Electron microscopies

The electron microscope has become a very important part of materials structure determination, yielding information on both the morphology. 

The principle involves using X-rays (beam of electrons) to image atomic structures. The very short wavelengths allow much higher resolution than with a standard optical microscope. There are several types of electron microscope which allow different types of image to be formed. They operate in either transmission mode (electrons pass through the sample) or reflection mode (electrons are reflected from the surface) 

This technique combines the features of the TEM (high resolution) and the SEM (surface scanning) to produce a superior instrument. The STEM can be used not only for imaging but also for analytical work since several scanning signals are collected simultaneously, the contrast is enhanced relative to the standard instruments. The high-resolution picture gives information on structure, absorbed species and depth. 

The X-rays emitted from the surface of a sample in both SEM and TEM can be used diagnostically to determine the partition of species, since they are characteristic of the elements in the sample. This process is known as EDAX (energy dispersive analysis of X-rays) and can be performed not only on areas of sample but also on individual crystallites, although light elements (with atomic numbers less than sodium) cannot be assessed as the X-rays produced are too soft and are easily absorbed. 

Electron microscopy is a fairly straightforward technique to determine the size and shape of supported particles. It can also reveal information on the composition and internal structure of the particles, for example by detecting the characteristic X-rays that are produced by the interaction of the electrons with matter, or by analyzing how the electrons are diffracted [1,9], 

the interaction of the primary beam with the sample provides a wealth of information on morphology, crystallography and chemical composition. 

Three types of electron microscopes are shown schematically in Fig. 7.3. Transmission electron microscopy (TEM) uses transmitted and diffracted electrons. A TEM instrument is in a sense similar to an optical microscope if one replaces optical by 

Dedicated SEM instruments have resolutions of about 5 nm. Simple versions of SEM with micron resolution are often available on Auger electron spectrometers, for the purpose of sample positioning. The main difference between SEM and TEM is that SEM sees contrast due to the topology and composition of a surface, whereas the electron beam in TEM projects all information on the mass it encounters in a two-dimensional image, which, however, is of subnanometer resolution. 

The scanning transmission electron microscope (STEM) combines the two modes of operation. Here, the scanning coils are used to illuminate a small area of 

As illustrated by Fig. 10.4, an electron microscope offers additional possibilities for analyzing the sample. Diffraction patterns (spots from a single-crystal particle and rings from a collection of randomly oriented particles) enable one to identify crystallographic phases as in XRD. Emitted X-rays are characteristic for an element and allow for a determination of the chemical composition of a selected part of the sample (typical dimension 10 nm). This technique is called electron microprobe analysis (EMA, EPMA) or, referring to the usual mode of detection, energy dispersive analysis of X-rays (EDAX or EDX). Also the Auger electrons carry information on sample composition, as do the loss electrons. The latter are potentially informative on the low Z elements, which have a low efficiency for X-ray fluorescence. 

For studying supported catalysts, TEM is the commonly applied form of electron microscopy. Detection of supported particles is possible provided that there is sufficient contrast between particles and support. This may impede applications of TEM on supported oxides. Determination of particle sizes or of distributions therein rests on the assumption that the size of the imaged particle is truly proportional to the size of the actual particle and that the detection probability is the same for all particles, independent of their dimensions. Semi in situ studies of catalysts are possible by coupling the instrument to an external reactor. After evacuation of the reactor, the catalyst can be transferred directly into the analysis position, without seeing air [12]. For reviews of TEM on catalysts we refer to refs. [11,12]. 

Electron microscopy can be divided into two areas transmission electron microscopy (TEM) involving thin specimens and the scanning electron microscope (SEM) involving bulk samples.However, whenever a polymer is exposed to a beam of electrons, energy is dissipated in the specimen, bonds are broken, and permanent chemical and physical changes result.The extent to which these effects prevent examination is then largely a matter of the material itself and information required.  

The book by Sawyer and (irubb provides a more detailed account of electron microscopy of polymers and in particular, an excellent overview of the different sample preparation techniques that have been devised. 

Electron microscopy is an imaging technique that uses an electron beam to probe a material. Since the wavelength of electrons is much smaller than the wavelength of visible light, diffraction effects occur at much smaller physical dimensions. The imaging 

Sample preparation Straightforward and fast Complex and slow 

Electron microscopy has also been employed as a means of elucidating coal structure granules as small as 200 to about 1000 J.m in diameter have been observed. In addition, it has been reported that particles range from 250 A in a low-rank coal to 100 A in a high-rank coal while two general ranges for the ultrafine structures have also been observed one 100 J.m and the other 100 pm with some of the particles in the form of polygonal platelets. 

Electron microscopy (EM) can be divided into the techniques of transmission electron microscopy and of direct imaging of surfaces. There are a number of reviews and monographs on the different techniques of EM [1, 2]. 

