The document is a detailed overview of the components and principles of optical microscopes, discussing parts such as the eyepiece, objective lenses, and focus knobs, as well as the processes of magnification and resolution. It explains various types of microscopy techniques, including bright field, phase contrast, and fluorescence microscopy, and highlights the importance of proper illumination and maintenance of the microscope for effective observation. Additionally, it addresses the characteristics and costs associated with different objective lenses, particularly achromat and apochromat lenses.

1. Kunal P. Deshmukh
Assistant Professor
School of Life Sciences
S.R.T.M.University Nanded.

2. • Eyepiece (ocular lens) (1)
• Objective turret, revolver, or
revolving nose piece (to hold
multiple objective lenses) (2)
• Objective lenses (3)
• Focus knobs (to move the
stage)
– Coarse adjustment (4)
– Fine adjustment (5)
• Stage (to hold the specimen)
(6)
• Light source (a light or a
mirror) (7)
• Diaphragm and condenser
(8)
• Mechanical stage (9)

3. Eyepiece (ocular lens)
The eyepiece, or ocular lens, is a cylinder containing two or more lenses; its function is to bring the image
into focus for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are
interchangeable and many different eyepieces can be inserted with different degrees of magnification. Typical
magnification values for eyepieces include 2×, 50× and 10×.
Objective turret (revolver or revolving nose piece)
Objective turret, revolver, or revolving nose piece is the part that holds the set of objective lenses.
Objective
At the lower end of a typical compound optical microscope, there are one or more objective lenses that collect
light from the sample. The objective is usually in a cylinder housing containing a glass single or multi-element
compound lens. Typically there will be around three objective lenses screwed into a circular nose piece which
may be rotated to select the required objective lens.Microscope objectives are characterized by two
parameters, namely, magnification and numerical aperture. The former typically ranges from 5× to 100× while
the latter ranges from 0.14 to 0.7, corresponding to focal lengths of about 40 to 2 mm, respectively. Objective
lenses with higher magnifications normally have a higher numerical aperture and a shorter depth of field in
the resulting image.

4. Oil immersion
Some microscopes make use of oil-immersion objectives
or water-immersion objectives for greater resolution at
high magnification. These are used with
index-matching material such as immersion oil or water
and a matched cover slip between the objective lens and
the sample. The refractive index of the index-matching
material is higher than air allowing the objective lens to
have a larger numerical aperture (greater than 1) so that
the light is transmitted from the specimen to the outer face
of the objective lens with minimal refraction. Numerical
apertures as high as 1.6 can be achieved.The larger
numerical aperture allows collection of more light making
detailed observation of smaller details possible. An oil
immersion lens usually has a magnification of 40 to 100×.

5. Focus knobs
Adjustment knobs move the stage up and down with separate adjustment for coarse and fine
focusing. The same controls enable the microscope to adjust to specimens of different
thickness.
Frame
The whole of the optical assembly is traditionally attached to a rigid arm, which in turn is
attached to a robust U-shaped foot to provide the necessary rigidity. The arm angle may be
adjustable to allow the viewing angle to be adjusted.
The frame provides a mounting point for various microscope controls. Normally this will
include controls for focusing, typically a large knurled wheel to adjust coarse focus, together
with a smaller knurled wheel to control fine focus. Other features may be lamp controls
and/or controls for adjusting the condenser.

6. Stage
The stage is a platform below the objective which
supports the specimen being viewed. In the center of
the stage is a hole through which light passes to
illuminate the specimen. The stage usually has arms to
hold slides (rectangular glass plates with typical
dimensions of 25×75 mm, on which the specimen is
mounted).
Light source
Many sources of light can be used. At its simplest,
daylight is directed via a mirror. Most microscopes,
however, have their own adjustable and controllable
light source – often a halogen lamp, although
illumination using LEDs and lasers are becoming a
more common provision.

7. Condenser
The condenser is a lens designed to focus light from the
illumination source onto the sample. The condenser may also
include other features, such as a diaphragm and/or filters, to manage
the quality and intensity of the illumination. For illumination
techniques like dark field, phase contrast and differential
interference contrast microscopy additional optical components
must be precisely aligned in the light path.
Magnification
The actual power or magnification of a compound optical
microscope is the product of the powers of the ocular (eyepiece)
and the objective lens. The maximum normal magnifications of the
ocular and objective are 10× and 100× respectively, giving a final
magnification of 1,000×.

