Manipulation of nanomaterials by transmission electron microscopy (TEM) and scanning electron microscopy (SEM)

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Manipulation of nanomaterials by transmission electron microscopy (TEM) and scanning electron microscopy (SEM)

Ssanning electron microscopy

Introduction

Minor defects often result in considerable damage. Small fractures or cracks in materials can have disastrous effects on the stability of buildings, tools, etc. once an accident has happened, its causes have to be found. A microscope examination of the fracture surface shows whether a material defect or a processing defect has caused the fracture. Light –optical and electro-optical microscopes are used for this purpose. Electron microscopes are advantageous in that a high degree of magnification as well as an excellent depth of focus can be achieved. As a rule, Surfaces of fracture are very rough so that a light-optical microscope often cannot produce a sufficiently clear enlargement of the relevant image section.SEM replace the optical microscope and provides highly magnified image of the specimen surface. Great improvement in microscope was made by C.W.Oatley and his group in Cambridge and resolution in the order of 250(angstrom).

PRINCIPLE:

          A finely focused electron beam scanned across the surface of the sample generates secondary electrons, backscattered electrons, and characteristic X-rays. These signals are collected by detectors to form images of the sample displayed on a cathode ray tube screen. Features seen in the SEM image may then be immediately analyzed for elemental composition using EDS spectrum.

INSTRUMENTATION AND FEATURES OF SEM:

A Scanning Electron Microscope is composed of: 

·          Electron Source

·          Electromagnetic and condenser  Lens

·          Electron Optical Column

·          Ray Diagrams

·          Electron Beam/Specimen Interactions

·          Vacuum and Specimen chamber

Computers will be integrated more and more into commercial SEM’s and is enormous potential for the growth of computer supported applications. An environmental SEM has been developed that uses differential pumping to permit the observation of the specimen at higher pressures. Photographs of the formation of ice crystals have been taken and the instrument has particular application to samples that are not vacuum compatible, such as biological samples.

Local magnetic fields affect the trajectories of the secondary electrons, making the SEM useful tool for observing the magnetic domains of ferromagnetic materials, magnetic tapes, and disk surfaces.

Pulsed electron beam generates both thermal and acoustic signals which can be imaged to provide mechanical property maps of materials.

Some semiconductors and oxides produce photons in UV regions and these cathodoluminescence signals provide the information about electronic properties of this matrerials.the application of this method to semiconductor lasers devices is probably self-evident.

The essential features of SEM are,

1.   An electron source

2.   A means of focusing tiny spot of electrons from  the source on the specimen

3.   A means of scanning the spot across the specimen

4.   A means of detecting the response from the specimen

5.   A display system, capable of being scanned in register with  the incident scan

6.   A means of transmitting the response from the specimen to the display system.

OPERATION:

The electron source like thermionic tungsten, LaB6, hot and cold field emission produce the electrons focus it to the sample. Recently field emission electron source are used, these tips are very sharp; the strong electric field are created at the tip extracts electrons from source even at low temperature. The beam from the source is focussed by series of magnetic lenses; each lens has an associated defining aperture that limits the divergence of electron beam. Top lenses are said to be condenser lens, by increasing current through condenser lens, the focus length is decreased and divergence increased. Increasing the current through the first lens reduce the size of image of the image produced. Smaller spot size ,often given higher dial numbers to correspond with the higher lens current required for better resolution, are attained with less current and small signal-noise ratio. Very higher magnification images therefore are inherent noisy.

The beam next arrives at the final lens-aperture combination. The final lens focuses the beam on to the surface of the sample. The sample is attached to the specimen stage that provides x-and y-motion, as well as tilt with respect to beam axis and rotation about axis normal to the specimen surface. A final “z” motion allows adjustment of distance between the final and lens and sample surface. Typically the convergence angle is few mrad and it can be decreased by using small final aperture or by increasing working distance. The smaller convergence angle, the most variation in      z-direction topography that can be tolerated with still remaining in focus to some prescribed degree. The large depth of focus contributes to the case of observation of topographical effect.