In transmission electron microscopy, the specimen is traversed by an electron beam, typically in an energy range between 80 and 200 kV. Electrons with these energies can penetrate specimens only up to a 

The magnification is attained from the ratio of the size of the display screen to the size of the scanned surface region on the sample, and it can 

There is an essential advantage of SEM that, in general, no special preparations are necessary to perform morphological investigations of specimens. However, for nonconducting materials such as polymers the deposition of a thin conducting layer of metal or carbon (by vacuum evaporation or sputtering) is necessary. 

E)ue to the interaction between the primary electron beam and sped- 

Electron microscopy of herbicide-treated tissues has often been carried out in order to show the intracellular damage caused by the inhibition of carotenogenesis or to demonstrate the redistribution of the pigments after such treatment, for example, the increased presence of plastoglobuli containing phytoene. Electron microscopy is most usefully carried out in conjunction with carotenoid analyses, especially or organelle fractions. In isolation, however, this technique cannot elucidate the primary mode of action of bleaching herbicides. 

Electron microscopy and diffraction are indicated whenever one dimension of the particle is 2000 A or less, or when fine surface or internal structure is in question. It is therefore preeminent for thin films or crystals and for very fine-particle characterization where other methods fail or are of limited utility. Bulk materials can also be examined by thinning down to foils. The chief limitation is that the electron beam can, in general, give information on materials only less than about 2000 A in thickness by transmission. Where samples are not thinned, information on surface structure can be gained by replica techniques and by reflection diffraction. Several books have appeared on the subject and these provide details on the various applications and capabilities of the technique.  

Most micrographs are taken at from 4000 x to 16,000 x, but it is possible to work from 300 x to about 250,000 x. Reported magnifications in electron microscopy have an accuracy of about 10 %, which is satisfactory for most problems. At higher magnifications, accuracy may be considerably reduced. 

Modern microscopes can give a resolution of about 5 A. With a great deal of time and effort, it is possible to show resolution of about 2 A using 200-kV electrons. Several Japanese microscopists claim 1.8 A on metals and Heidenreich has obtained 2.1 A on graphite. 

However, there are three different tests for resolution, and there is no agreement as to which method is best. Further, very few materials are suitable for resolution tests. In most problems, there is no need for resolutions of better than 5 A, and with most samples it would be difficult to prove resolution of below 10 A. Resolutions of 2-3 A were recently achieved for intercalation complexes of superconducting Ta 2, allowing direct observation of the crystalline lattice and its imperfections. Computer enhancement of high-resolution electron micrographs of stained and unstained catalase crystals show amazing 

Resolution in reflection microscopy is about 100 A at best, but 200 A is more common. In replicas, resolution is about 20 A, at best, with direct replicas. Two-stage replicas give about 50 A and three-stage replicas about 100 A resolution. Decoration replicas show, indirectly, faults that may be of unit cell size. 

In electron microscopy (EM), a focussed beam of electrons are used (replaeing visible light in optical microscope) to bombard the sample for getting information about its strueture and eomposition. This improves the possible magnifieation to -2,000,000. 

Electron bombardment in a material produces the effects as shown in Fig. 6.18. 

Main Observations Made with these Microscopes 

A focussed (using magnetic lenses) electron beam falls on the sample surface kept in vacumn. The beam then scans over the sample surface and the scattered electrons detected and collected to have the information about the sample surface. 

Although optical microscopy may be extended into the nanoregime, other techniques must be used to clearly discern components below 100 nm. Indeed, the current nanotechnology revolution that we are experiencing would not have been possible 

Near-Field Scatmiiffi Optical MiciOSCOpy (NSOM)  

Source Brightness (particles cm eV sr ) Elastic mean free path (A) Absorption length s (A) Minimum probe size (A) 

Data from http //ncem.lbl.gov/team/team background.htm 

The basic principles that govern electron microscopy are analogous to optical microscopy. Whereas optical microscopes use light and optical lenses to illuminate and magnify the sample, electron microscopes utihze high-energy electrons and 

The use of electron microscopy, nitrogen adsorption and mercury porosimetry for the characterization of porous materials have laeen treated extensively elsevhere and these methods will only briefly Ise reviewed here. Hcmever, the use of SEC for tlie determination of PSD has, until recently, been founded on enpirical ground only and therefore, this technique will be discussed in detail. The paragr ih is concluded with a ocnparison of the results obtained with the different methods. 

Another method to preserve the gel structure is to enised the support in an epoxy resin (56). The athedded gel is then sliced with an ultramicrotoroe and micrographs of the stained gel are produced. This approach also facilitates studies of the three dimensional structure by examination of slices taken at different levels of the support. Amsterdam et al. obtained information on the uiperturbed structure of an agarose gel (i.e., 

The surface pore structure of seme chronatogr hic supports is shown in Fig. 2. The agarose material was frozen in isopentane surrounded hy liquid nitrogen and the isopentane sublimed off in a freeze-dryer. One portion was dehydrated with acetone, flushed with liquid CD2 and then dried at the 

2 Scanning electron micrograidis of scjti jchrcinatogr dy supports  

The figures given above in the parentheses are the average corrected data of approximate pore size of each gel. Although these figures cue quite reasonable (58, 59) it should be realized that data are sparse. However, they serve to illustrate the potential of SEH for studies of the pore structure of supports for SEC. 