8. Principles of Microscopy
- Resolution –
Resolution is defined as the ability to distinguish two very small
and closely-spaced objects as separate entities. Resolution is best
when the distance separating the two tiny objects is small. Resolution
is determined by certain physical parameters that include the
wavelength of light, and the light-gathering power of the
objective and condenser lenses. A simple mathematical equation
defines the smallest distance (dmin) separating the two very small
objects:
dmin = 1.22 x wavelength / N.A. objective + N.A. condenser
This is the theoretical resolving power of a light microscope.
N.A. (Numerical Aperture) is a mathematical calculation of the light-
gathering capabilities of a lens. The N.A. of each objective lens is
inscribed in the metal tube, and ranges from 0.25-1.4. The higher
the N.A., the better the light-gathering properties of the lens, and
the better the resolution. Higher N.A. values also mean shorter
working distances (you have to get the lens closer to the object).
N.A. values above 1.0 also indicate that the lens is used with some
immersion fluid, such as immersion oil.

9. From the equation above, it should also be clear that shorter
wavelength light (bluer light) will provide you with better resolution
(smaller dmin values).
In the early 1950's, a UV microscope was designed, but required quartz
objectives and a specialized imaging device. The quartz lenses provided
slightly better resolution (dmin = 0.1 µm), The human eye is best adapted
for green light and our ability to see detail may be compromised
somewhat with the use of blue or violet. Most manufacturers of
microscopes correct their simplest lenses (achromats) for green light.
-

10. Magnification and Imaging –
Most microscopes in current use are known as compound
microscopes, where a magnified image of an object is produced
by the objective lens, and this image is magnified by a second
lens system (the ocular or eyepiece) for viewing. a standard
microscope will provide you with a final magnification range
of ~40X up to ~1000X.
Each objective lens consists of six or more pieces of glass
that combine to produce a clear image of an object. The six or
more lenses in the objective lens are needed to provide
corrections that produce image clarity. The interaction of light
with the glass in a lens produce aberrations that result in a loss
in image quality because light waves will be bent, or refracted,
differently in different portions of a lens, and different colors
of light will be refracted to different extents by the glass.
Spatial aberrations (e.g., spherical aberration) can be corrected
by using lenses with different curvature on their surfaces, and
chromatic (i.e., color) aberrations can be minimized by using
multiple kinds of glass in combination.

11. These corrections increase the cost of the lens to the extent that an
apochromatic objective lens exhibiting full color correction and
extremely high N.A. can cost several thousand dollars. This
objective lens is about the size of your thumb.
The objective lenses in most microscopes are achromats,(achromat
means= a lens that transmit light without separating it into
constituent colore) and best suited for imaging with green light.
Green filters narrow the bandwidth of the light, and make achromat
objectives reasonably effective for most routine uses. The achromat
lenses are not suitable for critical high-resolution imaging with
white light, because red and blue light do not focus in the same
plane as green light. Chromatic aberrations will degrade resolution
in color images obtained with achromatic objectives. Color
photomicrography aimed at the highest level of resolution and
image clarity should be performed with totally corrected
apochromatic objective lenses.( apochromatic is photographic or
other lens that has better correction of chromatic and spherical
abbreation than the much more common achromat lenses.

12. Apochromatic lenses are usually made up of three
elements and brings light of three different frequencies
to a common focus. Apochromatic glasses made up of
flouro crown glasses and flint glasses). Fluorite lenses,
offer intermediate levels of correction, better than
achromats but not as good as apochromats. Fluorite
lenses are well suited for fluorescence microscopy
because of their high transmittance of shorter
wavelength light. Higher levels of correction make
objective lenses more expensive; the price range for
apochromatic objectives goes from about $3,000 to over
$10,000. when you look into a microscope, the magnified
and corrected image you see through the oculars is
actually a virtual image (as opposed to a real image). The
ocular, designed to provide a corrected virtual image when
viewed by eye, is not suitable for the generation of
photographic or video images through the microscope. For
photography or video microscopy it is necessary to use a
projection lens that generates a corrected real image.

