ELECTRON MICROSCOPY - SEM & TEM 1

ELECTRON MICROSCOPY-SCANNING ELECTRON MICROSCOPE

-TRANSMISSION ELECTRON MICROSCOPEPRESENTED BY – SUMER PANKAJ

CLASS – MSC. EST ROLL NO. - 42

Institute of Science and Technology for Advanced Studies and Research

Affiliated to Sardar Patel University Recognized under section 2(f) and 12 (B) of UGC act 1956 Mota Bazaar, Vallabh Vidyanagar,

Anand, Gujarat 38812058PG Department of Environmental Science and Technology

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CONTENT• Microscopy and its types• Principle of electron

microscopy• Types of electron

microscopy• SEM vs. Tem• Recent advancements in

SEM & TEM• References

ELECTRON MICROSCOPY - SEM & TEM

SEM (Scanning Electron Microscope)• Principle• Components• Instrumentation• Working• Applications• LimitationsTEM (Transmission Electron Microscope)• Principle• Components• Instrumentation• Working• Checklist• Applications• Limitations

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INTRODUCTION• Microscopy is the technical field of

using microscopes to view objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron and scanning probe microscope.

• Optical and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation or electron beams interacting with the specimen, and the collection of the scattered radiation or another signal in order to create an image.

• Scanning probe microscopy involves the interaction of a scanning probe with the surface of the object of interest.

1..

2.

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Types of

Microscopy

Others

Optical Microsc

opy

Scanning Probe Microsc

opy

Electron

Microscopy

• Bright field• Oblique

illumination• Dark field• Dispersion

staining• Phase contrast• Differential

interference contrast

• Interference reflection

• Fluorescence• Confocal• Wide-field

multiphoton microscopy

• Transmission electron microscopy (TEM)

• Scanning electron microscopy (SEM)

• Atomic force microscope (AFM)

• Scanning tunnel microscope

• Photonic force microscope

• Recurrence tracking microscope

• Ultraviolet microscopy

• Infrared microscopy

• Digital holographic microscopy

• Digital pathology (virtual microscopy)

• Laser microscopy• Amateur

microscopy

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PRINCIPLE OF ELECTRON MICROSCOPE• An electron microscope is a microscope that uses a beam of

accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects. A transmission electron microscope can achieve better than 50 pm resolution and magnifications of up to about 10,000,000x whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x.

• Electron microscopes are used to investigate the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, electron microscopes are often used for quality control and failure analysis. Modern electron microscopes produce electron micrographs using specialized digital cameras and frame grabbers to capture the image.

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SCANNING ELECTRON MICROSCOPE (SEM)

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PRINCIPLE OF SEM• 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 known as secondary electrons that contain information about the sample's surface topography and composition.

• SEM can achieve resolution better than 1 nanometre. Specimens can be observed in high vacuum, in low vacuum, in wet conditions (in environmental SEM), and at a wide range of cryogenic or elevated temperatures.

•  A wide range of magnifications is possible, from about 10 times (about equivalent to that of a powerful hand-lens) to more than 500,000 times, about 250 times the magnification limit of the best light microscopes

ELECTRON MICROSCOPY - SEM & TEM

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Components of

SEM

Emission

chamber

Electromagnetic lenses

Specimen

chamber

• Electron gun :- which emits electron

• Condenser Lens system – (focus the electron beam to a narrower area).

• Objective Aperture – (directs the narrowed beam to objective lens).

• Objective lens – (captures the electron beam emerging form the object).

• The Detector – (detects the electrons deflected by the specimen).

• Television Picture Tube – (image is formed on the television screen).

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CONSTRUCTION OF SEM/INSTRUMENTATION

• Emission chamber - it is an electron generating arrangement used to generate an electron –beam in TEM called as electron gun.This electron gun has a tungsten filament which functions as cathode. It emits electron from its surface when it gets heated by passage of high voltage electricity.

Tungsten is normally used in electron guns because it has the highest melting point and lowest vapour pressure of all metals, and because of its low cost. Other types of electron emitters include lanthanum hexaboride (lab6) and zirconium oxide cathodes.

• Electromagnetic lens systems – In SEM’s a radially symmetrically coil of wire is used as the lens with current passing through it, such virtual lenses are known as EM lenses.