When a solid is bombarded with high energy electrons the interaction produces secondary electrons (elastic), back-scattered electrons (inelastic), low loss electrons. Auger electrons, photo electrons, electron diffraction, characteristic x-rays, x-ray continuum, light, hole electron pairs and specimen current. These interactions are used to identify the specimen and elements of the specimen and can also be used to physically characterize particulate systems. 

The skill level required to operate and prepare specimens for a TEM is significantly higher than that associated with a SEM [156]. 

For scanning electron microscopy (SEM) the two most important interactions are  

The generation of secondary electrons, which are the result of elastic collisions and typically less than 50 eV. The images formed by these are the most common and are marked by great depth of field. 

Backscattered electrons, which are less applicable to particle sizing but have niche uses the contrast is closely related to the atomic number of the sample and typically the voltage is greater than 50 eV. 

The images obtained from electron microscopy may be used to determine both the surface and the internal membrane structure, to examine the porous morphology and to estimate membrane porosity and porous size distribution, which will determine the selectivity of the membrane as well as its permeability. 

The staining technique for TEM uses a material that absorbs electrons and preferentially attaches itself to or reacts with certain regions of the polymer rather than other regions. Materials frequently used are uranyl acetate and osmium tetroxide. For polyethylene the technique of chloro-sulphonation can be used. In this method, which involves immersing the sample in chlorosulphonic acid, the electron-absorbing material becomes attached to lamellar surfaces, so that lamellae (see section 3.4.2) with their planes parallel to the direction of the electron beam become outlined in black in the micrographs. 

Another method for making the sample visible by using its effects on the phase of the transmitted wave comes from the realisation that it is only in the plane immediately behind the sample, or in the image of this plane, that the amplitude is constant. In any plane just outside the sample, interference between the undiffracted and diffracted waves gives rise to an intensity distribution that is not uniform and depends on the structme of the sample. It is possible to show that, if a particular plane just outside the sample is imaged, rather than the sample itself, a visible image showing the structures within the sample is formed (see fig. 2.20(a)). This method is related to, but is not identical to, the method of phase-contrast microscopy used with optical microscopes and is sometimes known by that name. 

If a solvent is available for the polymer, the replica is made by shadowing the surface with a metal and then coating it with carbon. The sample itself is then dissolved away to leave a replica of its surface. If no solvent is available a slightly more complicated two-stage process is used. The procedures are outlined in fig. 2.21. The replica produced by either 

All the methods described so far suffer from the disadvantage that they allow only the study of very thin films of polymer, by direct TEM, or of polymer surfaces, by replication. Thin enough films are difficult to make and neither they nor surfaces produced by casting or by fracture are necessarily typical of bulk material. It is therefore desirable to have a technique that can be used for any surface, including one cut from the interior of a larger sample. A technique that allows any type of surface to be prepared in a way suitable for the examination of the underlying structure is the use 

In a typical treatment of polyethylene, 1-2 pm is removed from the surface, with the reagent attacking preferentially the non-crystalline material. The surface can then be studied either by two-stage replication or by high-resolution scanning electron microscopy (SEM). The latter gives somewhat lower resolution than TEM, but is much easier to use than the production of replicas. In this technique scattered or secondary electrons emitted from the surface are collected as an electron beam is scanned across it in a raster like that used to produce a television picture and the image is built up in a similar way from the intensity detected. 

As compared to TEM, scanning electron microscopy (SEM) can avoid destroying the bulky specimen, because a focused electron beam is scanned during catalyst observation. SE and backscattered electron (BSE) signals can be collected separately or together in any ratios simultaneously. Stereoscopic morphologies can be obtained but with resolution much inferior to that of TEM. Similar to TEM, EDS could be applied as accessory to analyze microdomain composition and element distribution. 

The electron tomography (ET) method is another approach for reconstruction of nanomaterials space locations [34]. By tilting the specimen continuously and recording images simultaneously, three-dimensional (3D) structures of composite materials could be extracted vividly by combination and reconstruction of these images with specific software. For nanocatalysts, the spatial location of the active components could therefore be located with much ease [35]. 

Abbreviations SEM = scanning electron microscopy, TEM = transmission electron microscopy, AFM = atomic force microscopy, STM = scanning 

Sometimes, low optical contrast between different phases, particles or droplets can be improved by the use of fluorescence markers. One such example is the viewing of coating cross-sections, where, for example, leaching of biocides or binder degradation by sunlight can be more easily seen after contacting the coating surface with a suitable marker. 

Another microscopy technique, which is actually based on light scattered by colloids, is dark-field or ultra-microscopy. In this technique, an ordinary optical microscope is used, but the sample is illuminated in such a way that light does not enter the objective unless scattered by the object under investigation. This technique does now allow a direct observation of, for example, a particle, but is particularly useful for detecting the presence of particles and investigating the Brownian motion of colloids. An important requirement is that the refractive index of the coUoids 

More advanced (and expensive) microscopy techniques are differential interference contrast microscopy and laser scanning confocal microscopy. The latter can provide precise optical sectioning so that three-dimensional images of stmctures can be recorded. 