13. - Illumination -
An essential factor in producing a good image with the light microscope
is obtaining adequate levels of light in the specimen, or object plane.
It is not only necessary to obtain bright light around the object, but for
optimal imaging, the light should be uniform across the field of view.
The best way to illuminate the specimen involves the use of yet another
lens system, known as a condenser. The front element of the condenser
is usually a large, flattened lens that sits directly beneath the specimen.
Its placement on a movable rack provides you with the means to focus
the light beam coming past the object and maximixe the intensity and
control the uniformity of illumination. It may be necessary to center the
field aperture diaphragm, using the condenser centering screws. When
the microscope is properly illuminated, both the object and the edges of
the field aperture diaphragm should be in the same plane of focus and
the field iris diaphragm should be centered in the field of view.

14. Light Microscopy
The optical microscope, often referred to as the
"light microscope", is a type of microscope which
uses visible light and a system of lenses to magnify
images of small samples. The light microscope, so
called because it employs visible light to detect small
objects, is probably the most well-known and well-
used research tool in biology. The smallest objects
that are considered to be living are the bacteria. The
smallest bacteria can be observed and cell shape
recognized at a mere 100x magnification.


15. Types of light microscopes
Bright Field Microscopy
Bright
field illumination,
sample contrast comes
from absorbance of
light in the sample.

16. With a conventional bright field microscope, light from an
incandescent source is aimed toward a lens beneath the stage called
the condenser, through the specimen, through an objective lens, and
to the eye through a second magnifying lens, the ocular or eyepiece.
We see objects in the light path because natural pigmentation or
stains absorb light differentially, or because they are thick enough to
absorb a significant amount of light despite being colorless.
A Paramecium should show up fairly well in a bright field
microscope, although it will not be easy to see cilia or most
organelles. After passing through the specimen, the light is displayed
to the eye with an apparent field that is much larger than the area
illuminated Students are usually aware of the use of the coarse and
fine focus knobs, used to sharpen the image of the specimen. They
are frequently unaware of adjustments to the condenser that can
affect resolution and contrast. Some condensers are fixed in position,
others are focusable, so that the quality of light can be adjusted.

17. Steps while Using a bright field microscope
1) Mount the specimen on the stage
2) Optimize the lighting
3) Adjust the condenser
4) Think about what you are looking for
5) Focus, locate, and center the specimen
6) Adjust eyepiece separation, focus
7) Select an objective lens for viewing

18. The lowest power lens is usually 3.5 or 4x, and is
used primarily for initially finding specimens. We
sometimes call it the scanning lens for that reason.
The most frequently used objective lens is the 10x
lens, which gives a final magnification of 100x with a
10x ocular lens. For very small protists and for details
in prepared slides such as cell organelles or mitotic
figures, you will need a higher magnification.
Typical high magnification lenses are 40x and 97x or
100x. The latter two magnifications are used
exclusively with oil in order to improve resolution.
Higher magnification lenses must be physically closer
to the specimen itself, which poses the risk of
jamming the objective into the specimen. Be very
cautious when focusing

19. 8) Adjust illumination for the selected objective lens
9) When to use bright field microscopy
Bright field microscopy is best suited to viewing stained or naturally pigmented
specimens such as stained prepared slides of tissue sections or living photosynthetic
organisms. It is useless for living specimens of bacteria, and inferior for non-
photosynthetic protists or metazoans, or unstained cell suspensions or tissue
sections.
Prepared slides, stained - bacteria (1000x), thick tissue sections (100x, 400x), thin
sections with condensed chromosomes or specially stained organelles (1000x),
large protists or metazoans (100x).
Smears, stained - blood (400x, 1000x), negative stained bacteria (400x, 1000x).
Living preparations (wet mounts, unstained) - pond water (40x, 100x, 400x), living
protists or metazoans (40x, 100x, 400x occasionally), algae and other microscopic
plant material (40x, 100x, 400x). Smaller specimens will be difficult to observe
without distortion, especially if they have no pigmentation.
distortion, especially if they have no pigmentation.

