Historically, electron energy-loss spectroscopy (EELS) in the electron microscope has evolved from work performed in biological as well as materials science and physics laboratories. The task of developing EELS compositional imaging for biological applications has been particularly challenging for two reasons. First, biological structures are radiation-sensitive and second, elements and compounds of interest are distributed sparsely in a multi-component matrix. For example, the physiologically important element, calcium, is typically present in cells at only 10-100 atomic parts per million.3,5 Even, when individual molecular components of a cell are isolated, elemental concentrations can still be low. Thus, double-stranded DNA contains only six phosphorus atoms per nanometer of its length; and protein molecules typically contain less than 0.5 per cent sulfur. This demand for high sensitivity in biological applications has provided an impetus to improve electron optics, to develop flexible image acquisition methods and to optimize spectral processing.

Secondary ion mass spectrometry (SIMS) is based upon the energetic ion bombardment of surfaces resulting in in the emission of sputtered particles, including both atomic and molecular ions. The use of mass spectrometric detection provides a highly versatile and sensitive tool for surface and thin film microanalysis. The scope of the technique includes a diversity of analysis modes including:

1. 1. Elemental Depth Profiling (dynamic SIMS),

2. 2. Laterally Resolved Imaging (ion microprobe or ion microscope analysis),

3. 3. Image Depth Profiling (combination of modes 1 and 2 providing 3-D images),

4. 4. Molecular Monolayer Analysis and Imaging (static SIMS),

5. 5. Sputtered Neutral Mass Spectrometry (post-ionization).

Much of the early work in dynamic SIMS centered on depth profiling and imaging techniques, with an emphasis on applications to electronic materials. SIMS has made extensive contributions to semiconductor materials science since the 1960's, including the development of new devices and processes, and in failure analysis.

Many recent applications in light microscopy involve the use of multiple fluorophores or the delineation of signals arising from spectrally distinct sources. In microspectroscopy, it is always desirable to illuminate fluorescently-labeled microscopic specimens with monochromatic light as the narrowest possible excitation wavelength range usually results in the highest emission signal-to-noise ratio. Generation of polychromatic light from an arc lamp and selection of the excitation wavelength by interference filters or monochrometers are the most common techniques for excitation microspectrofluorometry. Emission spectroscopy is usually done with filter wheels, monochrometers, or interferometers inserted between the microscope detection port and the detector. This presentation will be directed toward other, less frequently-used, approaches for spectral scanning of the specimen in the light microscope. Three topics will be considered: 1) the use of acousto-optical tunable filters and lasers for rapid, narrow-band, excitation wavelength selection; 2) the use of holographic notch filters for rejection of unwanted excitation laser light; 3) using liquid-crystal tunable filters for emission scanning.

Equipping a transmission electron microscope with an energy-filter offers extraordinary advantages for the characterization of both materials science and biological samples. Besides improvements for TEM imaging and electron diffraction like better contrast and resolution, elemental mapping using inner-shell ionizations has become the main application of EFTEM. Elemental maps with resolution down to 1 nm and elemental sensitivities down to a single monolayer have been reported. Using a Philips CM20 equipped with a Gatan Imaging Filter (GIF) for our experimental work, we acquired elemental maps with the three window method (A.E-1r background extrapolation from two pre-edge windows) and jump ratio images (division of post-edge image by a pre-edge image). One important application of EFTEM is the detection of secondary phases in materials e.g. precipitates and grain boundary phases. For example, fig.la shows the TEM-image of a 10%Cr steel with the secondary phases mostly invisible.

Differential interference contrast (DIC) light microscopy, particularly when coupled with digital image processing, is a powerful tool for the high-resolution microscopy of unstained, transparent biological specimens and can equally well be applied to semiconductor measurements. We show analytically, and with images of diatoms, plant cells and protoplasts, that switching the polarization of the incident light by 90 degrees, changes the image highlights found in conventional DIC images into shadows and vice versa (1). Using a ferroelectric liquid-crystal modulator, this switching can be done at frame rates, synchronized to the camera. By subtracting alternate frames, a stream of difference DIC images is created. We call this technique Pol Mod DIC. Subtraction of alternate images is carried out efficiently by frame buffer operations and amounts to massively parallel synchronous detection. A similar method has been applied to confocal microscopy (2).

