PRESENTED BY: -DR. GAURAV S. SALUNKHE

2 N D MDSORAL & MAXILLOFACIAL

PATHOLOGY

SCANNING ELECTRON MICROSCOPE

Contents

IntroductionHistory Construction of SEMBasics of specimen preparationWhy images are visible

History

The development of a SEM began a few yrs after the invention of a TEM by Ruska in 1931, but the commercialization of the SEM required about 30 yrs.

In 1935, the original prototype of the SEM, which scans the specimen with an e- beam to obtain an image, was made by Knoll(Germany).

In 1942, Zworykin (USA), developed a SEM for observing a bulk specimens.

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

Construction of SEM

A) Electron optical system.(to produce e-) electron gun, condenser lens, objective lens,

scanning coil.B) Specimen stage (to place the specimen).C) Secondary e- detector (to collect secondary e-).D) Image display unit.E) Operating system.The electron optical system and a space

surrounding the specimen are kept at vacuum.

Construction of SEM

Electron Gun- it produces an e- beam.It is a thermionic emission gun. (TEG), the

thermo electrons are emitted from a filament (cathode) made of a thin tungsten wire, (about 0.1mm ) by heating the filament at high temperature (about 2800k).Tungsten filament

assembly

Construction of SEM

These e- are gathered as an e-beam, flowing into the metal plate (anode) by applying a positive voltage.

If the hole is made at the center of the plate the e- beam flows through this hole.

If an electrode (Wehnelt electrode)is placed between the cathode and anode and applied with the negative charge, the speed of the e- beam can be adjusted.

Construction of SEM

Condenser lens/Electromagnetic lens- placing a lens below the e- gun enables to adjust the diameter of the e- beam.

A fine e- beam is requires for SEM .

A typical Electro Magnetic Lens

Role of the condenser lens in formation of fine e- beam

The aperture is placed between the condenser lens and objective lens.

The aperture made of a thin metal plate, has a small hole.

The e- beam, which passed through the condenser lens, illuminates this aperture-plate.

The aperture allows a part of the e- beam to reach the objective lens.

Construction of SEM

If the excitation of the condenser lens is increased, the e- beam greatly broadens on the aperture and therefore the number of the electrons reaching the objective lens decreases.

To the contrary, if the excitation of the condenser lens is decreased, the e- bean doesn’t broaden very much and therefore, most of the electrons pass through the aperture and many electrons reaches the objective lens.

In this way the e- probe diameter and the probe current can be adjusted.

Construction of SEM

Objective lens- the objective lens is used for focusing.

Construction of SEM

Specimen stage- It supports the specimen.Moves smoothly.It can perform horizontal (x,y axis) & vertical

movements(z axis), tilting of specimen, and rotation.

The horizontal movement is used for selection of the field while the vertical movement is used to change the image resolution.

SEM opened sample chamber

Secondary e- detector- It is used for detecting the secondary e- emitted from the specimen.

A scintillator (fluorescent substance) is coated on the tip of the detector and a high voltage of about 10 kV is applied to it.

The secondary e- are attracted to this high voltage and then generate light when they hit the scintillator.

This light is directed to a photon-multiplier tube (PMT) through a light guide.

The light is converted to the electrons, and these are amplified as an electric signal.

Secondary electron detector

Everhard -Thornley Detector

A supplementary electrode, called the collector, is placed before the scintillator, and is applied with few hundreds of voltage.

This collector, helps the scintillator to acquire secondary electrons.

By changing the voltage the amount of secondary electrons to be collected can be adjusted.

This collector was originally developed by Everhart and Thornley, so this detector can also be called as E-T detector.

When SEM is equipped with a strongly excited objective lens for higher resolution a secondary electron detector is placed above the objective lens.

This type of detector is called TTL ie. (Through The Lens ) detector.

Image display unit- The out put signals from the secondary electron detector are amplified and then transferred to the display unit.

Since the scanning on the display unit is synchronized with the electron-probe scan, brightness variation, which depends on the number of the secondary electrons, appears on the monitor screen on the display unit, thus forming a SEM image.

In general, the scan speed of the electron probe can be changed in several steps.

An extremely fast speed is used for observation and a slow scan speed is used for saving of image.