20. Care of the microscope
EVERYTHING on a good quality microscope is unbelievably expensive, so be careful.
Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder, for
example.
Hold the plug (not the cable) when unplugging the illuminator.
Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done.
Always make sure the stage and lenses are clean before putting away the microscope.
NEVER use a paper towel, a kimwipe, your shirt, or any material other than good quality lens
tissue or a cotton swab (must be 100% natural cotton) to clean an optical surface. Be gentle!
You may use an appropriate lens cleaner or distilled water to help remove dried material.
Organic solvents may separate or damage the lens elements or coatings.
Cover the instrument with a dust jacket when not in use.
Focus smoothly; don't try to speed through the focusing process or force anything. For
example if you encounter increased resistance when focusing then you've probably reached a
limit and you are going in the wrong direction.

21. Phase Contrast Microscope

22. Phase contrast illumination, sample
contrast comes from
interference of different path
lengths of light through the sample.
The same cells imaged with
traditional bright field microscopy
(left) and with phase contrast
microscopy (right).

23. Digram - Phase contrast
microscopy

24. Frits Zernike (1888–1966) received a Nobel prize in 1953
for his discovery of phase contrast.
Microscopy
In positive phase contrast the object (e.g., cell component)
appears darker than the surrounding background.
In negative phase contrast the object appears brighter
than the background.

26. Phase annulus

28. Zernicke realized that if he could retard the light passing through biological
specimens without affecting the light passing through the surrounding medium,
he could generate changes in amplitude within living cells.

30. How phase contrast works
A compound microscope equipped for negative phase
contrast has two additional components: a “phase
plate” that retards light exactly 1
⁄4wavelength in a
centered, ring-shaped area located at the back focal
plane of the objective lens and a matching “phase
annulus” in the condenser consisting of a clear ring on
a black field (Figure 2-3B). The presence of the
annulus and matching phase plate causes the direct
(unmodified background) light to pass only through
the phase ring and thus be retarded 1
⁄4Ü.

32. The Invisible Can Be Seen
The phase contrast microscope is a vital instrument in biological and
medical research. When dealing with transparent and colorless
components in a cell, dyeing is an alternative but at the same time stops
all processes in it. The phase contrast microscope has made it possible to
study living cells, and cell division is an example of a process that has
been examined in detail with it.
The phase contrast microscope is able to show components in a cell or
bacteria, which would be very difficult to see in an ordinary light
microscope.
Phase contrast microscopy is particularly important in biology. It reveals
many cellular structures that are not visible with a simpler bright field
microscope, . These structures were made visible to earlier microscopists
by staining, but this required additional preparation killed the cells. The
phase contrast microscope made it possible for biologists to study living
cells and how they proliferate through cell division.

33. FLUORESCENT MICROSCOPY

34. A fluorescence microscope
is much the same as a
conventional light
microscope with added
features to enhance its
capabilities

35. Basic Concepts in Fluorescence
When organic or inorganic specimens absorb and subsequently reradiate light, the
process is typically a result of fluorescence or phosphorescence. Fluorescence
emission is nearly simultaneous with the absorption of the excitation light as the
time delay between photon absorption and emission is typically less than a
microsecond. When the emission persists long after the excitation light is
extinguished, the phenomenon is known as phosphorescence.
In certain classes of atoms and molecules, electrons absorb light, become
energized, and then rapidly lose this energy in the form of heat and light emission.
If the electron keeps its spin, the electron is said to enter a singlet state, and the
kind of light that is emitted as the electron returns to ground state is called
fluorescence. If the electron changes its spin when excited, it enters the triplet
state, and the kind of light that is emitted as the electron returns to ground state is
known as phosphorescence. Phosphorescence is much longer-lived than
fluorescence. Both fluorescence and phosphorescence emissions are of particular
wavelengths for specific excited electrons. Both types of emission are dependent
on specific wavelengths of excitation light, and for both types of emission, the
energy of excitation is greater than the energy of emission.