Sample preparation, including carbon or gold sputtering, used to be rather time consuming and examination had to take place in vacuum. However, modem environmental SEM (E-SEM) equipment employs a pressure chamber and short working distance and allows immediate observation without sputtering and vacuum. The maximum sample size that will fit in the measurement chamber is typically about 15 X 15 cm.  

As stated by de Broglie s duality principle, aU particles, especially electrons, can behave as waves under appropriate circumstances. This principle states in its simplest form that a particle of mass m moving at a speed v has a wavelength given by 

The development and the recent increase in availability of the scanning electron microscope with its considerable depth of field and reduced beam intensity has widened the range of samples which can be examined 

Replication avoids the problem of sample deterioration in the instrument, but it is destructive in that reaction of the material cannot be continued after the replica has been prepared. Transitory features cannot be detected unless a series of preparations are examined corresponding to increasing progress of the reaction considered. The textures of replicas have been shown [220] to be in satisfactory agreement with those of the original surface as viewed in the scanning electron microscope. The uses and interpretations of observations made through sample replication procedures are illustrated in the studies of decomposition of metal carboxyl-ates by Brown and co-workers [97,221—223]. 

Both heated stages [224] and ambient temperature gas environments [225,226] have been developed for use in electron microscopy and both are combined [227,228] in the controlled atmosphere instrument. Pressures of up to 30 kPa and temperatures up to 1500 K have been used in studies of a wide variety of solid—gas and catalytic reactions [ 229]. 

Robinson [230] has developed a specimen chamber for use in the scanning electron microscope whereby the surface charging of insulators is reduced by a relatively high water vapour pressure (1 kPa). 

Direct observations of the decompositions of a wide range of inorganic compounds [231—246], which are unstable in the electron beam, particularly azides and silver halides, have provided information concerning the mechanisms of radiolysis these are often closely related to the processes which operate during thermal decomposition. Sample temperatures estimated [234] to occur at low beam intensity are up to 470 K while, at higher intensity, 670 K may be attained. 

High-iesolution scanning and transmission electron microscopy (HRSEM, HRTEM) can provide very specific information about surface films on any kind of particles. A comparison between pristine particles and particles scraped from cycled electrodes can provide very comprehensive information. Using element analysis, STEM techniques, and selected area electron diffraction (SAED), it is possible to map surface species in a nanometric scale [30]. 

Tertiary structures beyond the cellular level can be observed by electron microscopy (EM). Both, macromolecular complexes of proteoglycan-like aggregation factors [55] and isolated glycosaminoglycan chains forming fibrillar structures have been observed by EM [19, 32]. 

The question of composition can be addressed in another manner by electron microscopy. Lattice imaging techniques, which involve the reconstruction of the direct image from the diffraction pattern of a particle, can allow for the measurement of lattice spadngs characteristic of the constituent phases, and thus provide constituent analysis based on the structures. [3, 194] 

The mechanism of image formation is different from the light microscopy. It is not absorption, reflection, or fluorescence but scattering of the electrons on the atoms forming the specimen. Elastic scattering on the atomic cores provides information about the specimen structure, morphology, and crystallinity. Inelastic scattering on the atomic shells provides information about its chemical composition and even oxidation state. A comprehensive theory and many examples and practical hints can be found in Refs. 30 and 31. 

FIGURE 4.13 TEM of polymersomes at pH 3 (a) and 10 (b). Membrane consists of protonable poly(diethylaminoethyl methacrylate) (PDEAEM), which is responsible for the swelling/deswelling properties of pol3mersome. Source Gaitzsch et al. [32]. Reproduced with permission of John Wiley Sons. 

The electron-optical techniques are increasingly often being applied under conditions of ultra high vacuum, allowing the study of surfaces under controlled circumstances. The most significant developments for electron microscopy however have been in the imaging mode where considerable enhancement of resolution has occurred, leading to various forms of operation. 

Scanning electron microscopy (SEM) utilizes a highly focused electron beam which is scanned over the surface of the specimen. Since penetration through the specimen is not essential for this instrument, thicker samples (cm range) and lower accelerating potentials (low kV range) are commonly used. The most popular mode of operation is the emissive mode which utilizes those electrons that have either been emitted by the 

In electron microscopy, generally intense electron beams are used which can severely damage the surface (however, at high energies the electron-atom cross sections become smaller). Often, large magnetic fields are also present that could affect the surface structure. 

Since the first electron-microscopical observation of a heavy atom on a surface different studies have looked at effects related to individual atomic adsorbates. These include diffusion along the surface (atoms can be tracked in real time), giving results in agreement with equivalent FIM observations, and pair spacing distributions, showing for example a peak in the distribution near 4-5 A for uranium atoms on a carbon surface Clustering can be studied in some cases as well. 