• The coil is made up of few thousand turns of the wire having a soft iron casting around it. An 1 amp. Current is passed through the coil to produce EM field and the outer iron casting concentrates the magnetic field.

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TYPES OF EM LENSi. Condenser lens – there are 2 condenser placed just

below the electron gun i.e. Primary condenser lens and secondary condenser lens. It’s main function is to focus the electron beam to a narrower part.

ii. Objective aperature – it directs the narrowed electron beam which has the thickness of 20nm on to the objective lens through the scanning coil.

iii. Scanning coil –  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.

iv. Objective lens – it focuses the scanning beam on the desired area of the specimen.

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SPECIMEN CHAMBER• The detectors - the electrons are detected

by an everhart-thornley detector, which is a type of scintillator-photomultiplier system. The secondary electrons are collected by a positively charged grid arrangement. In modern SEM’s multi-electronic detectors are used to develop coloured image instead of panchromatic images.

• Television picture tube – the secondary electron collected by thee positively charge grid is sent to the television picture tube where the final image is formed. The number of scanning lines in SEM are far more as compared to an ordinary television, this makes the picture very clear upto 100ºA.

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WORKING OF SEM• In a typical SEM, an electron beam is  emitted from an electron gun fitted

with a tungsten filament cathode. Tungsten is normally used in electron guns because it has the highest melting point and lowest vapour pressure of all metals, and because of its low cost. Other types of electron emitters include lanthanum hexaboride (lab6) and zirconium oxide cathodes. 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 of sample. The beam passes through pairs of scanning coils or pairs of deflector plates in the electron column. In the final lens, the deflected beam in the x and y axis so that it can be scanned in a raster fashion over a rectangular area of the sample surface.

• When the primary electron beam interacts with the sample, the electrons lose energy by repeated random scattering and absorption within a teardrop-shaped volume of the specimen known as the interaction volume, the interaction volume depends on the electron's landing energy, the atomic number of the specimen and the specimen's density. The energy exchange between the electron beam and the sample results in the reflection of high-energy electrons by elastic scattering, each of which can be detected by specialized detectors. The beam current absorbed by the specimen can also be detected and used to create images of the distribution of specimen current. Electronic amplifiers of various types are used to amplify the signals, which are displayed as variations in brightness on a computer monitor.

ELECTRON MICROSCOPY - SEM & TEM

APPLICATION OF SEM

Biotechnology/ life sciences• cell

morphology, • Development

of biocompatible materials,

• tissue engineering research,

• microbiology

Medical sciences/ Research• Determining

the receptors of cell.

• Identification of Antigen-antibody complex.

Semiconductor industry• Failure

Analysis of Integrated Circuits

• Advanced Packaging: Wire Bonding

• Circuit Edits• Micro-Electro-

Mechanical Systems

Material Sciences• Steels & Metal

Alloys• Polymers and

Composites• Materials for

Building and Civil Engineering

• Composition of - Wood, Textile.01/05/2023ELECTRON MICROSCOPY - SEM & TEM

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Geology and Earth sciences.• metamorphi

c petrology and mineralogy

• Ore processing

• Oil & Gas• Palaeontolo-

gy

LIMITATIONS OF SEM• The disadvantages of a scanning electron microscope start with the

size (not portable) and cost (expensive).• SEM’s are expensive, large and must be housed in an area free of

any possible electric, magnetic or vibration interference.• Maintenance involves keeping a steady voltage, currents to

electromagnetic coils and circulation of cool water.• Special training is required to operate an SEM as well as prepare

samples.• The preparation of samples can result in artifacts. The negative

impact can be minimized with knowledgeable experience researchers being able to identify artifacts from actual data as well as preparation skill. There is no absolute way to eliminate or identify all potential artifacts.

• In addition, SEM’s are limited to solid, inorganic samples small enough to fit inside the vacuum chamber that can handle moderate vacuum pressure.

• Finally, SEM’s carry a small risk of radiation exposure associated with the electrons that scatter from beneath the sample surface.

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TRANSMISSION ELECTRON MICROSCOPE (TEM)

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ELECTRON MICROSCOPY - SEM & TEM

PRINCIPLE OF TEM• Illumination source is a beam of

high velocity electrons accelerated under vacuum, focused by condenser lens (electromagnetic bending of electron beam) onto specimen.