36. Fluorescence is a member of the
ubiquitous luminescence family of processes in which
susceptible molecules emit light from electronically
excited states created by either a physical (for example,
absorption of light), mechanical (friction), or chemical
mechanism. Generation of luminescence through
excitation of a molecule by ultraviolet or visible light
photons is a phenomenon termed photoluminescence,
which is formally divided into two
categories, fluorescence and phosphorescence,
depending upon the electronic configuration of the excited
state and the emission pathway. Fluorescence is the
property of some atoms and molecules to absorb light at a
particular wavelength and to subsequently emit light of
longer wavelength after a brief interval, termed the
fluorescence lifetime. The process of phosphorescence
occurs in a manner similar to fluorescence, but with a
much longer excited state lifetime.

38. Fig. 1 Cut-away diagram of an upright microscope equipped both for transmitted light and epi-
fluorescence microscopy. The vertical illuminator in the center of the diagram has the light
source at one end (episcopic lamphouse) and the filter cube at the other

39. LIGHT SOURCES
.
The most common lamps are the mercury burners, ranging in wattage
from 50 to 200 W and the xenon burners ranging from 75 to 150 W.
These light sources are powered by a direct current (d.c.) supply,
furnishing enough start-up power to ignite the burner (by ionization of
the gaseous vapor) and to keep it burning with a minimum of flicker.
The power supply should have a timer to track the number of hours the
burner has been in use. Arc lamps lose efficiency and are more likely to
shatter, if used beyond their rated lifetime. The mercury burners do not
provide even intensity across the spectrum from UV to infrared and
much of the intensity of the
mercury burner is expended in the near UV. In recent years, there has
been increasing use of lasers, particularly the argon-ion and argon–
krypton-ion lasers as light sources. They have the virtues of small
source size, low divergence, monochromaticity, and high mean
luminance

40. FILTER TERMINOLOGY

41. Basically, there are three categories of filters: exciter filters,
barrier filters, and dichromatic beam splitters (dichroic
mirrors). Fluorescence filters were formerly almost exclusively
made of colored glass or colored gelatin sandwiched between
glass plates. Now, interference filters are used for exciter filters to
pass or reject wavelengths of light with great selectivity and high
transmission. Dichromatic beam splitters are specialized
interference filters. Barrier filters may be either made of colored
glass or interference filters

43. Application of flouroscence Microscopy
To utilize fluorescence, we need to label the specimen (a cell, a tissue,
or a gel) with a suitable molecule (a fluorochrome) whose distribution
will become evident after illumination. The fluorescence microscope is
ideally suited for the detection of particular fluorochromes in cells and
tissues. Early investigations showed that many specimens (minerals,
crystals, resins, crude drugs, butter, chlorophyll, vitamins, inorganic
compounds, etc.) fluoresce when irradiated with UV light. In the 1930s,
the use of fluorochromes began in biology to stain tissue components,
bacteria, or other pathogens. Some of these stains were highly specific
and they stimulated the development of the fluorescence microscope.
Fluorescence microscopy has become an essential tool in biology as well
as in materials science as it has attributes that are not readily available in
other optical microscopy techniques.

44. The use of an array of fluorochromes has made it possible to
identify cells and submicroscopic cellular components and
entities with a high degree of specificity amid nonfluorescing
material. The fluorescence microscope can reveal the presence
of a single fluorescing molecule. In a sample, through the use
of multiple staining, different probes can simultaneously
identify several target molecules. There are specimens that
autofluoresce when they are irradiated and this phenomenon is
exploited in the field of botany, petrology, and in the
semiconductor industry. fluorochromes (also called
fluorophores), which are excited by specific wavelength
irradiating light and emit light of useful intensity.
Fluorochromes are stains that attach themselves to visible or
subvisible structures, are often highly specific in their
attachment targeting, and have significant quantum yield (the
photon emission/absorption ratio). The growth in the use of
fluorescent microscopes is closely linked to the development
of hundreds of fluorochromes with known intensity curves of
excitation and emission and well-understood biological
structure
targets.