SEM IMAGING MODES:

The limiting sharpness and feature visibility of SEM micrographs are depending upon

·         The electron probe size d_p

·         the electron probe current i_p

·         the electron probe convergence angle α _p

·         the  electron beam accelerating voltage V₀

Each of these four beam parameters dominates one of the four imaging modes. That is       

Resolution mode:

For the higher resolution image, d_p   must be as small as possible while at the same time containing sufficient beam current to exceed the visibility threshold for the contrast produced by features of interest .to image the finest details of sample, the probe diameter must be comparable or smaller than the feature itself. Resolution mode is meaningful at high image magnifications (<10,000>X) where the small details can be observed because at low magnifications image sharpness is controlled by the display parameters.

High current mode:

For the best image visibility and quality, large beam current i_p    is required. This also necessary for successful microanalysis because only a small fraction of beam interactions results in X-ray emission. When working on bulk samples, x -ray spatial resolution is limited to about 1µ. It would therefore be to use electron probe diameter of100nm to 1µm

Depth-of-focus mode:

To obtain the high depth of focus, alpha must be reduced to as small a value as possible by using small aperture, long working distance or both for this mode the curves of probe size versus probe current are of little help expect for estimating the size of probe at particular condenser lens setting so that the maximum magnification can be calculated.

Low-ºelectrons mostly carry the information about the interior of the sample.

SAMPLE PREPARATION:

The use of SEM that requires very little in regard to sample preparation provided that the specimen is vacuum compatible.

If the sample is conducting the limitation is whether it will fit on to the stage or for that matter, in to the specimen chamber. For special application, very large stage –vacuum chamber combinations have been fabricated in to which large forensic sample or 8-in dia semiconductor can be placed.

If the sample is insulating, that is coated with thin10nm conducting film of like carbon, gold. To avoid artefact and distortion that could be produced by non uniform coatings. Uncoated insulating samples also studied by low primary beam voltages (<2keV)

If the sample is metal that has been coated with thin oxide layer higher accelerating voltage might improve the image. Because the high energy beam passes through the oxide it can create electron –hole pair in sufficient numbers to establish local conduction.

For the biological specimen has been examined at temperature ranging from 25 °C to 180°C, the material be quench-frozen by plugging specimen stubs with material mounted on liquid nitrogen at-196°C.

Silver is essential metal coating to use for polymers and fibres. If the specimens are kept very long periods of time gold or gold/pd alloys are recommended. When operating at low beam voltage the problems of charging are not as great and minimum amount of metal is required on the surface of the specimen to suppress the charging effect.

RESOLUTION:

If the image is formed solely by electrons from the surface of specimen object, then the spot size will be the major reason for loss of resolution. But in particularly low-density specimen penetration can occur in to the specimen and the secondary electrons are emitted from volume below the surface. Where increasing the penetration will effectively increase noise or accumulations of multiple images, which will be increasing out of focus, and so will lead to an obscuring of detail through loss of contrast due to this increased noise. the loss of contrast is depending on the relative size of zone and features of object, another effect is that continued penetration of the primary beam may lead, in relatively thin specimens, to other zones of secondary emission on the lower surface or on some other object, this gives double or triple images., whose sharpness will depend on depth of focus and extend to which the spot size is increased by scattering of primary beam.

High emission can lead to glare at the edge of the specimens. Electrostatic charging causes false contrast, due to its influence on emission and to image distortion. The specimen may be damaged by electron bombardment and it may move due to impact of the electron beam or external vibration

Shortly can Increasing the resolution by increasing beam voltage so as to reduce the spot size if this gives an undue increase in penetration. the compromise will  not involve only resolution and contrast, but also other factors such as depth of focus, exposure time, beam damage and so on, which will vary in importance with the circumstances.

Image construction (mapping):

The information flow from SEM consists of the scan location in x-y space and corresponding set of intensities from the set of detectors that monitor the beam specimen interactions, all of which can be monitored in parallel. This information can be displayed by two ways that is

Line scan: the beam is scanned along single vector on the specimen. The same scan generator signal is used to drive the horizontal scan of the CRT. The effective magnification factor M between the specimen space and the CRT space is given by ratio of length of the scans,

                             M=L_CRT/L_specimen

Image (area) scanning: Image formation is produced by mapping operation that collects the information from the specimen space and passes the information to the display space. The principle of intensity modulation is used to display the magnitude of signal produced by electron-specimen interaction at the location scanned. This scanning occurs part by part across the specimen like television rate display operation.

