Only 10 $ per sample for interpreting of your SEM/TEM/AFM micrograph
Payment Upon Completion

Send your micrographs...

The Scanning Electron Microscope (SEM) produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning).

There are two families of electron guns:

• Conventional thermionic emitters such as Tungsten (W) or Lanthanum hexaboride (LaB6) tipped filaments.
• Tungsten field emission gun (FEG) , warm or Cold FEG. A pointed emitter is held at several kilovolts (2000-7000 V) so that there is sufficient potential at the emitter surface to cause field electron emission.

Field emission gun (FEG) is used to produce an electron beam that is smaller in diameter, more coherent and up to three orders of magnitude greater current density or brightness.

scrollable

Energy of electrons is depending of Voltage: 1 Kev to 50KeV

Current (A): Number of electrons /unit of time

1 amp = 1 coulomb/sec 1 coulomb ~ 6 x10¹⁸ electrons

Example if the current measured at sample is around 10^-9A to 10^-12 A then the number of electrons is around 6X10⁶ to 6X10⁹ electrons/sec.

Environmental Scanning Electron Microscope (ESEM)

ESEM is a variety of SEM called environmental scanning electron microscope. It can produce images of sufficient quality and resolution with the samples being wet or contained in low vacuum or gas. This greatly facilitates imaging biological samples that are unstable in the high vacuum of conventional electron microscopes. The major disadvantage of transmission electron microscope is the need for extremely thin sections of the specimens, typically about 100 nanometers. Biological specimens are typically required to be chemically fixed, dehydrated and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic polymers and similar materials may require special treatment with heavy atom labels in order to achieve the required image contrast.

ESEM is especially useful for non-metallic, uncoated and biological materials. The presence of gas, mainly Argon, around a sample permits to work with pressure greater than 500 Pa compared to conventional SEM requirements samples under vacuum about 10-3 to 10-4 Pa. This vacuum level creates the possibility to operate on non-conductive samples without any preparation or hydrated specimens without charging.

Transmission Electron Microscope (TEM)

In a Transmission Electron Microscope (TEM), the electron beam is accelerated by an anode typically at +100 keV (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 fiber 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.

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. Generally, the image resolution of an SEM is at least an order of magnitude poorer than that of a TEM. However, because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to many centimeters in size and (depending on instrument design and settings) has a great depth of field, and so can produce images that are good representations of the three dimensional shape of the sample.

The Scanning Transmission Electron Microscope (STEM) rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The focusing action (and aberrations) occurs before the electrons hit the specimen in the STEM, but afterward in the TEM.

Focused ion beam (FIB)

Focused ion beam, also known as FIB, is a technique used particularly in the semiconductor industry, materials science and increasingly in the biological field for site-specific analysis, deposition, and ablation of materials. A FIB setup is a scientific instrument that resembles a scanning electron microscope (SEM). However, while the SEM uses a focused beam of electrons to image the sample in the chamber, a FIB setup uses a focused beam of ions instead. Unlike an electron microscope, FIB is inherently destructive to the specimen.

When the high-energy gallium ions strike the sample, they will sputter atoms from the surface. Gallium atoms will also be implanted into the top few nanometers of the surface, and the surface will be made amorphous. A FIB-SEM consists in a system with both electron and ion beam columns, allowing the same feature to be investigated using either of the beams. A FIB-SEM system uses a beam of Ga+ ion to mill into the surface to locate a feature or defect of interest. The integrated SEM then uses a focused beam of electrons to image the sample in the chamber.

Only 10 $ for interpretation of your SEM/TEM/AFM micrograph
Payment Upon Completion

Send your micrographs...

To satisfy this curiosity, many inventions have been devised. One of them is the optical microscope. The human eye can distinguish objects down to about 0.2 mm. Optical microscopes reveal small objects, which would be otherwise invisible to the human eye, by magnifying them with the help of a combination of glass lenses. If we raise the amplification rate (magnification) of an optical microscope higher and higher, can we see an atom?

Electron microscopes enable clear observation of micro-structures, which is not possible with optical microscopes. Moreover, they also make it possible to analyze substance structures and obtain atomic level information by using an electron beam. The electron microscope is an epoch-making invention used throughout the world to investigate an atomic world that we could hardly imagine.

The difference between Electron Beam and Light

fig1. Ripples caused by the difference in the magnitude of the wave

As explained above, the ability to distinguish a small structure, that is resolution, largely depends on the wavelength of the “wave” used to illuminate the specimen.

The nature of this “wave” may be easily understood by comparing it to the wave pattern arising when a small stone is thrown into a lake. Assume the waves on the water surface come into contact with a rock protruding above the surface. If the rock is larger than the length between the crests of the waves (wavelength), then the wave pattern does not continue behind the rock (Fig,1). This creates a shadow. If the rock is smaller than the wavelength, however, the wave pattern will not be interrupted behind the rock and there is no shadow. In this case, the existence of the rock cannot be detected.