45. Application of fluroscence Microscope
These microscopes are often used for -
Imaging structural components of small specimens, such as
cells
Conducting viability studies on cell populations (are they
alive or dead?)
Imaging the genetic material within a cell (DNA and RNA)
Viewing specific cells within a larger population with
techniques such as FISH

47. A scanning electron microscope (SEM) is a type of
electron microscope that produces images of a sample by
scanning it with a focused beam of electrons. The
electrons interact with atoms in the sample, producing
various signals that can be detected and that contain
information about the sample's surface topography
and composition. The most common mode of detection is
by secondary electrons emitted by atoms excited by the
electron beam.

49. Interaction with matter

52. Principles and capacities
The types of signals produced by a SEM include secondary
electrons (SE), back-scattered electrons (BSE),
characteristic X-rays, light (cathodoluminescence) (CL),
specimen current and transmitted electrons. Secondary
electron detectors are standard equipment in all SEMs, but it
is rare that a single machine would have detectors for all
possible signals. The signals result from interactions of the
electron beam with atoms at or near the surface of the sample.
In the most common or standard detection mode, secondary
electron imaging or SEI, the SEM can produce very high-
resolution images of a sample surface, revealing details less
than 1 nm in size. Due to the very narrow electron beam, SEM
micrographs have a large depth of field yielding a
characteristic three-dimensional

53. appearance useful for understanding the surface structure of a
sample. This is exemplified by the micrograph of pollen shown
above. Back-scattered electrons (BSE) are beam electrons that
are reflected from the sample by elastic scattering. BSE
are often used in analytical SEM along with the spectra made
from the characteristic X-rays, because the intensity of the
BSE signal is strongly related to the atomic number (Z) of
the specimen. BSE images can provide information about the
distribution of different elements in the sample. For the same
reason, BSE imaging can image colloidal gold immuno-labels
of 5 or 10 nm diameter, which would otherwise be difficult or
impossible to detect in secondary electron images in biological
specimens. Characteristic X-rays are emitted when the
electron beam removes an inner shell electron from the
sample, causing a higher-energy electron to fill the shell
and release energy. These characteristic X-rays are used to
identify the composition and measure the abundance of
elements in the sample.

54. Functional Principle

59. Biological samples
For SEM, a specimen is normally required to be completely dry, since the
specimen chamber is at high vacuum. Hard, dry materials such as wood,
bone, feathers, dried insects, or shells can be examined with little
further treatment, but living cells and tissues and whole, soft-bodied
organisms usually require chemical fixation to preserve
and stabilize their structure. Fixation is usually performed by incubation
in a solution of a buffered chemical fixative, such as glutaraldehyde,
sometimes in combination with formaldehyde and other fixativesa and
optionally followed by postfixation with osmium tetroxide.The fixed
tissue is then dehydrated. Because airdrying causes collapse and
shrinkage, this is commonly achieved by replacement of water in the cells
with organic
.


60. solvents such as ethanol or acetone, and replacement
of these solvents in turn with a transitional fluid such
as liquid carbon dioxide by critical point drying. The
carbon dioxide is finally removed while in a
supercritical state, so that no gas-liquid interface is
present within the sample during drying. The dry
specimen is usually mounted on a
specimen stub using an adhesive such as epoxy resin
or electrically conductive double-sided adhesive tape,
and sputter-coated with gold or gold/palladium alloy
before examination in the microscope

61. Scanning process and image formation
In a typical SEM, an electron beam is thermionically emitted from an
electron gun fitted with a tungsten filament cathode. Tungsten is
normally used in thermionic electron guns because it has the highest
melting point and lowest vapour pressure of all metals, thereby
allowing it to be heated for electron emission, and because of its low
cost.

The electron beam, which typically has an energy ranging from 0.2
keV to 40 keV, is focused by one or two condenser lenses to a spot
about 0.4 nm to 5 nm in diameter. The beam passes through pairs of
scanning coils or pairs of deflector plates in the electron column, typically
in the final lens, which deflect the beam in the x and y axes so that it scans
in a raster fashion over a rectangular area of the sample surface.

63. SEMs may have condenser and objective lenses, but their function is to focus
the beam to a spot, and not to image the specimen.