It would be interesting to extend such studies to other light-atom substrates, such as the metals beryllium and aluminium, and to investigate step effects. Heavier-atom surfaces can also be analyzed in the form of thin films of mono-atomic thickness on a lighter substrate, as has recently been done  

There are two main classes of electron micro.s-copy (EM) techniques. In the first class, the electron probe is a stationary beam incident along a fixed direction. This incident beam can be parallel [conventional transmission electron microscopy (CTEM), high-resolution transmission electron microscopy (HRTEM), high-voltage transmission 

The images produced in transmission electron microscopy are essentially due to local diffraction phenomena absorption contrast plays only a minor role. Not only is electron diffraction responsi- 

Electrons are scattered by atoms as a result of Coulomb interaction with the nucleus and the electron cloud. The atomic scattering factor/e(0) thus contains two terms of opposite sign 

The first term clearly relates to the nucleus, whereas the second term is due to the electron cloud. The interaction with matter is stronger (x 10 ) for electrons than for X rays or neutrons, which interact only with the electron cloud or with the nucleus, respectively. As a result multiple scattering will not be negligible in electron diffraction experiments. Moreover, electron scattering is oriented mainly in the forward direction. Tables of/e(0) for different atoms are given in [140]. 

Kinematic Diffraction by Crystals 29.2.2.3.1. Lattice, Reciprocal Lattice 

The techniques, instrumentation and underlying theory of optical microscopy for materials scientists have been well surveyed by Telle and Petzow (1992). One of the last published surveys including metallographic techniques of all kinds, optical and electronic microscopy and also techniques such as microhardness testing, was a fine book by Phillips (1971). 

The impact of electron-optical instruments in materials science has been so extreme in recent years that optical microscopy is seen by many young research workers as faintly fuddy-duddy and is used less and less in advanced research this has the unfortunate consequence, adumbrated above, that the beneficial habit of using a wide range of magnifications in examining a material is less and less followed. 

The difficulty, of course, was that electrons cannot be focused by a glass lens, and it was necessary to use either magnetic or electrostatic Menses . A German, Hans Busch, in 1926/27 published some seminal papers on the analogy between the effect 

In an excellent historical overview of these stages and the intellectual and practical problems which had to be overcome, Mulvey (1995) remarks that the first production microscopes pursued exactly the same electron-optical design as Ruska s first experimental microscope. The stages of subsequent improvement are outlined by Mulvey, to whom the reader is referred for further details. 

the interaction of the primary beam with the sample provides a wealth of information on morphology, crystallography and chemical composition. Using transmission electron microscopy to make a projection of the sample density is a routine way to study particle sizes in catalysts. 

Although experiments involving electrons generally have to be performed in a vacuum, recent experimental developments have made it possible to study the 

Elaborate synthetic approaches have been developed that enable significant control over the size and shape of palladium nanostructures. In order to understand the properties of the materials formed based on the preparation method, several characterization techniques have been used. These include electron microscopy, scanning probe microscopy (SPM), nuclear magnetic resonance (NMR) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, infrared (IR) spectroscopy, electrochemistry, X-ray diffraction (XRD), thermogravimetric analysis (TGA), electron diffraction, photoelectron spectroscopy, dynamic light scattering (DLS), extended X-ray absorption fine structure (EXAFS), BET surface area analysis andX-ray reflectivity (XRR). In the following section we will describe the information provided by each of these characterization techniques. 

Electron microscopy is a powerful technique for the characterization of Pd nanoparticles, and is capable of providing information of the morphology, topography and composition of the material under investigation. Here, we describe details of TEM and scanning electron microscopy (SEM), both of which are capable of providing detailed information about particle shape and size distribution. 

Pd nanoparticles [73] here, only the Pd core provides a contrast and not the surrounding ligand, such that TEM allows the determination of size distributions for each Pd NP sample. 

Advancements in electron microscopy have led to the development of high-resolution transmission electron microscopy (HRTEM), which allows the analysis of nanoparhcles with resolution up to O.SA. This is made possible by using a 

2 Scanning Electron Microscopy (SEM) The operating principles of SEM are very similar to those of TEM, except that a high-voltage (a few hundred eV to 

There are two main limitations with the common EM apparatus the sample must be placed in a vacuum chamber (thus imposing limits on the size and volatility) and it must be a conductor of charge (to prevent build-up of static electricity that will distort the impinging electron beam). The advent of e-SEM (environmental SEM) can alleviate the requirement for a vacuum in some cases. Electron microscopy is not strictly a photon counting technique, but as its main application is similar to that of an optical microscope, although on a different scale, we decided to include it in this section. 

FIGU RE 1.22 Electron microscope images of the tracks (a) irradiating from aparticle containing uranium (b) and the energy-dispersive x-ray spectrum of the particle after plasma ashing (c). (Adapted from Esaka, F. et al Anal. Chim. Acta, 721,122, 2012. With permission.) 

