Scanning Electron MicroscopeGiven by : Gautam parmar

Stream-M.Sc Industrial Biotechnology (Sem-1)E-mail id-gparmar183@gmail.com

Department of Biosciences

Preface

INTRODUCTION

• The earliest known work describing the concept of a Scanning Electron Microscope was by M. Knoll (1935) who, along with other pioneers in the field of electron optics, was working in Germany.

• Subsequently M. von Ardenne (1938) constructed a scanning transmission electron microscope (STEM) by adding scan coils to a transmission electron microscope.

• The first STEM micrograph was of a ZnO crystal imaged at an operating voltage of 23 kV at a magnification of 8000 times, and a spatial resolution between 50 and 100 nm.

• The first commercial scanning electron microscope became available in 1965 by Cambridge Scientific Instruments.

• 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 that can be detected and that contain information about the sample's surface topography and composition.

INTRODUCTION

• The electron beam is generally scanned in a raster scan pattern, and the beam's position is combined with the detected signal to produce an image.

• Due to the very narrow electron beam, SEM micrographs have a large depth of field yielding a characteristic three-dimensional appearance useful for understanding the surface structure of a sample.

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

• The types of signals produced by a SEM include secondary electrons (SE), back-scattered electrons (BSE), characteristic X-rays, light (cathodoluminescence) (CL), and transmitted electrons.

• 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.

Principle• 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.

• The key to how the scanning electron microscope works is that the beam scanning the specimen surface is exactly synchronised with the spot in the screen that the operator is watching.

• The electron detector controls the brightness of the spot on the screen - as the detector "sees" more electrons from a particular feature, the screen brightness is increased.

• When there are fewer electrons, the spot on the screen gets darker. These days, the screen is generally a digital monitor, not a glass crt, but the principle is the same.

• These signals include secondary electrons (that produce SEM images), backscattered electrons (BSE), diffracted backscattered electrons (EBSD that are used to determine crystal structures.

• There are different types of electron image. The two most common are the secondary electron image (sei) and the backscattered electron image (bei).

• The sei is used mainly to image fracture surfaces and gives a high resolution image. • The bei is used typically to image a polished section and for illustrating contrast in the composition in multiphase samples.• For example, lead will appear brighter than iron and calcium oxide will appear brighter than calcium carbonate. X-ray microanalysis• The principle of EDX is that the electron beam generates X-rays within the specimen. Many of these X-rays have energies

characteristic of the elements that emitted them.• There are three principal components to a basic EDX system: • the X-ray detector; a box of electronics called the "pulse processor" that measures the voltage pulses corresponding to the X-

ray energies, and a computer, typically a PC.• The X-ray detector is positioned to intercept X-rays emitted from the specimen. On entering the detector, an X-ray generates a

small current, which is then converted into a voltage pulse.• A computer measures the voltage pulses over a period of time, say 60 seconds and plots them as a histogram. • The histogram shows a spectrum of the X-ray energies that were measured; by examining the spectrum, the elements present

can be determined.

InstrumentationSEMs consist of the following components:•Electron Source-Thermionic Gun. Field Emission Gun•Electromagnetic Lenses•Vacuum chamber•Sample chamber and stage•Computer•Detectors (one or more)-Secondary Electron Detector (SED), Backscatter Detector•Diffracted Backscatter Detector (EBSD)•X-ray Detector (EDS)

• Electron Column- The electron column is where the electron beam is generated under vacuum, focused to a small diameter, and scanned across the surface of a specimen by electromagnetic deflection coils.

• The lower portion of the column is called the specimen chamber. The secondary electron detector is located above the sample stage inside the specimen chamber.

• Electron gun- Located at the top of the column where free electrons are generated by thermionic emission from a tungsten filament at ~2700K. Electrons are primarily accelerated toward an anode that is adjustable from 200V to 30 kV.

• Condenser Lenses- After the beam passes the anode it is influenced by two condenser lenses that cause the beam to converge and pass through a focal point. What occurs is that the electron beam is essentially focused down to 1000 times its original size.

• Apertures- The function of these apertures is to reduce and exclude extraneous electrons in the lenses. The final lens aperture located below the scanning coils determines the diameter or spot size of the beam at the specimen.