64. Detection of secondary electrons
The most common imaging mode collects low-energy (<50 eV) secondary
electrons that are ejected from the k-shell of the specimen atoms by
inelastic scattering interactions with beam electrons. Due to their low
energy, these electrons originate within a few nanometers from the sample
surface. The electrons are detected by an Everhart- Thornley detector
Detection of backscattered electrons
BSE detectors are usually either of scintillator or of semiconductor types.
X-ray microanalysis
X-rays, which are produced by the interaction of electrons with the sample,
may also be detected in an SEM equipped for energy-dispersive X-ray
spectroscopy or wavelength dispersive X-ray spectroscopy

66. Resolution
Factors affecting the resolution are:
i) The diameter of the electron beam
ii) Scattering within the specimen
iii) The signal-to-noise ratio
iv) External disturbances due to
1. Electric of magnetic fields
2. Mechanical vibrations

67. ADVANTAGES
High resolution and magnification
3-D Topographical imaging
Compatible with PC technologies and softwares
Fast Analysing
Store data in digital form
Easier sample preparation techniques
DISADVANTAGES
Can not analys fluid or gas compounds
Expensive Instrumentation
Wasting time on sample preparation
Constant voltage during analysing

68. RESULT
SEM uses electrons instead of light to form an
image.
developed new areas of study & still helping.
popular among researchers due to their wide
range of applications
CONCLUSION
SEM;
provides detailed surface data of solid samples
informs external morphology, chemical
composition, crystalline structure
 SAMPLE;
must be prepared before placed

69. Transmission Electron Microscope

70. Transmission electron microscopy (TEM) is a microscopy
technique in which a beam of electrons is transmitted
through an ultra-thin specimen, interacting with the
specimen as it passes through. An image is formed
from the interaction of the electrons transmitted
through the specimen; the image is magnified and
focused onto an imaging device,
such as a fluorescent screen, on a layer of
photographic film, or to be detected by a sensor such
as a CCD camera.

72. Working Principle
TEM works like a slide projector. A projector shines a beam of light which
transmits through
the slide. The patterns painted on the slide only allow certain parts of the
light beam to pass
through. Thus the transmitted beam replicates the patterns on the slide,
forming an enlarged
image of the slide when falling on the screen. TEMs work the same way
except that they shine a beam of electrons (like the light in a slide projector)
through the specimen (like the slide). However, in TEM, the transmission
of electron beam is highly dependent on the properties of material being
examined. Such properties include density, composition, etc. For example,
porous material will allow more electrons to pass through while dense
material will allow less. As a result, a specimen with a non-uniform density
can be examined by this technique. Whatever part is transmitted is
projected onto a phosphor screen for the user to see.

73. ELECTRON SOURCE (GUN):-

77. The first and basic part of the transmission electron microscope is the
source of electrons. It is usually a V-shaped filament made of LaB6 or W
(tungsten) that is wreathed with Wehnelt electrode (Wehnelt Cap). Due
to negative potential of the electrode, the electrons are emitted from a small
area of the filament (point source). A point source is important because it
emits monochromatic electrons (with similar energy). In this, a positive
electrical potential is applied to the anode, and the filament (cathode) is
heated until a stream of electrons is produced. The electrons are accelerated
by the positive potential down the column, and because of the negative
potential of cap, all electrons are repelled toward the optic axis. A
collection of electrons occurs in the space between the Filament tip and Cap
, which is called a space charge. Those electrons at the bottom of the space
charge (nearest to the anode) can exit the gun area through the small (<1
mm) hole in the Wehnelt Cap and then move down the column to be later
used in imaging.

78.  2) CONDENSER LENS:-
 The stream of the electron from the electron gun is then focussed to a small, thin,
coherent beam by the use of condenser lenses. The first lens determines the “spot
size”; the general size range of the final spot that strikes the sample. The second lens
actually changes the size of the spot on the sample.

 3) CONDENSER APERTURE:-
 A condenser aperture is a thin disk or strip of metal with a small circular through-
hole. It is used to restrict the electron beams and filter out unwanted scattered
electrons before image formation.

 4) SAMPLE: -
 The beam from the condenser aperture strikes the sample and the electron-sample
interaction takes place in three different ways. One is unscattered electrons
(transmitted beam), elastically scattered electrons (diffracted beam) and inelastically
scattered electrons.