Transmission Electron Microscope (TEM

The transmission electron microscope (TEM) operates on the same basic principles as the light microscope but uses electrons instead of light. What you can see with a light microscope is limited by the wavelength of light. TEMs use electrons as "light source" and their much lower wavelength makes it possible to get a resolution a thousand times better than with a light microscope.

We can see objects to the order of a few angstrom (10^-10 m). For example, we can study small details in the cell or different materials down to near atomic levels. The possibility for high magnifications has made the TEM a valuable tool in both medical, biological and materials research.

Magnetic Lenses Guide the Electrons

A "light source" at the top of the microscope emits the electrons that travel through vacuum in the column of the microscope. Instead of glass lenses focusing the light in the light microscope, the TEM uses electromagnetic lenses to focus the electrons into a very thin beam. The electron beam then travels through the specimen you want to study. Depending on the density of the material present, some of the electrons are scattered and disappear from the beam. At the bottom of the microscope the unscattered electrons hit a fluorescent screen, which gives rise to a "shadow image" of the specimen with its different parts displayed in varied darkness according to their density. The image can be studied directly by the operator or photographed with a camera.

Preparation of Specimen for TEM

In a TEM, the specimen you want to look at must be of such a low density that it allows electrons to travel through the tissue. There are different ways to prepare your material for that purpose. You can cut very thin slices of your specimen from a piece of tissue either by fixing it in plastic or working with it as frozen material. Another way to prepare your specimen is to isolate it and study a solution of for example viruses or molecules in the TEM.

We can also stain the specimen in different ways and use markers to locate specific things in the tissue. It can for example, be stained with heavy metals like uranium and lead, which scatters electrons well and improves the contrast in the microscope. Below are two examples described in more detail.

Sections of Embedded Material

Biological material contains large quantities of water. Since the TEM works in vacuum, the water must be removed. To avoid disruption as a result of the loss of water, you preserve the tissue with different fixatives. These cross-link molecules with each other and trap them together as stable structures. The tissue is then dehydrated in alcohol or acetone.

After that, your specimen can be embedded in plastic that polymerize into a solid hard plastic block. The block is cut into thin sections by a diamond knife in an instrument called ultramicrotome. Each section is only 50-100 nm thick.

The thin sections of your sample is placed on a copper grid and stained with heavy metals. The slice of tissue can now be studied under the electron beam.

Whole mounts

Small or very thin objects can be examined directly by mounting them onto a support film and introducing them directly into the electron beam. Contrast is provided by heavy metal precipitation in one of three ways.

1. Positive staining: The object is chemically stained with a solution of the metal salt and appears dark on a bright background.
2. Negative staining: The object remains unstained but is embedded in a dried film of the heavy metal salt. The specimen appears light on a dark background. This method of visualization has been used extensively in the study of virus particles but is also useful for cell fractions (e.g. coated vesicles).
3. Shadowing: A thin layer of heavy metal atoms is deposited on the specimen by evaporation in a vacuum chamber. Shadowing from one direction only produces a pseudo-three-dimensional image. Rotary shadowing, where the specimen is uniformly coated with heavy metal, is used to visualize nucleic acids and proteins. 

Ultrathin sectioning

The most popular technique for examining biological materials is to embed the material under study in plastic and cut ultrathin sections that can be examined in a TEM. The material is stabilized by chemical fixation (usually with aldehydes such as formaldehyde or gluteraldehyde), contrasted with solutions of heavy metal salts (osmium tetroxide and uranyl acetate), dehydrated in ethanol or acetone, and embedded in plastic (epoxy resin). Ultrathin sections (60 nm) cut with glass or diamond knives using an ultramicrotome are floated on water, transferred to specimen support grids and examined in the TEM. Often the sections are further contrasted with uranyl acetate and lead citrate prior to examination in the microscope.

