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MICROSCOPY
 Microscopy
is the technical
field of using microscopes
to view samples or objects.
There are three well-known
branches of microscopy,
optical, electron and
scanning probe microscopy.
 Optical
and electron microscopy
involve the diffraction, reflection, or
refraction of electromagnetic
radiation/electron beam interacting
with the subject of study, and the
subsequent collection of this
scattered radiation in order to build
up an image.
 The
optical microscope uses
visible light and a system of
lenses to magnify images of
small samples. Optical
microscopes are the oldest and
simplest of the microscopes.
 New
designs of digital
microscopes are now available
which use a CCD camera to
examine a sample and the
image is shown directly on a
computer screen without the
need for expensive optics such
as eye-pieces.
Chromatic aberration
 Chromatic
aberration is caused
by a lens having a different
refractive index for different
wavelengths of light (the
dispersion of the lens).
the focal length “ f ” of a
lens is dependent on the
refractive index “ n ”, different
wavelengths of light will be
focused on different positions.
 Since
Numerical aperture
 Numerical
aperture (NA) of
an optical system is a
dimensionless number that
characterizes the range of
angles over which the system
can accept or emit light.
 Where
’ n ‘ is the index of
refraction of the medium in
which the lens is working and
θ is the half-angle of the
maximum cone of light that
can enter or exit the lens.
 Refractive
Index
 1.0 for air, 1.33 for pure water,
and up to 1.56 for oils
 NA
is important because it
indicates the resolving power
of a lens. The size of the finest
detail that can be resolved is
proportional to λ / NA, where λ
is the wavelength of the light.
WAVELENGTH

Wavelength is the distance between
repeating units of a propagating wave
of a given frequency. It is commonly
designated by the Greek letter
lambda (λ).
A
lens with a larger numerical
aperture will be able to
visualize finer details than a
lens with a smaller numerical
aperture. Lenses with larger
numerical apertures also
collect more light and will
generally provide a brighter
image.
Optical resolution
 Optical resolution describes
the ability of an imaging
system to resolve detail in the
object that is being imaged.
The ability of a lens to resolve
detail is usually determined by
the quality of the lens but is
ultimately limited by diffraction
 The
resolution of a microscope
is defined as the minimum
separation needed between
two objects under examination
in order for the microscope to
discern them as separate
objects.
 This
minimum distance is
labeled δ. If two objects are
separated by a distance
shorter than δ, they will appear
as a single object in the
microscope.
DEPTH OF FIELD
 Depth
of field (DOF) is the
portion of a scene that
appears sharp in the image.
 The
DOF is determined by the
subject distance (that is, the
distance to the plane that is
perfectly in focus), the lens
focal length, and the lens fnumber (relative aperture).
Magnification
 Magnification is the process
of enlarging something only in
appearance, not in physical
size. Magnification is also a
number describing by which
factor an object was
magnified.
 When
this number is less than
one it refers to a reduction in
size, sometimes called
Minification.
Real image
A
real image is a
representation of an actual
object (source) formed by rays
of light passing through the
image.
 If
a screen is placed in the
plane of a real image, the
image will generally become
visible. Real images can be
produced by concave mirrors
and converging lenses.
Virtual image
 A virtual image is an image in
which the outgoing rays from a
point on the object never
actually intersect at a point. A
simple example is a flat mirror
where the image of oneself is
perceived at twice the distance
from yourself to the mirror.
 That
is, if you are half a meter
in front of the mirror, your
image will appear at a distance
of half a meter inside or behind
the mirror.
Oil Immersion Objective
 oil
immersion is a technique
used to increase the resolution
of a microscope.
 This
is achieved by immersing
both the objective lens and the
specimen in a transparent oil
of high refractive index,
thereby increasing the
numerical aperture of the
objective lens.
 The
refractive indices of the oil
and of the glass in the first
lens element are nearly the
same, which means that the
refraction of light will be small
upon entering the lens In
addition to improving
resolution.
 The
use of oil is also
advantageous in that it
reduces the reflective losses
as light enters the lens.
 Cedar wood Oil is used in Oil
immersion.
Stereo microscope
 The
stereo or dissecting
microscope is designed
differently , and serves a
different purpose.