The resolving power of a microscope is related, among other things, to the wavelength of the radiation used. With ordinary white light of effective wavelength 550 nm, the resolving power is around 200 nm. Beyond this, there seems no way of 

In scanning electron microscopy (SEM), a finely focussed electron beam probe moves from one point on the specimen to the next to form a raster pattern, just as in television imaging. The intensity of scattered or secondary electrons is continuously 

Analytical electron microscopy is the most sophisticated tool available for micro-structural analysis today. In this method, we can obtain both the high-resolution structure and elemental composition of a specimen. This is probably the best technique to obtain local elemental composition of small regions of heterogeneous solids. When a high-energy electron beam is incident on a specimen, we get elastically and inelastically 

The most important aspect of electron microscopy in solid state chemistry lies in its ability to elucidate problems that are beyond the capability of X-ray or neutron crystallography. High-resolution electron microscopic (HREM) images show local structures of crystals in remarkable detail in Fig. 2.10 we show the HREM image of Big, W03 obtained with the Cambridge University 500 kV microscope to show the improved resolution compared with the image obtained with a 200 kV microscope 

Once the experimental image is obtained using a thin specimen ( 100 A), the 

FIGURE 4.6 Diagram of the main parts of a light microscope. 

Resolution is the smallest separation of two points that are visible as distinct entities. The resolving limit of the human eye is 0.1 mm on the other hand, the resolving limit of the light microscope is 0.2 pm. 

Proteoglycan—bovine articular cartilage 1.28(PGI), 1.07(PGII), 0.24 x 106(PGIII)  

The identification of size, shape, and axial ratio can also be done by direct observation in the electron microscope (EM). This is accomplished by depositing single molecules (if they can be obtained) directly on polymer-coated copper grids and then shadowing them with heavy metals or making a replica of the molecular surface on mica. The sample can then be viewed in the transmission EM and photographs can then be taken after calibration of the magnification factor. 

The size and shape of a macromolecule can be determined by measuring the physical properties of isolated macromolecules in solution. Large rigid macromolecules that are derived from extended structures including the collagen triple helix result in rodlike rigid or semirigid structures. The size, shape, and physical parameters for macromolecules discussed in this book 

Springer Series in Wood Science Methods in Lignin Chemistry (Edited by S.Y. Lin and C.W. Dence) 

A glossary of symbols and descriptions used in this section is found below. 

UV absorbance of a 0.5 im section in morphological region i Transmission electron microscopy Scanning electron microscopy Energy dispersive X-ray analysis 

A variety of apertures have been used to deliver nanometer-sized spots of light. While early NSOM tips were fabricated out of etched quartz crystals and micropipettes, tapered optical fibers with tip diameters of ca. 100 nm are now typically used. A metallic thin film such as aluminum is usually applied around the sides of the tapered region of the NSOM tip to focus the light toward the sample. For optical fibers, the numerical aperature is related to the difference in the indices of refraction of the cladding and core (Eq. 2)  

Although optical microscopy may be extended into the nanoregime, other techniques must be used to clearly discern components below 100 nm. Indeed, the current nanotechnology revolution that we are experiencing would not have been possible if there were not suitable techniques in order to characterize nanomaterials. As we saw in the previous section, in order to improve resolution, we must use source radiation with as small a wavelength as possible. 

Images obtained from electron microscopy are due to the nature/degree of electron scattering from the constiment atoms of the sample. Table 7.1 provides a comparison between electron. X-ray, and neutron sources, pertaining to their utility 

Near-Field Scanning Optical Microscopy (NSOM)  

Source Brightness (particles cm eV steradian ) Elastic Mean 1 Free Path (A) Absorption Length (A) Minimum Probe Size (A) 

Gonzalez-Tejuca, K. Aika, S. Namba, and J. Turkevich, J. Phys. Chem., 1977, 

Other symbols occasionally used in the literature include (br) for branched materials and (iso), (syndio), and (a) for isotactic, syndiotactic, and atactic structures respectively. No symbol appears to exist for mechanical blends, although these materials are obviously important. Where necessary the symbol -m- will denote a mechanical blend, for example, poly(styrene-m-butadiene) for a mechanical blend of polystyrene with polybutadiene. 

An important subclass of the IPN s are the semi-IPN s, where one polymer is linear and the other is locked in network form. Two subclasses may be described, semi-IPN s of the first or second kind, depending on whether the polymer synthesized first, polymer I (D Agostino and Lee, 1972), or the polymer synthesized second, polymer II, is crosslinked. 

In many places throughout the text, we shall refer to the first polymer synthesized as polymer I and the second polymer synthesized as polymer II. Their monomers, where appropriate, will also bear the designations I and II, respectively. Thus, in graft copolymers, the backbone chain is usually polymer I, and the side chains comprise polymer II. 

Scientists and engineers working in the fields of polyblends and block copolymers have realized for many years that phase separation of the two components takes place, and that this is indeed important to the development of the mechanical behavior characteristic of these materials. However, it was not until the development of the electron microscope that the structure of any but the coarsest mechanical blends could be discerned, and even then lack of contrast between the two phases remained serious. This problem was solved in 1965 by Kato (1966, 1968), who discovered that osmium tetroxide preferentially stains polymer molecules containing carbon-carbon double bonds, such as in polybutadiene and polyisoprene. The osmium tetroxide also hardens the rubbery phase, allowing convenient ultramicrotoming of specimens to 500 A thickness. 