In some cases, macromolecules can be specifically labelled prior to embedding and sectioning. For example, the location of some enzymes can be visualized by incubating the tissue with a substrate whose reaction with the enzyme leads to the local deposition of electron opaque material. Alternately, antibodies can be coupled to such enzymes, and the electron opaque reaction product is used to localize the antigens recognized by the antibodies. Some embedding resins (e.g. Lowicryl resins and LR White resin) have been designed to enable antibodies and electron opaque markers (such as colloidal gold particles) to be applied to the ultrathin sections. In this way, subcellular antigens recognized by the antibodies can be localized with the TEM.

Another sectioning technique that is increasing in popularity is cryosectioning (the sectioning of vitrified, frozen material). After chemical fixation, the tissue is immersed in cryo-protectant (usually sucrose) and then quickly frozen in liquid nitrogen. The cryo-protectant allows the biological material to be frozen without the formation of ice crystals, which would damage ultrastructure. This type of freezing, or vitrification, is possible in the absence of cryo-protectants but is technically demanding. Sections cut from the vitrified block can be thawed and incubated with antibodies specific to subcellular antigens. Electron opaque markers allow the antibodies to be seen in the TEM.

Colloidal gold coupled to protein A (a protein from bacterial cell walls which binds to the Fc portion of some immunoglobulins) has been used extensively in recent years to localize antibodies on resin and frozen sections of biological materials. The ability to produce homogeneous populations of colloidal gold with different particle sizes has enabled researchers to use these probes to colocalize different structures on the same section.

Cryofixation

It is possible to freeze biological material fast enough to vitrify the water present inside the cells. Vitrification of water occurs when the freezing has occurred so fast that ice crystals have no time to form. Vitrified biological material can be sectioned at low temperatures. Thin films of vitrified water and sections of vitrified material can be examined in transmission electron microscopes that are equipped with specimen stages that can be kept cold.

Rapid Freezing Methods

There are seven main rapid freezing methods presently available. They are

1. immersion freezing - the specimen is plunged into the cryogen.
2. slam (or metal mirror) freezing - the specimen is impacted onto a polished metal surface cooled with liquid nitrogen or helium.
3. cold block freezing - two cold, polished metal blocks attached to the jaws of a pair of pliers squeeze-freeze the specimen.
4. spray freezing - a fine spray of sample in liquid suspension is shot into the cryogen (usually liquid propane).
5. jet freezing - a jet of liquid cryogen is sprayed onto the specimen.
6. high pressure freezing - freezing the specimen at high pressure to subcool the water.
7. excision freezing - a cold needle is plunged into the specimen, simultaneously freezing and dissecting the sample.

Freeze-fracture followed by freeze etch and replication

If, for some reason, the object to be studied cannot be examined in the TEM, then a thin replica can be made. This is usually made by evaporating a thin layer of a heavy metal (usually platinum) onto the specimen and then coating this with a thin layer of carbon. The object and the replica are separated either by floating off the replica or by digesting away the object. There are four basic steps to follow

1. The specimen is frozen (often without regard to vitrification).
2. The specimen is fractured, while still frozen, under vacuum.
3. The fractured specimen can then be etched by leaving it frozen and under vacuum. Depending on the time of exposure, more or less water sublimes from the specimen (freeze drying).
4. A replica of the fractured surface is made which is then examined in the electron microscope.

A recent modification of this method employs rapid freezing achieved by slamming cells against a copper block cooled to -269°C with liquid helium. If these frozen cells are then exposed to extensive freeze drying (deep etching), very impressive images of the internal structures of cells are uncovered.