 It
uses two separate optical
paths with two objectives and
two eyepieces to provide
slightly different viewing angles
to the left and right eyes.
 In
this way it produces a threedimensional visualization of
the sample being examined
Stereo microscope
 The
stereo microscope is often
used to study the surfaces of
solid specimens or to carry out
close work such as sorting,
dissection, microsurgery,
watch-making, small circuit
board manufacture or
inspection, etc.
 Unlike
compound microscopes,
illumination in a stereo
microscope most often uses
reflected (episcopic) illumination
rather than transmitted
(diascopic) illumination, that is,
light reflected from the surface of
an object rather than light
transmitted through an object.
Digital Microscope
 Low power microscopy is also
possible with digital
microscopes, with a camera
attached directly to the USB
port of a computer, so that the
images are shown directly on
the monitor.
 Often
called "USB"
microscopes, they offer high
magnifications (up to about
200×) without the need to use
eyepieces, and at very low
cost.
Digital microscope
A
digital microscope uses optics
and a charge-coupled device
(CCD) camera to output a digital
image to a monitor. A digital
microscope differs from an optical
microscope in that there is no
provision to observe the sample
directly through an eyepiece.
 Since
the optical image is
projected directly on the CCD
camera, the entire system is
designed for the monitor
image
Digital microscope
 Resolution
of the image is
dependent on the CCD used in
the camera. Using a typical 2
Megapixel CCD, an image with
1600 x 1200 pixels is
generated. The resolution of
the image is dependent on the
field of view of the lens used
with the camera.
 The
approximate pixel
resolution can be determined
by dividing the horizontal field
of view (FOV) by 1600. Most
common instruments have a
relatively low resolution of 1.3
Megapixels, but higher
resolution cameras are
available.
 The
images can be recorded and
stored in the normal way on the
computer. The camera is usually
fitted with a light source, although
extra sources (such as a fibreoptic light) can be used to
highlight features of interest in the
object. They also offer a large
depth of field, a great advantage
at high magnifications.
Electron microscope
 The
electron microscope uses
a particle beam of electrons to
illuminate a specimen and
create a highly-magnified
image.
 Electron
microscopes have
much greater resolving power
than light microscopes and
can obtain much higher
magnifications of up to 2
million times, while the best
light microscopes are limited to
magnifications of 2000 times.
First EM - Ruska 1933
EM New Version
EM Image - Pollen
EM Image- Ant Head
EM Image- Cell
Transmission Electron
Microscope (TEM)
 The
original form of electron
microscope, the transmission
electron microscope (TEM)
uses a high voltage electron
beam to create an image.
 The
electrons are emitted by
an electron gun, commonly
fitted with a tungsten filament
cathode as the electron
source.
 The
electron beam is
accelerated by an anode
typically at +100keV (40 to 400
keV) with respect to the
cathode, focused by
electrostatic and
electromagnetic lenses.
 The
Electron beam is then
transmitted through the
specimen that is in part
transparent to electrons and in
part scatters them out of the
beam
Scanning Electron Microscope
(SEM)

Unlike the TEM, where electrons of
the high voltage beam carry the
image of the specimen, the electron
beam of the Scanning Electron
Microscope (SEM) does not at any
time carry a complete image of the
specimen.
 The
SEM produces images by
probing the specimen with a
focused electron beam that is
scanned across a rectangular
area of the specimen (Raster
scanning).
 At
each point on the specimen the
incident electron beam loses
some energy, and that lost energy
is converted into other forms,
such as heat, emission of lowenergy secondary electrons, light
emission (cathodoluminescence)
or x-ray emission.
 The
display of the SEM maps
the varying intensity of any of
these signals into the image in
a position corresponding to the
position of the beam on the
specimen when the signal was
generated.
SEM Image – Insect coated
with Gold
Reflection Electron Microscope
(REM)
 In
the Reflection Electron
Microscope (REM) as in the TEM,
an electron beam is incident on a
surface, but instead of using the
transmission (TEM) or secondary
electrons (SEM), the reflected
beam of elastically scattered
electrons is detected.
Scanning Transmission Electron
Microscope (STEM)
 The STEM rasters a focused
incident probe across a
specimen that (as with the
TEM) has been thinned to
facilitate detection of electrons
scattered through the
specimen.