• Image formation is due to loss and scattering of electrons by individual parts of the specimen. Emergent electron beam is focused by objective lens. Final image forms on a fluorescent screen for viewing.

• Image is captured on negative or by digital camera

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Components of

TEM

Emission

chamber

Electromagnetic lenses

Specimen

chamber

Photographic

arrangement

Vacuum

system

• Electron gun :- which emits electron • Condenser

Lens system – (focus the electron beam on the object).

• Objective lens – (captures the electron beam emerging form the object).

• Additional Lens – (Magnifies the changes in the object).

• Projector Lens – (Collects the magnified image and projects it on the florocent screen)

• Air Lock system – (Maintains the vaccum in the specimen chamber).

• Microgrid – (Holds the specimen in its place).

• Specimen Holder – (Ensures the alingment of specimen in the path of electron)

• Rotary Pump – (Creats initial low vaccum).

• Oil Diffusion Pump – (maintains the high vaccum).

• Coldfingure – (Establishes high vaccum).

• A plate camera – (enlarges the image produced by the camera).

• 35 mm film camera or digital camera (captures the image projected on the screen).

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CONSTRUCTION OF TEM/ INSTRUMENTATION

1. EMISSION CHAMBER – It is an electron generating arrangement used to generate

an electron –beam in TEM called as electron gun

This electron gun has a tungsten filament which functions as cathode. It emits electron from its surface when it gets heated by passage of high voltage electricity. It is safely housed in a metallic chamber known as cathode shield.

The anode consist of a metal plate with a hole drilled in the centre. The circular hole of the anode is exactly in the line with the circular hole of the cathode shield. Thus the path of the electron beam is defined

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ELECTROMAGNETIC LENSES • In TEM a radially symmetrically coil of wire is

used as the lens with current passing through it, such virtual lenses are known as EM lenses.

• The coil is made up of few thousand turns of the wire having a soft iron casting around it. An 1 amp. Current is passed through the coil to produce EM field and the outer iron casting concentrates the magnetic field.

• The focal length of the lens can be adjusted by adjusting the current passing through it.

• There are various types of EM lenses systems are used in tem like condenser lens system, objective lens, additional lens, projector lens etc.

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TYPES OF EM LENS SYSTEMS i. Condenser lens system – it is located just below the electron

gun assembly. And its main function is to condense (focus) the electron beam on the object. In earlier versions there were only one condenser lens placed but in Morden TEM’s there are 2 condenser lenses.

ii. Objective lens – it is places just below the specimen chamber and its main function is to capture the electron beam emerging from the objects.

iii. Additional lenses – these are also called as intermediate lenses which are placed below the objective lens, these lenses allows the magnification of the diffraction pattern of the electrons caused by the structure of the specimen.

iv. Projector lens – it resembles to that of a eye piece of a light microscope. It collects the magnified image from the intermediate and objective lenses and projects it on a florescent screen.

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SPECIMEN CHAMBER • The specimen is to be place exactly in the

centre between the condenser and objective lens.

• In an electron microscope the changing of specimen should be carried out without breaking the vacuum present in the column of the microscope, for this the specimen chamber is provided with an air-lock system.

• The sections used in tem must be ultra thin having the thickness less than 1 µ. These thin sections are to be mounted on a microgrid, which is placed on the specimen holder.

• The specimen holder is a metallic block which assures that the specimen mounted on the microgrid is in the electron path.

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VACUUM SYSTEM• Vacuum is developed at two levels – a standard rotary pump is used

to develop initial low vacuum and for high vacuum a metal piece is inserted in the vacuum system to produce high vacuum and is known as cold-fingure.

• This metal piece before inserting is treated with liquid nitrogen which traps the air molecules and gasses on its cool surface.

• Oil diffusion pumps are used to maintain this high vacuum. This vacuum generated provides an air-lock column where the specimen is mounted. PHOTOGRAPHIC

ARRANGEMENT

• It consist of a plate camera which is situated below the florescent screen as it enlarges the photographed image on the screen.

• An additional 35mm film camera or a digital camera is fixed to capture the image projected on the screen.

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WORKING OF TEM• The original form of electron microscope, the transmission electron microscope

 (TEM) uses a high voltage electron beam to illuminate the specimen and create an image. The electron beam is produced 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 +100 ev (40 to 400 kev) with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the "image") may be viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. Alternatively, the image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a digital camera. The image detected by the digital camera may be displayed on a monitor or computer.