The image is recorded in a digital format (electrical file), It is easier to process image and convenient to send or receive image information.

Vacuum system- The electron optical system and the specimen chamber must be kept at a high vacuum of 10-3 to 10-4 Pa.

Thus , these component is evacuated by diffusion pump. If a user desire an oil-free environment, a turbo molecular pump may be used.

Sample Preparation

1. Cleaning the surface of the specimen.2. Stabilizing the specimen.3. Rinsing the specimen.4. Dehydrating the specimen.5. Drying the specimen.6. Mounting the specimen.7. Coating the specimen.

Cleaning the surface of the specimen. The proper cleaning of the surface of the

specimen is important, because the surface contains many unwanted deposits, such as dust, mud, soil etc. depending upon the source of the sample/specimen.

Stabilizing 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.Fixation cab be achieved by :-1. Perfusion or microinjection.2. Immersions.3. With vapours.

 Fixation is usually 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.

Rinsing the specimen. After the fixation step, the sample must be

rinsed in order to remove excessive fixatives.

Dehydrating the specimen.All water must be removed from the samples

because the water would vaporize in the vacuum.

The fixed tissue is then dehydrated. Because 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 is performed with a graded series of ethanol or acetone.

Drying the specimen.For SEM, a specimen is normally required to

be completely dry, since the specimen chamber is at high vacuum. 

Otherwise the sample will be destroyed in the electron microscope chamber.

Mounting the specimen.After the specimen has been cleaned, fixed, rinsed,

dehydrated and dried, using an appropriate protocol, specimen has to be mounted on the holder that can be inserted into the scanning electron microscope.

All samples must also be of an appropriate size to fit in the specimen chamber and are generally mounted rigidly on a specimen holder called a specimen stub.

The dry specimen is usually 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.

Coating the specimen.The idea of coating the specimen is to

increase the conductivity of the specimen and to prevent the high voltage charge on the specimen by conducting charge to the ground.

These specimen are coated with thin layer ie.20nm-30nm of conductive metal.

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

All non-metals need to be made conductive by covering the sample with a thin layer of conductive material.

This is done by using a device called a "sputter coater.”

 Conductive materials in current used for specimen coating includes gold, gold-palladium alloy , platinum, osmium , iridium, tungsten, chromium and graphite.

“Sputter Coater”

Advantages Disadvantages

1. Thermal conductivity is increased.

2. Damaged to the sample is reduced.

3. The quantity of secondary electrons is increased.

1. The shape & size of nano particle is lost or altered.

2. Specimen information about elemental composition & surface potential may be lost.

Metal Coating

Why Images Are Visible?

The SEM image appears as if it is been observed by the naked eye.

Interaction of electrons with specimens: when e- enters the specimen, they are scattered with in the specimen and gradually lose their energy.

The scattering range of the electrons inside the specimen is different depending on the electron energy, atomic number, and the density of the constituent atom.

If the atomic number and density are larger, the scattering range is smaller.

If the electron energy is higher then the scattering range is larger.

How Does SEM Works

To further understand how does SEM works, we must begin with the electrons. In a light microscope, light from a source (usually an incandescent light) is focused through lenses onto the sample. The image is formed when the sample reflects and absorbs different wavelengths of this light which is detected by our eyes and formed into an image by our brains. An electron microscope works in a similar fashion. Electrons from a source are focused on the sample. These electrons reflect off the sample, they are then picked up by an electron detector and then processed into an image which is projected onto a CRT that our eyes can see.

To begin our understanding of how an SEM works, let's begin with the source of electrons, the electron gun.

Most SEMs have what is called a hot cathode source, usually a tungsten filament similar to that in a light bulb.

When such a filament is heated by passing current through it, it not only emits light, but an electron cloud forms around the filament.

Left on their own, they remain in the cloud and are reabsorbed into the filament when the current is removed.

Placing a positively charged plate (an anode) near the filament and the electrons (being negatively charged) will be attracted to it.

Problem is, the electrons would not be well directed and would probably jump over to the anode plate in a series of arcs.

But by placing a negatively charged cathode plate near the filament (which they are repelled by) with a hole in it and a positively charged anode (which they are attracted to) under this with another hole in it and we have the makings of an electron gun.

