle
 Normally a conventional (wide-field) Microscope uses different wavelengths
from a light source, to visualize and illuminate a large area of a specimen,
forming fuzzy, murky, and crowded images, because cell sample images are
captured from all directions, without a focal point.
 To avoid these issues, a Confocal Microscope is used. In wide-field or
Fluorescent microscopes, the whole specimen receives light, receiving
complete excitement and emitting light which is detected by a photodetector
on the microscope. However, with the confocal microscope, point illumination
is the principle working mechanism.
 A specimen is stained with fluorochrome is examined. When a beam of light is
focused at a particular point of the fluoro-chromatic specimen, it produces an
illumination that is focused by the objective lens to a plane above the
objectives. The objective has an aperture on the focal plane located above it,
which primarily functions to block any stray light from reaching the specimen.
 A measure of the illumination point is about 0.25 to 0.8 um in diameter,
determined by the objective numerical aperture and 0.5 to 1.5 um deep, with
the brightest intensity.
 The specimen normally lies between the camera lens and the perfect point of
focus, known as the plane of focus. Using the laser from the microscope, the
laser scans over a plane on the specimen (beam scanning0 or by moving the
stage (stage scanning). A detector then will measure the illumination
producing an image of the optical section. scanning several optical sections,
they are collected in a computerized system as data, forming a 3D image. The
image can be measured and quantified.
 Its outcome is also favored by the aperture found above the objective which
blocks stray light.
 Images produced by the confocal microscope have a very good contract and
resolution capacity despite the thickness of the specimen. Images are stored in
the high-resolution 3D image of the cell complexes including their structures.
 The main characteristic of the Confocal Microscope is that it only detects what
is focused and anything outside the focus point, appears black.
 The image of the specimen is formed when the microscope scanner, scans the
focused beam across a selected area with the control of two high-speed
oscillating mirrors. Their movement is facilitated by galvanometer motors. One
mirror moves the beam from left to right on the lateral X-axis while the second
mirror translates the beam along the Y-axis. After a scan on the X-axis, the
beam moves rapidly back to the starting point to start a new scan, a process
known as flyback. No information is collected during the flyback process,
therefore the point of focus, which is the area of interest is what is illuminated
by the laser scanner.

Applications
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.
3. It is used in pharmaceutical industries, to ensure the maintenance of thin-film
pharmaceuticals, allowing control of the quality and uniformity of drug
distribution.
4. It is used to retrieve data from some 3D optical storage systems. This has
helped in quantifying the age of Magdalen’s papyrus.

ELECTRON MICROSCOPY

Electron microscope uses electron beam to visualize the object. These are mainly
of two types Scanning electron microscope (SEM) and Transmission electron
microscope (TEM).

SCANNING ELECTRON MICROSCOPY

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
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 OF 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, 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

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
1. They are easy to operate and have user-friendly interfaces.
2. They are used in a variety of industrial applications to analyze surfaces of solid
objects.
3. Some modern SEMs are able to generate digital data that can be portable.
4. It is easy to acquire data from the SEM, within a short period of time of about
5 minutes.

Disadvantages
1. They are very expensive to purchase
2. They are bulky to carry
3. They must be used in rooms that are free of vibrations and free of
electromagnetic elements
4. They must be maintained with a consistent voltage
5. They should be maintained with access to cooling systems
 TRANSMISSION ELECTRON MICROSCOPY (TEM)
This is a powerful electron microscope that uses a beam of electrons to focus on a
specimen producing a highly magnified and detailed image of the specimen.
The magnification power is over 2 million times better than that of the light
microscope, producing the image of the specimen which enables easy
characterization of the image in its morphological features, compositions and
crystallization information is also detailed.

Principle
 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 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:

 Electron gun
 Image producing system
 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
 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.

Preparation of Sample:
 The specimen to be viewed under the TEM must undergo a special preparation
technique to enable visualization and creation of a clear image.

 Electrons are easily absorbed and easily scattered on solid elements, showing
poor visualization for thick specimens. And therefore, very thin specimens are
used for accurate and clear visualization forming a clear image as well. The
specimen should be about 20-100nm thin and 0.025-0.1nm diameter, as small
as that of a bacterial cell. Thin specimens allow interaction with electrons in a
vacuumed space, are able to maintain their innate structure.
 To get thin slice specimens, the specimen is first fixed on a plastic material
with glutaraldehyde or osmium tetraoxide. These chemical agents stabilize the
structure of the cell and maintain its originality. The addition of an organic
solvent like alcohol such as ethanol will dehydrate the cell completely for
embedding the specimen to the plastics.
 The specimen is then permeated by adding an unpolymerized liquid epoxy
plastic making it hardened like a solid block. This is where thin sections are cut
from using a glass knife with a piece of special equipment known as an
ultramicrotome.
 The specimen is then stained appropriately (with the appropriate stain) for the
uniform scattering of electrons. The thin sections are then soaked in heavy
metallic elements such as lead citrate and uranyl acetate allowing the lean and
aluminum ions to bind to the cell structures. This forms an opaque layer
against the electrons on the cell structures to increase contrast.
 The stained thin sections are then mounted on copper grids for viewing.
 The primary staining techniques that are applied for viewing under the TEM is
Negative staining coupled with heavy metallic elements coating. The metallic
coating scatters electrons which appears on the photographic film while
 uncoated sections and used to study bacterial, viral cell morphologies and
structures.
 Freeze-itching treatment:
 To reduce the possible dangers of artifacts, freeze-itching is used especially for
the treatment of microbial cells, unlike chemical fixation, dehydration, and
embedding, where most specimens get contaminated.

 Microbial cell organelles undergo special treatment known as Freeze-itching

whereby the specimens are prepared with liquid nitrogen and then warmed at
-100°C in a vacuum chamber.
 The sections are then cut with a precooled knife in liquid nitrogen at -196°C.
After warming up the sectioned specimen in a high vacuum for about 2
minutes, it can then coated ith platinum and carbon layer forming replicas.
 These are then be viewed under the TEM displaying more detailed internal
structures of the cell in 3D.
 This step of treatment with Liquid nitrogen is known as freeze-itching.

Applications
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
It has a very powerful magnification of about 2 million times that of the Light
microscope. It can be used for a variety of applications ranging from basic Biology
to Nanotechnology, to education and industrial uses.
1. It can be used to acquire vast information on compounds and their structures.
2. It produces very efficient, high-quality images with high clarity.
3. It can produce permanent images.
4. It is easy to train and use the Transmission Electron Microscope

Disadavantages
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.
FLUORESCENCE MICROSCOPY

CONFOCAL MICROSCOPY
SEM IMAGE

TEM IMAGE