 The
high resolution of the TEM
is thus possible in STEM. The
focusing action occur before
the electrons hit the specimen
in the STEM, but afterward in
the TEM
Sample preparation in EM
 Chemical
Fixation for biological
specimens aims to stabilize the
specimen's mobile
macromolecular structure by
chemical cross linking of proteins
with aldehydes such as
formaldehyde and glutaraldehyde,
and lipids with osmium tetroxide.
Cryofixation
 When freezing a specimen so
rapidly, to liquid nitrogen or
even liquid helium
temperatures, the water forms
vitreous (non-crystalline) ice.
This preserves the specimen
in a snapshot of its solution
state.
Dehydration
 Freeze
drying, or replacement
of water with organic solvents
such as ethanol or acetone,
followed by critical point drying
or infiltration with embedding
resins.
Embedding, biological
specimens
 After
dehydration, tissue for
observation in the
transmission electron
microscope is embedded so it
can be sectioned ready for
viewing.
 To
do this the tissue is passed
through a 'transition solvent'
such as epoxy propane and
then infiltrated with a resin
such as Araldite epoxy resin;
tissues may also be
embedded directly in watermiscible acrylic resin.
 After
the resin has been
polymerized (hardened) the
sample is thin sectioned (ultra
thin sections) and stained - it is
then ready for viewing.
Sectioning
 Produces
thin slices of
specimen, semitransparent to
electrons. These can be cut on
an ultra microtome with a
diamond knife to produce ultra
thin slices about 60-90nm
thick.
 Disposable
glass knives are also
used because they can be made
in the lab and are much cheaper.
Staining
 Uses
heavy metals such as lead,
uranium or tungsten to scatter
imaging electrons and thus give
contrast between different
structures, since many (especially
biological) materials are nearly
"transparent" to electrons (weak
phase objects).
 Typically
thin sections are
stained for several minutes
with an aqueous or alcoholic
solution of uranyl acetate
followed by aqueous lead
citrate.
Negative Staining
 Negative
staining is usually done
with heavy metal salts commonly
derived from molybdenum,
uranium, or tungsten. Heavy ions
are used since they will readily
interact with the electron beam
and produce phase contrast.
A
small drop of the sample is
deposited on the carbon
coated grid, allowed to settle
for approximately one minute,
blotted dry if necessary, and
then covered with a small drop
of the stain (for example 2%
uranyl acetate).
 After
a few seconds, this drop
is also blotted dry, and the
sample is ready for viewing.
Freeze-fracture or freeze-etch
A
preparation method
particularly useful for
examining lipid membranes
and their incorporated proteins
in "face on" view.
 The
fresh tissue or cell
suspension is frozen rapidly
(cryofixed), then fractured by
simply breaking or by using a
microtome while maintained at
liquid nitrogen temperature.
 The
cold fractured surface
(sometimes "etched" by
increasing the temperature to
about -100°C for several minutes
to let some ice sublime) is then
shadowed with evaporated
platinum or gold at an average
angle of 45° in a high vacuum
evaporator
Conductive Coating
 An
ultra thin coating of
electrically-conducting
material, deposited either by
high vacuum evaporation or by
low vacuum sputter coating of
the sample.
 This
is done to prevent the
accumulation of static electric
fields at the specimen due to
the electron irradiation
required during imaging.
 Such
coatings include gold,
gold/palladium, platinum,
tungsten, graphite etc. and are
especially important for the
study of specimens with the
scanning electron microscope.
ESEM
 The
accumulation of electric
charge on the surfaces of nonmetallic specimens can be
avoided by using environmental
SEM in which the specimen is
placed in an internal chamber at
higher pressure than the vacuum
in the electron optical column.
 Positively
charged ions
generated by beam
interactions with the gas help
to neutralize the negative
charge on the specimen
surface.
 Environmental
Scanning
Electron Microscope is used to
study Wet and Oily specimens
Fluorescence microscopy
 The
absorption and subsequent
re-radiation of light by organic and
inorganic specimens is typically
the result of well-established
physical phenomena described as
being either fluorescence or
phosphorescence.
FLUORESCENCE MICROSCOPE
FLUORESCENCE MICROSCOPEWORKING
Endothelial cells under the
fluorescent microscope
Yeast cell membrane
 The
emission of light through
the fluorescence process is
nearly simultaneous with the
absorption of the excitation
light due to a relatively short
time delay between photon
absorption and emission,
ranging usually less than a
microsecond in duration.
