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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).
 Since the focal length “ f ” of a
 lens is dependent on the
 refractive index “ n ”, different
 wavelengths of light will be
 focused on different positions.
     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).
Alens 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.
 Thisminimum 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 f-
 number (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


Areal image is a
representation of an actual
object (source) formed by rays
of light passing through the
image.
 Ifa 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

 oilimmersion is a technique
 used to increase the resolution
 of a microscope.
 Thisis 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.
 Ituses two separate optical
  paths with two objectives and
  two eyepieces to provide
  slightly different viewing
  angles to the left and right
  eyes.
   this way it produces a three-
 In
 dimensional 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.
 Oftencalled "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.
 Sincethe 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 fibre-
 optic 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.
 TheSEM produces images by
 probing the specimen with a
 focused electron beam that is
 scanned across a rectangular
 area of the specimen (Raster
 scanning).
 Ateach 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 low-
 energy 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)
 Inthe 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
      dehydration, tissue for
 After
 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 water-
 miscible acrylic resin.
 Afterthe 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).
 Typicallythin 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.
Asmall 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).
 Aftera 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
 Anultra 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 non-
 metallic 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.
 Positivelycharged 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 MICROSCOPE-
        WORKING
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.
 Forexample, 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
 Incontrast, the study of animal
 tissues and pathogens is often
 complicated with either
 extremely faint or bright,
 nonspecific autofluorescence.
 Forthe 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 three-
 dimensional images by using a
 spatial pinhole to eliminate out-of-
 focus 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-by-
 point and reconstructed with a
 computer, allowing three-
 dimensional reconstructions of
 topologically-complex objects.
        Image formation
 Ina 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.
 Afterpassing 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

 TheAtomic 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 non-
 destructive, 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


 Darkfield 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.
 Allsuch 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)

 Itis 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 probe-
 surface 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.
Aconductive probe, usually
only a few nanometers wide at
the tip, is placed in full contact
with an operational electronic
or optoelectronic sample.
 Byconnecting 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.
 Ifthe 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
 UltrasonicForce 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 image-
  based information environment
  enabled by computer
  technology that allows for the
  management of information
  generated from a digital slide.
 Digitalpathology 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.

				
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