The electron cloud is attracted to the anode plate enough that they will travel through the hole in the cathode. But in doing so, they gain enough speed that most of them travel right through the hole in the anode plate.

Now we have an electron gun. The speed of the electrons emitted from this gun is controlled by the amount of potential (voltage) applied to the cathode and anode plates.

The electrons from the gun come out in almost a spray pattern, so we may have a flow of electrons, but this could hardly be called a beam.

We need lenses to control the flow of electrons, however, the glass lenses of a light microscope will not work. Instead, an electron microscope uses electromagnetic lenses.

spray pattern

An electromagnetic lens An electromagnetic lens is a relatively simple device. By applying current to wire coiled around an iron cylindrical core, a magnetic field is created which acts as a lens.

The advantage of an electromagnetic lens in an electron microscope is that by varying the current through the wires, the lens can have a variable focal length.

We now can arrange the electron gun and lenses in a column mounted on a sample chamber.

 The condenser lens controls the size of the beam, or the amount of electrons traveling down the column.

The objective lens focuses the beam into a spot on the sample. This is necessary to have an image in proper focus.

So far, the column we have designed will just focus the electron beam into a spot on the sample.

This is fine for welding or if we wanted the beam to pass through the sample as in a TEM, but for an SEM to work, we need the beam to scan.

By placing sets of plates around the beam and varying the potential between them, the electron beam can be deflected. If these plates are attached to a scan generator, the beam can be made to scan lines across the sample

But this scan generator is not only controlling the scan coils, but is also controlling the beam of a CRT, the image formed on the CRT will be synched to the electron beam scanning the sample.

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

But how is the image formed?

To understand this, we need to know what happens when the electron beam interacts with the atoms of the sample.

The incident beam electrons (from the electron gun) do not simply reflect off the sample surface.

As the beam travels through the sample it can do three things:

1. It can pass through the sample without colliding with any of the sample atoms (matter is mostly space).

2. It can collide with electrons from the sample atoms, creating secondary electrons.

3. It can collide with the nucleus of the sample atom, creating a backscattered electron.

How secondary e- are formed

The incident beam is composed of highly energized electrons. If one of these electrons collides with a sample atom electron, it will knock it out of its shell. This electron is called a secondary electron and is weak in energy. If these secondary electrons are close enough to the sample surface, they can be collected to form a SEM image.

The incident beam electron loses little energy in this collusion. In fact, a single electron from the beam will produce a shower of thousands of secondary electrons until it doesn't have the energy to knock these electrons from their shells.

How backscattered e- are formed

If the incident beam collides with a nucleus of a sample atom, it bounces back out of the sample as a backscattered electron.

These electrons have high energies and because a sample with a higher density will create more of them, they are used to form backscattered electron images, which generally can discern the difference in sample densities.

Are used to determine crystal structures and orientations of minerals

An electron detector is placed in the sample chamber. By having a 10 keV positive potential on its face, it attracts the secondary electrons emitted from the sample surface.

Detection of Secondary Electrons

Secondary electrons hit against the scintillator for conversion into the optical signal, which are reconverted into electrons on the photoelectric conversion face .

These electrons are accelerated with the electric field and hit against the first dynode.

Detection of Secondary Electrons

These electrons are then led to next dynode to produce a large number of secondary electrons.

Thus the number of secondary electrons increases sequentially and finally then taken out as a signal current.

So how is the contrast formed?

In secondary imaging mode, as the incident beam scans across the sample's surface topography, secondary electrons are emitted from the sample.

If the beam travels into a depression or hole in the sample, the amount of secondary electrons that can escape the sample surface is reduced and the image processing places a corresponding dark spot on the screen.

Conversely, if the incident beam scans across a projection or hill on the sample, more secondary electrons can escape the sample surface, and the image processing places a bright spot on the screen.

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

In backscattered imaging mode, as the incident beam scans across the sample's surface topography, backscattered electrons are emitted from the sample.

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.

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.

Secondary image showing surface morphology

Backscattered image showing compositional inhomogeneity

Image of a cement

Light’ region is made up predominantly ofFe. (i.e. the heaviest element)

‘Grey’ region is made up predominantly ofCa.

