Electron Microscopy

Submitted by:-DecoyB.Tech(B.T.)-4th sem

Pt nanoparticles

The Central player - (e)

• The electron “e” is an elementary particle• Also called corpuscle • carries a negative charge. • the electron was discovered by J. J. Thompson

in 1897• e is a constituent of the atom • 1000 times smaller than a hydrogen atom. • the mass of the electron 1/1836 of that of a

proton.

The milestones

The Wave Properties• In 1924, the wave-particle dualism waspostulated by de Broglie (Nobel Prize 1929).

• All moving matter has wave properties with the wavelength λ being inversely related to the momentum p by

λ = h / p = h / mv(h : Planck constant; m : mass; v : velocity)

The Wavelength• Resolving power of EM is from Wave properties of

electrons• Limit of resolution is indirectly proportional to the

wavelength of the illuminating light• ie, longer the wavelength, lesser is the resolution• λ = √150 / V, where • λ – wavelength in Angstroms, V – accelerating

voltage in volts

The electron wave

• The generation of a monochromatic and coherent electron beam is important

• Design of modern electron microscopes is based on this concept

Scheme of electron-matter interactions arising

from the impact of an electron beam onto a specimen.

A signal below the specimen is

observable if thethickness is small enough to allow

some electrons topass through

Elastic Electron Interactions

• no energy is transferred from the electron to the sample.

• These signals are mainly exploited in - Transmission Electron Microscopy and - Electron diffraction methods.

Inelastic Electron Interactions

- Energy is transferred from the electrons to the specimen

- The energy transferred can cause different signals such as - X-rays, - Auger electrons- secondary electrons, - plasmons, - phonons, - UV quanta or cathodoluminescence.

• Used in Analytical Electron Microscopy … SEM

What is Electron Microscopy?

• Electron microscopy is a diagnostic tool with diversified combination of techniques ……

• that offer unique possibilities to gain insights into - structure,- topology, - morphology, and - composition of a material.

What is an Electron Microscope ?

• A special type of microscope having a high resolution of images, able to magnify objects in nanometers, which are formed by controlled use of electrons in vacuum captured on a phosphorescent screen

Why were the EMs ad vented?

• To study objects of < 0.2 micrometer• For analysis of sub cellular structures• Intra cellular pathogens - viruses• Cell metabolism• Study of minute structures in the

nature

Greater resolving power of the EMs than light microscope

• An EM can magnify structures from 100 – 250000 times than light microscopy

The novelty of EMs from others

• Beam of Electrons …… instead of a beam of light

• Electro-magnetic lens ………..instead of Ground glass lenses

• Cylindrical Vacuum column - Electrons should travel in vacuum to avoid collisions with air molecules that cause scattering of electrons distorting the image

Comparison of lens system of Light & Electron Microscope

Commonly used EMs in biology

• Transmission Electron Microscope• Scanning Electron Microscope “ mainly for various life forms and microbes”

• Scanning tunneling microscope• Atomic Force Microscope“ Actual visualization of molecules and

individual atoms, also in motion”

Transmission Electron Microscopy

The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this group developing the first TEM with resolving power greater than that of light in 1933 and the first commercial TEM in 1939.

TEM - DefinitionTEM is a microscopy technique whereby 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.

Applications• TEMs are capable of imaging at a significantly higher resolution

than light microscopes, owing to the small de Broglie wavelength of electrons.

• To examine fine detail—even as small as a single column of atoms, which is tens of thousands times smaller than the smallest resolvable object in a light microscope.

• Application in Biological sciences like cancer research, virology, materials science as well as pollution, nanotechnology, and semiconductor research.

• Application in chemical & physical sciences like in chemical identity, crystal orientation, electronic structure and sample induced electron phase shift as well as the regular absorption based imaging.

High resolution TEM - HRTEM• Crystal structure can also be investigated by high-resolution

transmission electron microscopy (HRTEM), • HRTEM is also known as phase contrast. • In a specimen of uniform thickness, the images are formed

due to differences in phase of electron waves, which is caused by specimen interaction.

• Image formation is given by the complex modulus of the incoming electron beams.

• The image is dependent on the number of electrons hitting the screen,

• it can be manipulated to provide more information about the sample as in complex phase retrieval techniques.

• By taking multiple images of a single TEM sample at differing angles,

• typically in 1° increments, • a set of images known as a "tilt

series" can be collected.

• This methodology was proposed in the 1970s by Walter Hoppe.

• Under absorption contrast conditions, this set of images can be used to construct a three-dimensional representation of the sample.

Gold particles on E. coli appear as bright white dots due to the higher percentage of backscattered electrons compared to the low atomic weight elements in the specimen

Scanning TEM (STEM)• Modified type of TEM • by the addition of a system that raster the beam across

the sample to form the image, combined with suitable detectors.

• The STEM uses magnetic lenses to focus a beam of electrons

• The image is formed not by secondary electrons as in SEM but by primary electrons coming through the specimen

Limitations of TEM• Many materials require extensive sample preparation • Difficult to produce a very thin sample

• relatively time consuming process with a low throughput of samples.

• The structure of the sample may change during the preparation process.

• Small field of view may not give conclusive result of the whole sample.

• sample may be damaged by the electron beam, particularly in the case of biological materials.

Definition of SEM

• An electron microscope that produces images of a sample by scanning over it with a focused beam of electrons.

• The incident electrons interact with electrons in the sample, producing various signals that can be detected and

• contain information about the sample's surface topography and composition.

The electron beams • The types of signals produced by a SEM

include - secondary electrons, - back-scattered electrons (BSE), - X-rays, - light rays (cathodoluminescence),

- A standard SEM uses Secondary electrons & Back scattered electrons

Salient features• Electrons are used to create images of the surface of

specimen - topology• Resolution of objects of nearly 1 nm• Magnification up to 500000 x (250 times > light microscopes)• secondary electrons (SE), backscattered electrons (BSE) are

utilized for imaging• specimens can be observed in high vacuum, low vacuum and • In Environmental SEM specimens can be observed in wet

condition.• Gives 3D views of the exteriors of the objects like cells,

microbes or surfaces

Scanning tunneling microscopy• 1980 – Gerd Bennig and Heinrich Rohrer invented STM• Also called Scanning Probe microscopes

• Object resolution 0.1-0.01 nm• Thin wire probe made of platinum - iridium is used to trace the

surface of the object

• Electrons from the probe overlap with electron from the surface- tunnel into one another’s clouds- tunnels create a current as the probe moves on the uneven

surface of the specimen

The applications• The STM can be used in ultra high vacuum, air, water, and various

other liquid or gaseous environments

• and at temperatures ranging from near zero to a few hundred degrees Celsius

• First movie made using STM – “individual fibrin molecule forming a clot”

• Live specimen examination – as in “Virus infected cells exploding and releasing new viruses”

• Visualization of intra cellular changes

Recent advances The effect of metallic nanoparticles on cells was

probed by treating two cell lines with C-coated Cu nanoparticles. The up-take of these nanoparticles was shown by HAADF-STEM that reveal them as bright patches inside the cells (s. image).

Nanoparticle Cytotoxicity Depends on Intracellular Solubility: Comparison of Stabilized Copper Metal and Degradable Copper Oxide Nanoparticles

References:-• A. M. Studer, L. K. Limbach, L. Van Duc, F. Krumeich, E. K. Athanassiou, L. C. Gerber,

H. Moch, and W. J. StarkToxicology Lett. 197 (2010) 169-174 DOI

• www.Wikipedia.en.us/electron microscopy

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