Whereas the wavelength of visible light is 400 to 800 nm (1 nanometer is one 100,000th of 0.1mm), the wavelength of the electron beam, which is used as a light source in the electron microscope, varies depending on the accelerating voltage. The accelerating voltages commonly used are 100 to 200 kV (corresponding to wavelengths of 0.0037nm to 0.0025nm).

This wavelength is far shorter than that of light, and sufficient to distinguish the arrangements of atoms (several nanometers). For the optical microscope the combination of the lens is varied to alter the magnification. In contrast, for the electron microscope, the intensity of the electric current passed to the electromagnets is varied to change the intensity of the magnetic field. This corresponds to the changing the thickness of a convex lens. In fact, by manipulating the electric current, the magnification can be freely controlled.

Another characteristic “electron diffraction”

TEMs employ a high voltage electron beam in order to create an image. An electron gun at the top of a TEM emits electrons that travel through the microscope’s vacuum tube. Rather than having a glass lens focusing the light (as in the case of light microscopes), the TEM employs an electromagnetic lens which focuses the electrons into a very fine beam. This beam then passes through the specimen, which is very thin, and the electrons either scatter or hit a fluorescent screen at the bottom of the microscope. An image of the specimen with its assorted parts shown in different shades according to its density appears on the screen. This image can be then studied directly within the TEM or photographed.  Figure 1 shows a diagram of a TEM and its basic parts. 

Fig. 1 Simplified diagram of a transmission electron microscope.  Drawing by Graham Colm, courtesy of Wikimedia Commons.

What Are the Differences Between a TEM and a Light Microscope?

Although TEMs and light microscopes operate on the same basic principles, there are several differences between the two. The main difference is that TEMs use electrons rather than light in order to magnify images. The power of the light microscope is limited by the wavelength of light and can magnify something up to 2,000 times. Electron microscopes, on the other hand, can produce much more highly magnified images because the beam of electrons has a smaller wavelength which creates images of higher resolution. (Resolution is the degree of sharpness of an image.) Figure 2 compares the magnification of a light microscope to that of a TEM. 

Fig. 2 [left] Cotton stem; area in the circle is the phloem tissue. Light microscope x250. Photo by K. Esau.  [right] Enlarged image of cotton phloem tissue showing a sieve element (top cell) and a companion cell (bottom cell), TEM x8,000. Photo by J. Thorsch.

How Are TEM Specimens Prepared?

Specimens must be very thin so that electrons are able to pass through the tissue. This may be done by cutting very thin slices of a specimen’s tissue using an ultramicrotome.  The tissue must first be put in a chemical solution to preserve the cell structure.  The tissue must also be completely dehydrated (all water removed). 

                                 Fig. 3 Ultramicrotome.  Photo by J. Thorsch.                                                             Fig. 4 Microtome grid.  Image by Laurie Hannah

Once preserved and dehydrated, tissue samples are placed in hard, clean plastic.  The plastic supports the tissue while it is being thinly cut with the ultramicrotome (Fig. 3).

After sections are cut and mounted on grids, (tiny circular disks with openings,) a solution of lead is used to stain the tissue (Fig. 4).  The lead provides contrast to the tissue by staining certain cell parts.  When placed in the electron microscope, the electrons are scattered by the lead.  They do not penetrate the tissue or hit the fluorescent screen, leaving those areas dark. 

Esau’s Work With the TEM

Esau started using the TEM in her research in the early 1960s.  When she moved to UC Santa Barbara in 1963, the campus purchased a Siemens electron microscope for her. She then received a grant from the National Science Foundation in 1969 for another new microscope which she used for the remainder of her career in Santa Barbara. The TEM significantly improved her understanding of the relationship between plants and viruses. Electron microscopy also aided in clarifying the functioning of sieve elements, the food conducting cells in plants. Without the TEM, much of this research would not have been possible. 

Only 10 $ for interpretation of your SEM/TEM/AFM micrograph
Payment Upon Completion

Send your micrographs...

The scanning electron microscope (SEM) uses a focused beam of high-energy electrons to generate a variety of signals at the surface of solid specimens. The signals that derive from electron-sample interactions reveal information about the sample including external morphology (texture), chemical composition, and crystalline structure and orientation of materials making up the sample.

In most applications, data are collected over a selected area of the surface of the sample, and a 2-dimensional image is generated that displays spatial variations in these properties. Areas ranging from approximately 1 cm to 5 microns in width can be imaged in a scanning mode using conventional SEM techniques (magnification ranging from 20X to approximately 30,000X, spatial resolution of 50 to 100 nm). The SEM is also capable of performing analyses of selected point locations on the sample; this approach is especially useful in qualitatively or semi-quantitatively determining chemical compositions (using EDS), crystalline structure, and crystal orientations (using EBSD). The design and function of the SEM is very similar to the EPMA and considerable overlap in capabilities exists between the two instruments.