Osmium tetroxide staining can be accomplished by exposing a sample to osmium tetroxide vapor for a week, or by soaking overnight in a 1 % 

All 48 alveolar macrophages that were seen in the serially sectioned human alveoli fixed in the inflated state were found within or bordering on alveolar junction zones (Parra et al. 1986). A computer-reconstruction showed the predilection of alveolar macrophages and type II pneumocytes located in septal jxmction zones. Superposing alveolar macrophages and type II cells in such a reconstruction obliterated almost all gaps in the basement membranes, except for those produced by pores of Kohn when these were free of cells. 

The size of the alveolar macrophage varies from 15 pm to 30 pm (Bowden 1971). The cytoplasm is faintly basophilic. The nuclei of the smaller cells are round but in the larger cells they may be deeply indented. Their chromatin is much finer than that of the lymphocytes. Nucleoli are inconspicuous. 

Rat alveolar macrophages obtained from unstimulated lungs by endobronchial lavage showed heterogeneity with respect to cell size (88-20 pm diameter), surface morphology and cytochemistry 

The first three methods listed involve the measurement of morphological or structural-related parameters whereas the last method is a typical penneation-ielatBd technique. 

Electron microscopy (EM) is one of the techniques that can be used for membrane characterisation. Two basic techniques can be distinguished scanning electron microscopy (SEM) and transmission electron microscopy (TEM). 

Of these two techniques, scanning electron microscopy provides a very convenient and simple method for characterising and investigating the porous structure of microfUtration 

The latter method is probably the mote simple one. Water has a high surface tension (Y = 72.3 10 N/m), and on replacing it by another liquid with a much lower surface tension this also reduces the capillary forces acting during drying. The choice of the liquid used depends on the membrane structure, since all the liquids must be non-solvents for the membrane. An example of a typical sequence of liquids is water, ethanol, butanol, pentane or hexane. The last solvent in this sequence, an alkane, has a very low surface tension (hexane Y= 18.4 10 N/m) and can be easily removed. 

In summary, U can be stated that scanning electron microscopy is a very simple and useful technique for charaaerising microfiltration membranes. A clear and concise picture of the membrane can be obtained in terms of the top layer, cross-section and bottom layer. In addition, the porosity and the pore size distribution can be estimated from the photographs. Care must be taken that the preparation technigue does not influence the actual porous structure. 

Early stages of polymer oxidation, as for UHMWPE/HMWPE blends, can be detected by CSFM. A comparison between CSFM and chemiluminescence imaging (ICL) for this purpose is still lacking. Applications of CLSM in in situ (i.e. non-invasive) mapping have been reviewed [90]. 

To obtain real-space information about the morphology of polymeric materials, various optical microscopic methods such as OM and CLSM are available (cfr. Chp. 5.3). Use of electrons as a light source for microscopy opens other perspectives [124]. Electron microscopy (EM) provides structural information in both the real and reciprocal space. Electron 

Conventional SEM (developed originally with thermionic emitters) operates typically in high-vacuum conditions and at high accelerating voltage e.g. 10- 0 keV), offers an image resolution of some 

Specimen type Bulk (oo) Thick Ultrathin Thin 

Features Surface topography Radiation sensitive Microstructure Microstructure 

9 A TEM image of a cross-section of a plant ceU showing chloroplasts, organelles responsible for the reactions of photosynthesis (Chapter 12). Chloroplasts are typically 5 pm long. (Dr Jeremy Burgess/ Science Photo Library.) 

A consequence of these stringent experimental requirements is that electron microscopy cannot be used to study living cells. In spite of these hmitations, the technique is very useful in studies of the internal structure of cells (Fig. 9.9). 

---

Transmission electron microscopy (TEM) can resolve features down to about 1 nm and allows the use of electron diffraction to characterize the structure. Since electrons must pass through the sample however, the technique is limited to thin films. One cryoelectron microscopic study of fatty-acid Langmuir films on vitrified water [13] showed faceted crystals. The application of TEM to Langmuir-Blodgett films is discussed in Chapter XV. 

SEM Scanning electron microscopy [7, 10, 14] A beam of electrons scattered from a surface is focused Surface morphology... 

R. E. Lee, Scanning Electron Microscopy and X-Ray Microanalysis, PTR Prentice Hall, Englewood Cliffs, NJ, 1993. 

P. R. Thornton, Scanning Electron Microscopy, Chapman and Hall, 1968. See also Scanning Electron Microscopy Systems and Applications, The Institute of Physics, London, 1973. 