Sample Preparation For Transmission Electron Microscopy

General Schedule For Plant Tissue

1	Chemical	Temperature	Time	Repetitions
Primary fixation	2.5% glutaraldehyde in buffer	room or 0-4°C	2-4 hours or microwave*	1
Wash	buffer	room or 0-4°C	30 minutes	3-5
Secondary fixation	1-4% osmium tetroxide in buffer	room or 0-4°C	2-4 hours	1
Wash	buffer or distilled water**	room or 0-4°C	30 minutes	3-5
en bloc staining (optional)***	0.5% uranyl acetate	0-4°C	overnight	1
Wash after en bloc staining	distilled water	room or 0-4°C	10-15 minutes	2
Dehydration	25% ethanol 50% ethanol 70-75% ethanol 90-95% ethanol 100% ethanol Transition solvent if embedding resin is not miscible with ethanol	room or 0-4°C	20 minutes 20 minutes 20 minutes 20 minutes 30 minutes	1 1 1 1 2
Infiltration	1 part resin/2 parts solvent 1 part resin/1 part solvent (optional) 2 parts resin/1 part solvent 100% resin	room room room room	1 hour-overnight 1 hour-overnight 1 hour-overnight 1 hour	1 1 1 1
Embedding	Place in 100% resin in suitable container	1	1	11
Degassing (optional)	Place in vacuum desiccator or vacuum oven	room-60° C	3-30 minutes
Polymerization	1	60-70° C	> 8 hours

* See instructions for microwave fixation.

**Wash in water if en bloc staining is used.

*** If omitting en bloc staining, proceed to dehydration at end of washes.

 SCANNING ELECTRON MICROSCOPE (SEM)

The SEM is a microscope that uses electrons instead of light to form an image.  Since their development in the early 1950's, scanning electron microscopes have developed new areas of study in the medical and physical science communities.  The SEM has allowed researchers to examine a much bigger variety of specimens.

The scanning electron microscope has many advantages over traditional microscopes.  The SEM has a large depth of field, which allows more of a specimen to be in focus at one time.  The SEM also has much higher resolution, so closely spaced specimens can be magnified at much higher levels.  Because the SEM uses electromagnets rather than lenses, the researcher has much more control in the degree of magnification.  All of these advantages, as well as the actual strikingly clear images, make the scanning electron microscope one of the most useful instruments in research today.

The SEM is an instrument that produces a largely magnified image by using electrons instead of light to form an image.  A beam of electrons is produced at the top of the microscope by an electron gun.  The electron beam follows a vertical path through the microscope, which is held within a vacuum.  The beam travels through electromagnetic fields and lenses, which focus the beam down toward the sample.  Once the beam hits the sample, electrons and X-rays are ejected from the sample.

Detectors collect these X-rays, backscattered electrons, and secondary electrons and convert them into a signal that is sent to a screen similar to a television screen.  This produces the finalimage.

Sample preparation for SEM

Because the SEM utilizes vacuum conditions and uses electrons to form an image, special preparations must be done to the sample.  All water must be removed from the samples because the water would vaporize in the vacuum.  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."

The sputter coater uses an electric field and argon gas.  The sample is placed in a small chamber that is at a vacuum.  Argon gas and an electric field cause an electron to be removed from the argon, making the atoms positively charged.  The argon ions then become attracted to a negatively charged gold foil. The argon ions knock gold atoms from the surface of the gold foil.  These gold atoms fall and settle onto the surface of the sample producing a thin gold coating.

Sample Preparation for Scanning Electron Microscopy

General Schedule for Plant Tissue

1	Chemical	Temperature	Time	Repetitions
Primary fixation	2.5% glutaraldehyde in distilled water	room or 0-4°C	2-4 hours or microwave*	1
Wash	distilled water	room or 0-4°C	30 minutes	3-5
Secondary fixation	1-4% osmium tetroxide in distilled water	room or 0-4°C	2-4 hours	1
Wash	distilled water	room or 0-4°C	30 minutes	3-5
Dehydration	25% ethanol 50% ethanol 70-75% ethanol 90-95% ethanol 100% ethanol	room or 0-4°C	20 minutes 20 minutes 20 minutes 20 minutes 30 minutes	1 1 1 1 2
Critical point dry
Mount on specimen stub with silver paste or graphite
Coat with gold/palladium alloy
Store stubs in desiccator

* See instructions for microwave fixation.

 

Microwave Fixation

·         Place glass and grid sheet in microwave with numbered coordinates aligned with left edge of plate.

·         Place 400-ml beaker with 300 ml distilled water in back left corner of the oven.

·         Heat on COOK 1-2 min.

·         Replace heated beaker with fresh beaker of water (same amount and same position in microwave)

·         Place 3 ml of sample in fixative in flat bottom glass tube.

·         Place tube on square at X-18 coordinates.

·         Heat 15 sec on COOK 1.