‘Dark’ region is made up predominantly of Si and Al. (i.e. the lightest elements)

Fig. 1 Light micrograph showing the hard palatine mucosa. Stratified squamous keratinized epithelium (E), lamina propria (*) and connective tissue papillae are shown (arrow). Fig. 2 SEM image showing the surface of the palatine mucosa, squamous epithelium (E) and lamina propria (*).  Fig. 3 image of hard palatine mucosa. Shows transverse palatine plicae (*) and epithelial projections (arrows).  Fig. 4 Shows elongated protrusions on the surface of the palatine mucosa (*).  Fig. 5 image showing polygonal desquamated epithelial cells. Fig. 6 Surface of polygonal epithelial cells.

SEM Applications

SEMs have a variety of applications in a number of scientific and industry-related fields, especially where characterizations of solid materials is beneficial.

In addition to topographical, morphological and compositional information, a Scanning Electron Microscope can detect and analyze surface fractures, provide information in microstructures, examine surface contaminations, reveal spatial variations in chemical compositions, provide qualitative chemical analyses and identify crystalline structures.

Presence of extensive areas of resorption can be noticed on the

lingual aspect of all roots.

Ravindran Sreeja,Chaudhary Minal; Tumsare Madhuri; Patil Swati; Wadhwan Vijay J. Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009 A scanning electron microscopic study of the patterns of external root resorption under different conditions

Mandibular permanent molar undergoing root resorption due to

an associated periapical granuloma

Ravindran Sreeja,Chaudhary Minal; Tumsare Madhuri; Patil Swati; Wadhwan Vijay J. Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009 A scanning electron microscopic study of the patterns of external root resorption under different conditions

Mandibular first premolar undergoing pressure resorption during the course of orthodontic

treatment

Ravindran Sreeja,Chaudhary Minal; Tumsare Madhuri; Patil Swati; Wadhwan Vijay J. Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009 A scanning electron microscopic study of the patterns of external root resorption under different conditions

Human blood was obtained by venous puncture.

The RBCs were isolated by centrifugation.

Fixation- 1% glutaraldehyde

Washed- phosphate buffer.

Mounting Sputter coating- with

gold

SCANNING ELECTRON MICROSCOPIC IMAGE OF THE TONGUE

Scanning electron micrographs of the early human embryo

SEMs can be as essential research tool in fields such as life science, biology, gemology, medical and forensic science, metallurgy.

In addition, SEMs have practical industrial and technological applications such as semiconductor inspection, production line of miniscule products and assembly of microchips for computers.

SEM Advantages

Advantages of a Scanning Electron Microscope include its wide-array of applications, the detailed three-dimensional and topographical imaging and the versatile information garnered from different detectors.

SEMs are also easy to operate with the proper training and advances in computer technology and associated software make operation user-friendly.

This instrument works fast.

SEM Disadvantages

The disadvantages of a Scanning Electron Microscope start with the size and cost.

SEMs 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, SEMs are limited to solid, inorganic samples small enough to fit inside the vacuum chamber that can handle moderate vacuum pressure.

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

 Difference between SEM and TEM TEM SEM

 Based on transmitted electrons.  scattered electrons

 focuses on  internal composition.  sample’s surface

 image 2D image 3D image

 shows  morphology, crystallization, stress or even magnetic domains.

 morphology of samples.

 sample  cut thinner  no such need

 resolution  much higher low

Size of sample only small amount of sample can be analysed

allows for large amount of sample to be analysed

 pictures are shown on fluorescent screens  is shown on monitor

Sample preparation Is  very important  need special sample preparation.

References

1. Theory & practice of histological techniques: John Bancroft, M Gamble.

2. Scanning Electron Microscopy, Dr H. Bagshaw3. Introduction to SEM. By Rodney Herring 4. Hortolà, P. (2010). "Using digital colour to increase

the realistic appearance of SEM micrographs of bloodstains". Micron 41 (7): 904–908. 

5. "Introduction to Electron Microscopy" . FEI Company. p. 15. Retrieved 12 December 2012

6. Ravindran Sreeja,Chaudhary Minal. -J. Appl. Oral Sci. vol.17 no.5 Bauru Sept./Oct. 2009-A scanning electron microscopic study of the patterns of external root resorption under different conditions

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