• Scanning System- Images are formed by rastering the electron beam across the specimen using deflection coils inside the objective lens. The stigmator or astigmatism corrector is located in the objective lens and uses a magnetic field in order to reduce aberrations of the electron beam.

Fixation- This is done to preserve the sample and to prevent further deterioration so that it appears as close as possible to the living state, although it is dead now. It stabilizes the cell structure.

Rinsing- The samples should be washed with a buffer to maintain the pH. For this purpose, sodium cacodylate buffer is often used which has an effective buffering range of 5.1-7.4. The sodium cacodylate buffer thus prevents excess acidity which may result from tissue fixation.

Post fixation- A secondary fixation with osmium tetroxide (OsO4), which is to increase the stability and contrast of fine structure. OsO4 binds phospholipid head regions, which creating contrast with the neighbouring protoplasm (cytoplasm). OsO4 helps in the stabilization of many proteins by transforming them into gels without destroying the structural features.Dehydration- Following osmium fixation, water is chemically extracted from the specimen using a graded series of ethanol (30%, 20%, 10%, 70%, 100%). It is used so that the epoxy resin used in infiltration and embedding step are not miscible with water.Drying- In order to prevent damage to the specimens during air drying, the critical point drying technique is frequently employed. A critical point drier (CPD) is used to replace all of the ethanol with liquid carbon dioxide under pressure.

Sample preparation

• Infiltration- Epoxy resin is used to infiltrate the cells. It penetrates the cells and fills the space to give hard plastic material which will tolerate the pressure of cutting.

• Polymerization- Next is polymerization step in which the resin is allowed to set overnight at a temperature of 60 degree in an oven.

• Sectioning- The specimen must be cut into very thin sections for electron microscopy so that the electrons are semi-transparent to electrons. These sections are cut on an ultra microtome which is a device with a glass or diamond knife. For best resolution the sections must be 30 to 60 nm.

SEM vs TEMSEM TEM

Based on scattered electron capture Based on electron transmission technique

Used to study sample surface and its morphology

Used to study detailed internal composition (morphology, magnetic domains etc

Sample size is thick Sample size is thin

Low resolution High resolution

Large amount and multiphase sample analysis is possible

Small amount of sample can analysed.

Images are shown on monitor or picture tube.

3-D structure can be observed

Used for surfaces, powders, polished & etched microstructures

Images are shown on fluorescent screen

2-D structure

Imaging dislocations, tiny ppt, grain boundaries, defects in solids

ApplicationsApplications• Forensic Science- superior performance the SEM is used in an increasing number of various

applications and provides valuable results for instance in the following applications:• Gunshot residue analysis• Firearms identification (bullet markings comparison)• Investigation of gemstones and jewellery• Examination of paint particles and fibres• Filament bulb investigations at traffic accidents• Handwriting and print examination / forgery• Counterfeit bank notes• Trace comparison• Examination of non-conducting materials• High resolution surface imaging

Nanomaterial characterization

•The surface structure of polymer Nano composites, fracture surfaces, Nano fibres, nanoparticles and Nano coating can be imaged through SEM with great clarity.

•Electro spun nylon 6 Nano fibres decorated with surface bound silver nanoparticles used for antibacterial air purifier can be characterized using SEM

•SEM can also be employed for viewing the dispersion of nanoparticles such as carbon nanotubes, nanoclay and hybrid POSS Nano fillers in bulk and on the Nano composite fibres.

• The composition or the amount of nanoparticles near and at the surface can be estimated using the EDX, provided they contain some heavy metal ions. Eg- Au, Pd, Ag, nanoparticles on surface can be easily detected easily using EDX technique.

Archaeological studies • SEM-EDS is one of the most versatile analytical techniques in archaeology, applicable to the study of

a wide range of inorganic and organic residues and archaeological materials.Tephra chronology- • Geochronologists often use SEM-EDS and EMPA-WDS to analyse volcanic

glass shards and chemically match them to a specific volcanic eruption This dating procedure, called tephra chronology.

Industrial application• The combination of SEM and energy dispersive X-ray microanalysis (EDS) is used particularly for

quality control and verification of material composition. Samples of cement and cement additives (powdered gypsum and fly ash)

• EDS analysis is performed using Bruker’s Quantax system to determine the chemical composition of the fly ash particles in cement.