 When
emission persists longer
after the excitation light has
been extinguished, the
phenomenon is referred to as
Phosphorescence
 Fluorescence
microscopy is
a rapid expanding technique,
both in the medical and
biological sciences. The
technique has made it possible
to identify cells and cellular
components with a high
degree of specificity.
 For
example, certain
antibodies and disease
conditions or impurities in
inorganic material can be
studied with the fluorescence
microscopy.
Autofluorescence
 A variety of specimens exhibit
autofluorescence ( emission
of visible light) when they are
irradiated
 In
contrast, the study of animal
tissues and pathogens is often
complicated with either
extremely faint or bright,
nonspecific autofluorescence.
 For
the latter studies,
fluorochromes are added
(also termed fluorophores),
which are excited by specific
wavelengths of irradiating light
and emit light of defined and
useful intensity
Fluorophores

It is a component of a molecule
which causes a molecule to be
fluorescent. It is a functional
group in a molecule which will
absorb energy of a specific
wavelength and re-emit energy at
a different (but equally specific)
wavelength.
 The
amount and wavelength of
the emitted energy depend on
both the fluorophore and the
chemical environment of the
fluorophore.
 1.
Energy is absorbed by the
atom which becomes excited.
 2. The electron jumps to a higher
energy level.
 3. Soon, the electron drops back
to the ground state, emitting a
photon (or a packet of light) - the
atom is fluorescing
Confocal microscopy
 Confocal
microscopy is an optical
imaging technique used to
increase micrograph contrast
and/or to reconstruct threedimensional images by using a
spatial pinhole to eliminate out-offocus light
Confocal Microscope
Confocal Microscope Image
Confocal Microscope Ray path
3D Confocal Microscope Ray path
Confocal Microscope Image
Basic concept
 The principle of confocal
imaging was patented by
Marvin Minsky in 1957. In a
conventional fluorescence
microscope, the entire
specimen is flooded in light
from a light source.
 Due
to the conservation of
light intensity transportation, all
parts of the specimen
throughout the optical path will
be excited and the
fluorescence detected by a
photodetector or a camera.
 In
contrast, a Confocal
microscope uses point
illumination and a pinhole in an
optically conjugate plane in
front of the detector to
eliminate out-of-focus
information.
Confocal laser scanning
microscopy (CLSM or LSCM)
 Confocal
laser scanning
microscopy (CLSM or LSCM)
is a technique for obtaining
high-resolution optical images.
 The
key feature of confocal
microscopy is its ability to
produce in-focus images of
thick specimens, a process
known as optical sectioning.
 Images
are acquired point-bypoint and reconstructed with a
computer, allowing threedimensional reconstructions of
topologically-complex objects.
Image formation
 In
a Confocal laser scanning
microscope, a laser beam passes
through a light source aperture
and then is focused by an
objective lens into a small (ideally
diffraction limited) focal volume
within a fluorescent specimen.
A
mixture of emitted
fluorescent light as well as
reflected laser light from the
illuminated spot is then
recollected by the objective
lens.
A
beam splitter separates the
light mixture by allowing only
the laser light to pass through
and reflecting the fluorescent
light into the detection
apparatus.
 After
passing a pinhole, the
fluorescent light is detected by
a photodetection device (a
photomultiplier tube (PMT) or
avalanche photodiode),
transforming the light signal
into an electrical one that is
recorded by a computer.
Atomic de Broglie microscope
 The
Atomic de Broglie
microscope is an imaging
system which is expected to
provide resolution at the
nanometer scale using neutral
He atoms as probe particles.
 Such
a device could provide
the resolution at nanometer
scale and be absolutely nondestructive, but it is not
developed so well as optical
microscope or an electron
microscope.
Atomic Force Microscope
Atomic Force Microscope
Atomic Force Microscopy Image
Dark field microscopy
 Dark
field microscopy is a
technique for improving the
contrast of unstained,
transparent specimens.
 Dark
field illumination uses a
carefully aligned light source to
minimize the quantity of
directly-transmitted
(unscattered) light entering the
image plane, collecting only
the light scattered by the
sample.