79.  5) OBJECTIVE LENS: -
 The main function of the objective lens is to focuses the transmitted electron from the sample
into an image.

 6) OBJECTIVE APERTURE:-
 Objective aperture enhances the contrast by blocking out high-angle diffracted electrons.

 7) SELECTED APERTURE:-
 It enables the user to examine the periodic diffraction of electron by ordered
 arrangements of atoms in the sample.

 8) PROJECTOR LENS:-
 The projector lens are used to expand the beam onto the phosphor screen.

 9) SCREEN:-
 Imaging systems in a TEM consists of a phosphor screen, which may be made of fine
 (10-100 micro meter) particulate zinc sulphide, for direct observation by the operator.

 10) IMAGE PATTERN:-
 The image strikes the phosphor screen and light is generated, allowing the users to see the
image. The darker areas of the image represent those areas of the sample that fewer electrons
are transmitted. The lighter areas of the image represent those areas of the sample that more
electrons were transmitted.

80.  Sample Preparation
 Sample preparation is important for electron microscopy. There are three main steps for sample
preparation: Processing, embedding and polymerization.

 Processing
 This includes: fixation, rinsing, post fixation, dehydration and infiltration.
 Fixation
 It stabilizes the cell structure. There is minimum alteration to cell morphology and volume.
Glutaraldehyde is
 often used as the fixative in TEM. As a result of glutaraldehyde fixation the protein molecules
are covalently cross linked to their
 neighbors.

 2) Rinsing
 The samples should be washed with a buffer to maintain the pH. For this purpose, sodium
cacodylate buffer is often used which has an effective buffering range of 5.1-7.4. The
sodium cacodylate buffer thus prevents excess acidity which may result from tissue fixation
during microscopy.


81.  3) Post fixation
 A secondary fixation with osmium tetroxide (OsO4), which is to increase the
stability and contrast of fine structure. OsO4 binds phospholipid head regions,
which creating contrast with the neighbouring protoplasm (cytoplasm). OsO4 helps
in the stabilization of many proteins by transforming them into gels without
destroying the structural features. Tissue proteins, which are stabilized by OsO4
and does not coagulated by alcohols during dehydration

 4) Dehydration
 The water content in the tissue sample should be replaced with an organic solvent
since the epoxy resin used in infiltration and embedding step are not miscible with
water.

 5) Infiltration
 Epoxy resin is used to infiltrate the cells. It penetrates the cells and fills the space to
give hard plastic material which will tolerate the pressure of cutting.

 6) Embedding:
 After processing the next step is embedding. This is done using flat molds.


82. 7) Polymerization

Next is polymerization step in which the resin is allowed to set overnight at
a temperature of 60 degree in an oven..

8) Sectioning
The specimen must be cut into very thin sections for electron microscopy so
that the electrons are semitransparent to electrons. These
sections are cut on an ultramicrotome which is a device with a glass or
diamond knife. For best resolution the sections must be 30 to 60 nm. The
resin block can be made ready for the sectioning by trimming it at the tip
with a razor blade or black trimmer so that the smallest cutting face is
available. Fix the block to a microtome and cut the sections. Sections float
onto a surface of liquid held in trough and remain together in a form of
ribbon. Freshly distilled water is generally used to fill the trough. These
sections are then collected onto a copper grid and viewed under the
microscope.


83.  Advantages
 TEMs find application in cancer research, virology, materials science as well
as pollution, nanotechnology, and semiconductor research.

 TEM is however a significant achievement of quantum mechanical
understanding of electron. It was a major step in magnification studies and
obtaining better resolution than optical microscopes and hence is a very
important building block in study of application of quantum mechanics.
Moreover it is very commonly used in studies of material structures and
properties and for other experimental purposes.

84.  Limitations
 There are a number of drawbacks to the TEM technique. Many materials require
extensive sample preparation to produce a sample thin enough to be electron
transparent, which makes TEM analysis a relatively time consuming process with a
low throughput of samples. The structure of the sample may also be changed during
the preparation process. Also the field of
 view is relatively small, raising the possibility that the region analysed may not be
characteristic of the whole sample. There is potential that the sample may be
damaged by the electron beam, particularly in the case of biological materials.