Biological application

Human Embryo Development by SEM- •SEM can be used for the study of a lower limb of a human embryo after 10 weeks’ development. The presence of a tactile metatarsal prominence can be seen on the distal tips of fingers. In addition, one can see how, at this stage of development, the fingers are already independent of each other.Plants diseases •Sclerotinia is a plant pathogenic fungus that cause a disease called cotton rot or watery soft rot in several crops, like lettuce. This pathogen has the ability to produce a black resting structure known as sclerotia.

X

Parasite study by SEM-•SEM allows us to visualize external morphological characteristics and is a very useful tool for obtaining data on systematic and taxonomic studies of parasites in general. •The parasitological studies of these hosts allow us to learn the composition of their communities and thus the parasite biodiversity, providing as well interesting information as to the complex life cyclesErythrocytes morphology with amino acid-based surfactants-The surfactants interact with the erythrocytes membrane causing changes in the structure of membrane proteins and lipids, which can induce some alterations in the external surface of the cells which affects the oxygen transporting efficiency of the cells.

Future potential• The Scanning Ion Microscope (SIM)-Helium is used as source of illumination. Requires low vacuum condition. Ions are generated using

field emission gun. Ion optical lenses are usually of the simple ‘Einzel’ electrostatic type but offer competitive aberration characteristics.

• The Virtual Microscope - An SEM simulator (or “virtual microscope”) need only consist of a personal computer and appropriate software to emulate instrument operation.

• A virtual SEM should include a user interface, comparable if not identical to actual SEM user interfaces so that the user learns how to use the microscope control software as well as the microscope.

• The Intelligent Microscope- XpertEze requires the operator to indicate general sample type (conductor, semiconductor, insulator, biological or unknown), the required detection mechanism (secondary electron, backscattered electron, etc.,) and either an exact desired magnification or a magnification range.

• It initialises the SEM in terms of core parameters (such as accelerating voltage, probe current, gun alignment, screen brightness, etc.) using the constraints specified by the operator and drawing upon its own knowledge of what settings are appropriate for a given sample at a certain magnification using a particular detector.

• Optical technologies capable of storing thousands of images per disc will handle image archiving needs and next-generation intelligent database management systems.

• For image display, the unwieldy and bulky cathode ray tube will be superseded by slim liquid crystal display panels with reduced power consumption, electromagnetic pollution and space requirement.

• The technology already exists for voice controlled microscopes - voice control will become omnipresent in home and business computers in the next five years and hence will find its way into the microscopy mainstream soon thereafter.

• Instrument performance in terms of achievable resolution and signal-to-noise ratio will improve by an order of magnitude.

Limitations• Maintenance involves keeping a steady voltage, currents to electromagnetic coils and circulation of cool

water. • SEMs are expensive, large and must be housed in an area free of any possible electric, magnetic or

vibration interference • In addition, SEMs are limited to solid, inorganic samples small enough to fit inside the vacuum chamber

that can handle moderate vacuum pressure.• SEMs carry a small risk of radiation exposure associated with the electrons that scatter from beneath the

sample surface.• Detailed account of cellular components is not possible as it is based on electron capture technique rather

than electron transmission. • Special training is required to operate an SEM as well as prepare samples.

References• C.Brandon, Introduction to Scanning Electron Microscopy, Materials Engineering Department at San Jose State University,• C Joy David, Protons, 2010, Ions, electrons and the future of the SEM, University of Tennessee, Knoxville, page 5,6.• B.C. Breton, 1999, SEM in next the Millennium, Department of Engineering, University of Cambridge, page-1,5.• A. Bogner et al, 2007, A history of scanning electron microscopy developments: Towards ‘‘wet-STEM’’ imaging, page 1,3• Iolo ap Gwynn,2006, Limitations and Potential of the Scanning Electron Microscope (SEM), The University of Wales

Bioimaging Laboratory, page 1.• Nestor J. Zaluzec, Introduction to Transmission/Scanning Transmission Electron Microscopy and Microanalysis, page 5,6.• Bob Hafner, 2007, Scanning Electron Microscopy Primer, University of Minnesota, page -3,5,7,9.

• Ellery Frahm, Scanning Electron Microscopy (SEM) Applications in Archaeology, University of Sheffield, Sheffield page 4,5,6,1.

• Wikipedia.org

THANX A LOT