Dark field Microscopy - Image
Bright field Microscopy - Image
Infrared microscopy
 The
term infrared microscope
covers two main types of
diffraction-limited microscopy.
The first provides optical
visualization plus IR
spectroscopic data collection.
 The
second (more recent and
more advanced) technique
employs focal plane array
detection for infrared chemical
imaging, where the image
contrast is determined by the
response of individual sample
regions to particular IR
wavelengths selected by the user.
Scanning probe microscopy
This is a sub-diffraction technique.
Examples of scanning probe
microscopes are the atomic force
microscope (AFM), the Scanning
tunneling microscope and the
photonic force microscope.
 All
such methods imply a solid
probe tip in the vicinity (near
field) of an object, which is
supposed to be almost flat.
Scanning tunneling
microscopy (STM )
 Scanning
tunneling microscopy
(STM) is a powerful technique for
viewing surfaces at the atomic
level. Its development in 1981
earned its inventors, Gerd Binnig
and Heinrich Rohrer (at IBM
Zürich), the Nobel Prize in
Physics in 1986
 STM
probes the density of
states of a material using
tunneling current. For STM,
good resolution is considered
to be 0.1 nm lateral resolution
and 0.01 nm depth resolution.
 The
STM can be used not only
in ultra high vacuum but also
in air and various other liquid
or gas ambients, and at
temperatures ranging from
near zero kelvin to a few
hundred degrees Celsius
 The
STM is based on the concept
of quantum tunneling. When a
conducting tip is brought very
near to a metallic or semi
conducting surface, a bias
between the two can allow
electrons to tunnel through the
vacuum between them
Scanning tunneling microscopy
(STM )
Scanning Probe Microscopy
(SPM)
 It
is a type of microscopy that
forms images of surfaces
using a physical probe that
scans the specimen.
 An
image of the surface is
obtained by mechanically
moving the probe in a raster
scan of the specimen, line by
line, and recording the probesurface interaction as a
function of position.
PROBE FORCE MICROSCOPY
Scanning voltage microscopy
(SVM)
 It is also called
Nanopotentiometry. It is a
scientific experimental technique
based on atomic force
microscopy.
A
conductive probe, usually
only a few nanometers wide at
the tip, is placed in full contact
with an operational electronic
or optoelectronic sample.
 By
connecting the probe to a
high-impedance voltmeter and
rastering over the sample's
surface, a map of the electric
potential can be acquired.
 SVM
is generally
nondestructive to the sample
although some damage may
occur to the sample or the
probe if the pressure required
to maintain good electrical
contact is too high.
 If
the input impedance of the
voltmeter is sufficiently large,
the SVM probe should not
perturb the operation of the
operational sample.
 SVM
is particularly well suited to
analyzing microelectronic devices
(such as transistors or diodes) or
quantum electronic devices (such
as quantum well diode lasers)
directly because nanometer
spatial resolution is possible.
SVM can also be used to verify
theoretical simulation of complex
electronic devices.
Ultrasonic force Microscopy
 Ultrasonic
Force Microscopy
(UFM) has been developed in
order to improve the details
and image contrast on "flat"
areas of interest where the
AFM images are limited in
contrast.
 The
combination of AFM-UFM
allows a near field acoustic
microscopic image to be
generated.
 The
AFM tip is used to detect
the ultrasonic waves and
overcomes the limitation of
wavelength that occurs in
acoustic microscopy. By using
the elastic changes under the
AFM tip, an image of much
greater detail than the AFM
topography can be generated.
Stimulated Emission Depletion
microscopy, or STED

Stimulated Emission Depletion
microscopy, or STED microscopy, is
a technique that uses the non-linear
de-excitation of fluorescent dyes to
overcome the resolution limit imposed
by diffraction with standard confocal
laser scanning microscopes and
conventional far-field optical
microscopes
NMR MICROSCOPY
Phase contrast Microscope Image
Phase contrast Microscope Image
Digital Pathology (virtual
microscopy)
 Digital Pathology is an imagebased information environment
enabled by computer
technology that allows for the
management of information
generated from a digital slide.
 Digital
pathology is enabled in
part by virtual microscopy,
which is the practice of
converting glass slides into
digital slides that can be
viewed, managed, and
analyzed.