Fundamental Principles of Scanning Electron Microscopy (SEM)

Accelerated electrons in an SEM carry significant amounts of kinetic energy, and this energy is dissipated as a variety of signals produced by electron-sample interactions when the incident electrons are decelerated in the solid sample. These signals include secondary electrons (that produce SEM images), backscattered electrons (BSE), diffracted backscattered electrons (EBSD that are used to determine crystal structures and orientations of minerals), photons (characteristic X-rays that are used for elemental analysis and continuum X-rays), visible light (cathodoluminescence–CL), and heat. Secondary electrons and backscattered electrons are commonly used for imaging samples: secondary electrons are most valuable for showing morphology and topography on samples and backscattered electrons are most valuable for illustrating contrasts in composition in multiphase samples (i.e. for rapid phase discrimination). X-ray generation is produced by inelastic collisions of the incident electrons with electrons in discrete ortitals (shells) of atoms in the sample. As the excited electrons return to lower energy states, they yield X-rays that are of a fixed wavelength (that is related to the difference in energy levels of electrons in different shells for a given element). Thus, characteristic X-rays are produced for each element in a mineral that is “excited” by the electron beam. SEM analysis is considered to be “non-destructive”; that is, x-rays generated by electron interactions do not lead to volume loss of the sample, so it is possible to analyze the same materials repeatedly.

Scanning Electron Microscopy (SEM) Instrumentation – How Does It Work?

Essential components of all SEMs include the following:

• Electron Source (“Gun”)
• Electron Lenses
• Sample Stage
• Detectors for all signals of interest
• Display / Data output devices
• Infrastructure Requirements:
  • Power Supply
  • Vacuum System
  • Cooling system
  • Vibration-free floor
  • Room free of ambient magnetic and electric fields

SEMs always have at least one detector (usually a secondary electron detector), and most have additional detectors. The specific capabilities of a particular instrument are critically dependent on which detectors it accommodates.

Applications

The SEM is routinely used to generate high-resolution images of shapes of objects (SEI) and to show spatial variations in chemical compositions: 1) acquiring elemental maps or spot chemical analyses using EDS, 2)discrimination of phases based on mean atomic number (commonly related to relative density) using BSE, and 3) compositional maps based on differences in trace element “activitors” (typically transition metal and Rare Earth elements) using CL. The SEM is also widely used to identify phases based on qualitative chemical analysis and/or crystalline structure. Precise measurement of very small features and objects down to 50 nm in size is also accomplished using the SEM. Backescattered electron images (BSE) can be used for rapid discrimination of phases in multiphase samples. SEMs equipped with diffracted backscattered electron detectors (EBSD) can be used to examine microfabric and crystallographic orientation in many materials.

Strengths and Limitations of Scanning Electron Microscopy (SEM)?

Strengths

There is arguably no other instrument with the breadth of applications in the study of solid materials that compares with the SEM. The SEM is critical in all fields that require characterization of solid materials. While this contribution is most concerned with geological applications, it is important to note that these applications are a very small subset of the scientific and industrial applications that exist for this instrumentation. Most SEM’s are comparatively easy to operate, with user-friendly “intuitive” interfaces. Many applications require minimal sample preparation. For many applications, data acquisition is rapid (less than 5 minutes/image for SEI, BSE, spot EDS analyses.) Modern SEMs generate data in digital formats, which are highly portable.

Limitations

Samples must be solid and they must fit into the microscope chamber. Maximum size in horizontal dimensions is usually on the order of 10 cm, vertical dimensions are generally much more limited and rarely exceed 40 mm. For most instruments samples must be stable in a vacuum on the order of 10^-5 – 10^-6 torr. Samples likely to outgas at low pressures (rocks saturated with hydrocarbons, “wet” samples such as coal, organic materials or swelling clays, and samples likely to decrepitate at low pressure) are unsuitable for examination in conventional SEM’s. However, “low vacuum” and “environmental” SEMs also exist, and many of these types of samples can be successfully examined in these specialized instruments. EDS detectors on SEM’s cannot detect very light elements (H, He, and Li), and many instruments cannot detect elements with atomic numbers less than 11 (Na). Most SEMs use a solid state x-ray detector (EDS), and while these detectors are very fast and easy to utilize, they have relatively poor energy resolution and sensitivity to elements present in low abundances when compared to wavelength dispersive x-ray detectors (WDS) on most electron probe microanalyzers (EPMA). An electrically conductive coating must be applied to electrically insulating samples for study in conventional SEM’s, unless the instrument is capable of operation in a low vacuum mode.

User’s Guide – Sample Collection and Preparation

Sample preparation can be minimal or elaborate for SEM analysis, depending on the nature of the samples and the data required. Minimal preparation includes acquisition of a sample that will fit into the SEM chamber and some accommodation to prevent charge build-up on electrically insulating samples. Most electrically insulating samples are coated with a thin layer of conducting material, commonly carbon, gold, or some other metal or alloy. The choice of material for conductive coatings depends on the data to be acquired: carbon is most desirable if elemental analysis is a priority, while metal coatings are most effective for high resolution electron imaging applications. Alternatively, an electrically insulating sample can be examined without a conductive coating in an instrument capable of “low vacuum” operation.