Protein adsorption has been studied with a variety of techniques such as ellipsome-try [107,108], ESCA [109], surface forces measurements [102], total internal reflection fluorescence (TIRE) [103,110], electron microscopy [111], and electrokinetic measurement of latex particles [112,113] and capillaries [114], The TIRE technique has recently been adapted to observe surface diffusion [106] and orientation [IIS] in adsorbed layers. These experiments point toward the significant influence of the protein-surface interaction on the adsorption characteristics [105,108,110]. A very important interaction is due to the hydrophobic interaction between parts of the protein and polymeric surfaces [18], although often electrostatic interactions are also influential [ 116]. Protein desorption can be affected by altering the pH [117] or by the introduction of a complexing agent [118]. 

The effect is more than just a matter of pH. As shown in Fig. XV-14, phospholipid monolayers can be expanded at low pH values by the presence of phosphotungstate ions [123], which disrupt the stmctival order in the lipid film [124]. Uranyl ions, by contrast, contract the low-pH expanded phase presumably because of a type of counterion condensation [123]. These effects caution against using these ions as stains in electron microscopy. Clearly the nature of the counterion is very important. It is dramatically so with fatty acids that form an insoluble salt with the ion here quite low concentrations (10 M) of divalent ions lead to the formation of the metal salt unless the pH is quite low. Such films are much more condensed than the fatty-acid monolayers themselves [125-127]. 

The specific surface area of a solid is one of the first things that must be determined if any detailed physical chemical interpretation of its behavior as an adsorbent is to be possible. Such a determination can be made through adsorption studies themselves, and this aspect is taken up in the next chapter there are a number of other methods, however, that are summarized in the following material. Space does not permit a full discussion, and, in particular, the methods that really amount to a particle or pore size determination, such as optical and electron microscopy, x-ray or neutron diffraction, and permeability studies are largely omitted. 

With certain critical Pco/Poi ratios, structural oscillations can be observed [306]. Patterns of stationary and/or traveling waves can actually be seen by means of photoemission electron microscopy (see Ref. 313, and note Section XVIII-7B. Such behavior can be modeled mathematically (e.g.. Refs. 214, 314). 

It has also been shown that sufiBcient surface self-diflfiision can occur so that entire step edges move in a concerted maimer. Although it does not achieve atomic resolution, the low-energy electron microscopy (LEEM) technique allows for the observation of the movement of step edges in real time [H]. LEEM has also been usefiil for studies of epitaxial growth and surface modifications due to chemical reactions. 

Figure A3.14.il. Spiral waves imaged by photoelectron electron microscopy for the oxidation of CO by O2 on a Pt(l 10) single crystal under UHV conditions. (Reprinted with pennission from [35], The American Institute of Physics.)...

Joy D C 1986 The basic principles of EELS Principles of Analytical Electron Microscopy ed D C Joy, A D Romig Jr and J I Goldstein (New York Plenum)... 

Thomas G and Goringe M J 1981 Transmission Electron Microscopy of Materials (New York Wiiey)... 

The history of EM (for an overview see table Bl.17,1) can be interpreted as the development of two concepts the electron beam either illuminates a large area of tire sample ( flood-beam illumination , as in the typical transmission electron microscope (TEM) imaging using a spread-out beam) or just one point, i.e. focused to the smallest spot possible, which is then scaimed across the sample (scaiming transmission electron microscopy (STEM) or scaiming electron microscopy (SEM)). In both situations the electron beam is considered as a matter wave interacting with the sample and microscopy simply studies the interaction of the scattered electrons. 

Reimer L 1993 Transmission Electron Microscopy (Berlin Springer)... 

Plattner H 1989 Electron Microscopy of Subcellular Dynamics (London CRC)... 

Knoll G and Plattner H 1989 Ultrastructural analysis of biological membrane fusion and a tentative correlation with biochemical and biophysical aspects Electron Microscopy of Subcellular Dynamics ed H Plattner (London CRC) pp 95-117... 

Hyatt M A 1981 Changes in specimen volume Fixation for Electron Microscopy ed M A Hyatt (New York Academic) pp 299-306... 

Thust A and Rosenfeid R 1998 State of the art of focai-series reconstruction in HRTEM Electron Microscopy 1998 14th Int. Cent, on Electron Microscopy (Cancun) voi 1 (Bristoi institute of Physics Pubiishing) pp 119-20... 

Frank J 1996 Three-Dimensional Electron Microscopy of Macromolecular Assemblies (New York Aoademio)... 

Ruiz T 1998 Conferenoe talk Gordon Conf. on Three-Dimensional Electron Microscopy... 

Butler E P and Hale K F 1981 Practical Methods in Electron Microscopy vol 9, ed A Glauert (New York North-Holland)... 

Berriman J and Unwin N 1994 Analysis of transient structures by cryo-electron microscopy combined with rapid mixing of spray droplets Ultramicroscopy 56 241-52... 

Sheppard C J R 1987 Scanning optical microscopy Adv. Opt. Electron Microscopy 10 1-98 Cooke P M 1996 Chemical microscopy Anal. Chem. 68 333-78... 

Peachey L D, Ishikawa H and Murakami T 1996 Correlated confocal and intermediate voltage electron microscopy imaging of the same cells using sequential fluorescence labeling fixation and critical point dehydration Scanning Microsc. (SuppI) 10 237-47... 

---