SCANNING ELECTRON MICROSCOPE (SEM)

Electron MicroscopeElectron Microscopes are scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale.

This examination can yield information about the:• Topography • Morphology• Composition • Crystallographic information

Scale

Types of EMMainly 2 types:

• Transmission Electron Microscope (TEM) - allows one the study of the inner structures.

• Scanning Electron Microscope (SEM) - used to visualize the surface of objects.

Differences between SEM and TEMTEM SEM

Electron beam passes through thin sample.

Electron beam scans over surface of sample.

Specially prepared thin samples are supported on TEM grids.

Sample can be any thickness and is mounted on an aluminum stub.

Specimen stage halfway down column.

Specimen stage in the chamber at the bottom of the column.

Image shown on fluorescent screen. Image shown on TV monitor.

Image is a two dimensional projection of the sample.

Image is of the surface of the sample

Compound microscope image TEM image

Budding yeast cell

E. coli bacteria

Compound microscope image TEM image SEM image

SEM image

First Electron Microscope

Invented by Ernst Ruska

Year-1933

He was awarded the Nobel Prize for physics for his invention in 1986

History of sem First SEM – debuted in 1938 by

Manfred Von Ardenne

In 1965, Cambridge Scientific Instrument (UK) & JOEL (Japan) first commercialized SEM individually.

SEM Produces images of a sample by

scanning it with a focused beam of electrons in a raster scan pattern

Electrons interact with atoms --produces various signals that contain information about the sample's surface topography and composition. 

1.Electron cannon.

2. Electro-magnetic lenses to focus the

electron beam .

3. Vacuum pumps system

.

4.Opening to insert the object into the high-vacuum observation

chamber.

5. Operation panel with focus, alignment and

magnification tools and a joystick for positioning

of the sample.

6. Screen for menu and image display

7.Cryo-unit to prepare frozen material before insertion in the observation chamber in Cryo-SEM mode

PARTS OF SEM

Electron gun consisting of cathode and anode.

The condenser lens controls the amount of electrons travelling down the column

The objective lens focuses the beam into a spot on the sample.

Deflection coil helps to deflect the electron beam.

SED attracts the secondary electrons.

Additional sensors detect backscattered electrons and X-rays.

SEM SAMPLE PREPARATIONAppropriate size samples --- fit in the specimen chamber Mounted rigidly on a specimen holder-- specimen stub

aluminium stubs

SEM SAMPLE PREPARATIONFor imaging in the SEM, specimens must be 

Electrically conductive

Electrically grounded 

SEM SAMPLE PREPARATION1. Cleaning the surface of the specimen2. Stabilizing the specimen3. Rinsing the specimen4. Dehydrating the specimen5. Drying the specimen6. Mounting the specimen7. Coating the specimen

SEM SAMPLE PREPARATIONCleaning the surface of the specimen

Very important Surface contains many unwanted

deposits, such as dust, mud, soil etc

SEM SAMPLE PREPARATIONStabilizing the specimen

Hard, dry materials such as wood, bone, feathers, dried insects, or shells can be examined with little further treatment, but living cells and tissues and whole, soft-bodied organisms usually require chemical fixation to preserve and stabilize their structure.

Stabilization is typically done with fixatives.

SEM SAMPLE PREPARATIONFixation -- performed by incubation in a

solution of a buffered chemical fixative, such as glutaraldehyde, sometimes in combination with formaldehyde and other fixatives.

Fixatives that can be used are:-1. Aldehydes.2. Osmium tetroxide.3. Tanic acid.4. Thiocarbohydrazides.

SEM SAMPLE PREPARATIONRinsing the specimen Sample must be rinsed -- remove

excessive fixatives.

SEM SAMPLE PREPARATIONDehydrating the specimenWater must be removed Air-drying causes collapse and

shrinkage, this is commonly achieved by replacement of water in the cells with organic solvents such as ethanol or acetone.

Dehydration -- performed with a graded series of ethanol or acetone.

SEM SAMPLE PREPARATIONDrying the specimen

Specimen should be completely dry

Otherwise the sample will be

destroyed

SEM SAMPLE PREPARATIONMounting the specimenSpecimen has to be mounted on the

holder Mounted rigidly on a specimen holder

called a specimen stub Dry specimen -- mounted on a

specimen stub using an adhesive such as epoxy resin or electrically conductive double-sided adhesive tape.

Charge-up

This charge-up phenomenon can be prevented by coating the non-conductor sample with metal (conductor).

Sample coating is intended to prevent charge-up phenomenon by allowing the charge on the specimen surface go to ground through the coated conductive film.

SEM SAMPLE PREPARATIONCoating the specimen To increase the conductivity of the

specimen and to prevent the high voltage charge on the specimen

Coated with thin layer i.e., 20nm-30nm of conductive metal.

All metals are conductive and require no preparation before being used.

SEM SAMPLE PREPARATIONCoating the specimen Non-metals need to be made conductive Done by using a device called a

"sputter coater.”Conductive materials   Gold Gold-palladium Alloy Platinum Osmium Iridium Tungsten Chromium Graphite

“Sputter Coater”

A spider coated in gold

Electron beam-sample interactions The incident electron beam is scattered in the sample,

both elastically and inelastically This gives rise to various signals that we can detect Interaction volume increases with increasing acceleration

voltage and decreases with increasing atomic number

Images: Smith College Northampton, Massachusetts

So now we have a beam that is scanning across the sample surface and this beam is synched to the beam of a  CRT.

Detectors

Image: Anders W. B. Skilbred, UiO

Secondary electron detector

Backscattered electron detector

• 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.

A low atomic weight area of the sample will not emit as many backscattered electrons as a high atomic weight area of the sample.

In reality, the image is mapping out the density of the sample surface.

How do we get an image? 156 electrons!

Image

Detector

Electron gun288 electrons!

SCANNING ELECTRON

MICROSCOPIC IMAGE OF THE TONGUE

Scanning electron micrographs of the early human embryo

This form of image processing is only in gray scale which is why SEM images are always in black and white.

These images can be colorized through the use of feature-detection software, or simply by hand editing using a hand graphic editor.

This is usually for aesthetic effects, for clarifying structure, or for adding a realistic effect to the sample.

Pollen and Stamens Wool fibers

So how does a SEM change the magnification of an image?

By reducing the size of the area scanned by the scan coils, the SEM changes the magnification of the image.

Vacuum Chamber SEMs require a vacuum to operate.

Without a vacuum, the electron beam generated by the electron gun would encounter constant interference from air particles in the atmosphere.

Not only would these particles block the path of the electron beam, they would also be knocked out of the air and onto the specimen, which would distort the surface of the specimen.

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

SEM Advantages Gives detailed 3D and topographical

imaging and the versatile information garnered from different detectors.

Works very fast. Modern SEMs allow for the generation

of data in digital form. Most SEM samples require minimal

preparation actions.

SEM Disadvantages SEMs are expensive and large. Special training is required to operate an

SEM. Preparation of samples can result in artifacts. Limited to solid samples. Carry a small risk of radiation exposure

associated with the electrons that scatter from beneath the sample surface.

Thank you