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CHECKLIST FOR TEM Various lenses and the electron gun should be accurately

lined up on a common axis. The specimen should be less than 1µ in size.

After placing the specimen in the specimen chamber fine micrometre screws are to be used to move the specimen in the line of electron beam.

The focal length of the EM lenses varies with the wavelength of electron and intensity of the electric current flowing through so the electric flow should be stabilized using a stabilizer.

The electric current can also be maintained by creating resistance for the flow.

Morden TEM’s have aberration correctors which reduces the distortion in the image. They also have a monochromaters to reduce the energy spread of the incident electron beam less than 0.15ev.

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ICAT

ION

S O

F TE

M Material Sciences • morphology, structure, and local chemistry of

metals• investigation of crystal structures, orientations

and chemical compositions of phases, precipitates and contaminantsGeology, Environmental Science

• To study the mineral composition.• To study the toxic effect at molecular level.• Study of composition of Mineral from ore

Medical and life Sciences• To study the structure and composition of

viruses (virology)• To study the composition of cell.

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LIMITATIONS• Many materials require

extensive sample preparation to produce a sample of 1µ which is time consuming.

• The sample preparation may bring structural changes in the original structure.

• As the field of view is very small so the area or the region of the sample observed may not represent the whole.

• Biological samples may get damaged on prolonged exposure to electron beam.

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WHICH ONE IS BETTER ?

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SEM’s• SEM images are artificially coloured

these days by false colour contrast (FCC) process. This may be done for aesthetic effect, to clarify structure or to add a realistic appearance to the sample and generally does not add information about the specimen.

• The third dimension images can be reconstructed by collaging some series of stereo pairs captured from with different angles.

• The environmental scanning electron microscope  is a scanning electron microscope (SEM) that allows for the option of collecting electron micrographs of specimens that are "wet," uncoated, or both by allowing for a gaseous environment in the specimen chamber.

TEM’s• TEM’s are combined with SEM’s

band dark field imaging has been reported in the generally low accelerating beam voltage range used in SEM, which increases the contrast of unstained biological specimens at high magnifications with a field emission electron gun.

• Earlier version of TEM’s had only one condenser lens but in Morden TEM’s there are 2 condenser lens followed aberration correctors which reduces the distortion in the image.

• They also have a monochromater to reduce the energy spread of the incident electron beam less than 0.15ev.

Advancements in TEM & Sem

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REFERENCES• HTTPS://

EN.WIKIPEDIA.ORG/WIKI/ENVIRONMENTAL_SCANNING_ELECTRON_MICROSCOPE• HTTPS://EN.WIKIPEDIA.ORG/WIKI/SCANNING_ELECTRON_MICROSCOPE• HTTP://

WWW.MICROSCOPEMASTER.COM/TRANSMISSION-ELECTRON-MICROSCOPE.HTML

• HTTP://WWW.SEM-APPLICATIONS.COM/• HTTPS://

WWW.GOOGLE.CO.IN/SEARCH?Q=SEM&ESPV=2&BIW=1242&BIH=557&TBM=ISCH&SOURCE=LNMS&SA=X&VED=0AHUKEWIIU6TCIMFRAHUIQ48KHZJOAJAQ_AUIBIGB

• HTTPS://WWW.GOOGLE.CO.IN/SEARCH?Q=SEM&ESPV=2&BIW=1242&BIH=557&TBM=ISCH&SOURCE=LNMS&SA=X&VED=0AHUKEWIIU6TCIMFRAHUIQ48KHZJOAJAQ_AUIBIGB#TBM=ISCH&Q=TEM

• HTTPS://WWW.GOOGLE.CO.IN/SEARCH?Q=SEM&ESPV=2&BIW=1242&BIH=557&TBM=ISCH&SOURCE=LNMS&SA=X&VED=0AHUKEWIIU6TCIMFRAHUIQ48KHZJOAJAQ_AUIBIGB#TBM=ISCH&Q=IMAGE+IN+SEM+AND+TEM

• WWW.PATHO.HKU.HK/DOCS/MMEDSC/MMS2KEM5.PPT

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01/05/2023ELECTRON MICROSCOPY - SEM & TEM 30Thank you…