Shishu Ananta Mahavidyalaya

Department of Botany
Bijay kumar Dash
+3 Final year Botany(Hons.)
BS16-101
1602010290140060
 TOPIC:
IMAGING OF TISSUE SPECIMEN-ELECTRON
MICROSCOPY
Contents:
 What is my presentation ?
 Microscopy and its importance
 Discovery of electron microscope
 Classification
 Mechanism of TEM and SEM
 Application
 Conclusion
What is my presentation ?

 My presentation is completely based on electron microscopy.

 The electron microscope is a type of microscope that uses electrons
to create an image of the target.
 It has much higher magnification or resolving power than a normal
light microscope.
 The electron microscope is an integral part of many laboratories.
 Researchers use it to examine biological materials (such as
microorganisms and cells), a variety of large molecules, medical
biopsy samples, metals and crystalline structures, and the
characteristics of various surfaces.
Microscopy :

 The word microscope is derived from the Greek word mikros (small)
and Skopeo(look at).
 A microscope is an instrument used to see objects that are too small
to be seen by the naked eye.
 Microscopy is the science of investigating small objects and
structures using such an instrument. Microscopic means invisible to
the eye unless aided by a microscope.
 Importance of Microscope:
 Nowadays, many important fields of science can not progress without the aid of
microscopy. It is important in areas like engineering, biology, physics and many
other areas of knowledge.
 In biology electronic microscopy has brought the possibility to see things we
could only wonder about. Of course the researchers were not guessing out of the
blue, but being able to see processes in first hand most certainly helps. An
example is the ability to see cellular processes like phagocytosis or mitosis with
the aid of optical microscopes.
Discovery of Electron Microscope:
 In the early 20th century a significant alternative to the light microscope was
developed, an instrument that uses a beam of electrons rather than light to
generate an image.
 The German physicist, Ernst Ruska, working with electrical engineer Max Knoll,
developed the first prototype electron microscope in 1931, a transmission
electron microscope (TEM).
 The transmission electron microscope works on similar principles to an optical
microscope but uses electrons in the place of light and electromagnets in the
place of glass lenses. Use of electrons, instead of light, allows for much higher
resolution.
 Development of the transmission electron microscope was quickly followed in
1935 by the development of the scanning electron microscope by Max Knoll.
Although TEMs were being used for research before WWII, and became popular
afterwards, the SEM was not commercially available until 1965.
Classification:

 Electron microscope is mainly of two types;

a) Scanning electron microscope
b) Transmission electron microscope
What is TEM ?

 TEM is a unique tool in characterization of materials crystal structure

and microstructure simultaneously by diffraction and imaging
techniques.
 Beam of electrons is transmitted through an ultra thin specimen.
 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
 DEVELOPER
OF
TEM

ERNEST RUSKA(1931)
What is SEM ?
 DEVELOPER
OF
SEM

Manfred von Ardenne(1938)

 Mechanism of TEM:
 Specimen is bombarded by a beam of electrons, the primary electrons . The
bombarding electrons are focused to a bundle onto the object.
 In areas in the object where these electrons encounter atoms with a heavy atomic
nucleus, they rebound.
 In regions where the material consists of lighter atoms , the electron are able to pass
through.
 The fine pattern of electrons leaving the object , reaches the objective lens forms the
image
 It is then greatly enlarged by projector lens.
 Eventually, the traversing electrons (transmission) reach the scintillator plate at the
base of the column of the microscope.
 The scintillator contains phosphor compounds that can absorb the energy of the
stricking electrons and convert it to light flashes.
 Thus a contrasted image is formed on this plate.
 Mechanism of SEM:
 The electron gun produces an electron beam when tungsten wire is heated by current.
 This beam is accelerated by the anode.
 The beam travels through electromagnetic fields and lenses, which focus the beam down
toward the sample.
 A mechanism of deflection coils enables to guide the beam so that it scans the surface of
the sample in a rectangular frame.
 When the beam touches the surface of the sample, it produces:
– Secondary electrons (SE)
– Back scattered electrons (BSE)
– X - Rays...
 The emitted SE is collected by SED and convert it into signal that is sent to a screen which
produces final image.
 Additional detectors collect these X-rays, BSE and produce corresponding images.
 A secondary electron detector attracts the scattered electrons and, depending
on the number of electrons that reach the detector, registers different levels of
brightness on a monitor.
 By reducing the size of the area scanned by the scan coils, the SEM changes the
magnification of the image.
 BIOLOGICAL APPLICATIONS OF TEM:
 In medicine as a diagnostic tool – important in renal biopsies.
 Cellular tomography
– Tomography refers to imaging by sectioning, through the use of any kind of
penetrating wave.
– Information is collected and used to assemble a three dimensional image of
the target.
– Used for obtaining detailed 3D structures of subcellular macromolecular
objects.
 Cancer research - studies of tumor cell ultrastructure .
 Toxicology – to study the impacts of environmental pollution on the different
levels of biological organization.
 BIOLOGICAL APPLICATIONS OF SEM:
 Virology - for investigations of virus structure
 Cryo-electron microscopy – Images can be made of the surface of frozen
materials.
 3D tissue imaging -
– Helps to know how cells are organized in a 3D network
– Their organization determines how cells can interact.
 Forensics - SEM reveals the presence of materials on evidences that is otherwise
undetectable
 SEM renders detailed 3-D images
– extremely small microorganisms
– anatomical pictures of insect, worm, spore, or other organic structures
 RECENT DEVELOPMENTS:
 Three famous physicists, Harald H. Rose , Knut W. Urban and Maximillian Haider
have received the Wolf-prize in physics 2011 for the realization of aberration-
corrected electron microscopy.
 Aberrations are intrinsic imperfections of electron lenses.
 Those aberrations are reduced by installing in a microscope a set of specially
designed auxiliary "lenses" which are called aberration correctors.
 They designed a novel aberration corrector thereby improving resolution of
transmission electron microscope.
 Conclusion:
 Since its invention, electron microscope has been a valuable tool in the
development of scientific theory
 It has contributed greatly to biology, medicine and material sciences.
 This wide spread use because they permit the observation of materials on a
nanometer (nm) to micrometer (μm) scale.
 Although SEMs and TEMs are large, expensive pieces of equipments, they remain
popular among researchers due to the high resolution and detailed images they
produce.