GaN and the related AlN semiconducting materials have recently attracted considerable attention because of their versatile applications for optoelectronics. Large stresses are present in the GaN/AlN thin-film heterostructure and can exceed GPa's. They originate from a large lattice mismatch between the substrate(sapphire)/GaN (14%) and the AlN/GaN interface (-2.7%) in quantum well structures. The strain is expected to induce local piezoelectric fields in these polar materials. It is essential for a further development of GaN based thin films to fully understand the formation and the local strength of these fields. Previous studies show that there are unusual large fluctuations of the patterns in lattice images present across GaN/AlxGa1-xN and GaN/InyGa1-yN quantum wells. They are attributed to compositional fluctuations and have been studied by quantitative high resolution electron microscopy. Differences between the strain profiles and the electrostatic (scattering) potential profiles were observed.

Data from digital electron probe x-ray microanalysis (EPXMA) images can be used to generate high resolution profiles of Ca binding within sarcomeres of vertebrate striated muscle. While ratios of elemental peak to bremstrahlung at each point (Hall method) have been used in many studies to allow comparison of data taken from different samples, this procedure has limitations for our application. A different normalization procedure is described here which provides a means of assessing variation among elemental and bremstrahlung profiles obtained from different images and samples.

Freeze dried cryosections of chemically skinned frog or rabbit skeletal muscle were prepared as previously described. Digital EPXMA images were collected for 24 to 36 hours using a Zeiss EM910 STEM and an Oxford ExL2 microanalysis system. Calcium and bremstrahlung counts were summed within one pixel wide masks placed at successive positions along each half sarcomere, as previously described, using an automated routine in IPLAB on a Power Mac 7100.

Since the first demonstration by Cosslett and Duncumb, X-ray mapping by an electron probe microanalyzer (EPMA) has become a most popular approach in microanalysis because elemental distributions of constituents in a bulk sample can be displayed visually. The major disadvantage of EPMA mapping is poor spatial resolution (∼ 1 μm). The spatial resolution of X-ray microanalysis can be improved to a few nanometers using electron transparent thin-specimens in the analytical electron microscope (AEM). However, X-ray count rates from thin specimens are strictly limited because of the improved spatial resolution (i.e. smaller interaction volume) and the poor collection efficiency of X-ray. To obtain reasonable counts for accurate quantification in the AEM, extraordinarily long mapping times are required. Therefore, quantitative X-ray mapping is rarely attempted in the AEM. However, these limitations can be overcome by use of intermediate-volt age instruments combined with field-emission guns to increase the beam current-density, careful stage design to maximize the X-ray collection efficiency and the peak-to-background ratio, and ultrahigh vacuum system to reduce contamination.

Here we demonstrate sub- 2Å resolution, in compositionally sensitive Z-contrast imaging, for a 200 kV field-emission transmission electron microscope (FEGTEM). This method has shown great promise for determining the atomic structure of interfaces in such materials as ceramics, semiconductors and high-Tc superconductors.

The Z-contrast technique involves focusing an electron probe at a specimen surface (which is oriented with a zone-axis along the beam direction). The transmitted signal, at an annular detector, is displayed on a VDU, rastered synchronously with the probe. Coherent, contrast reversal effects are suppressed in this geometry; images approximate to a convolution of the probe intensity profile with the square of the projected specimen potential. The Z2 dependence means that images can, with care, be interpreted as chemically sensitive maps of the projected structure. Furthermore, electron energy loss spectroscopy (EELS) can be performed simultaneously with Z-contrast imaging to yield additional compositional information.

Experiments and theoretical work on spinodal decomposition of thin films of binary polymer blends indicate large changes in phase behavior and dynamics relative to the bulk behavior due to the complex interactions of the polymer-polymer, polymer-air, and polymer-substrate interfaces. Currently used techniques are able to sense growing domain sizes but are not always able to sense local two and three dimensional variations within domains. We are using a new characterization approach to these thin films utilizing the Scanning Transmission X-ray Microscope (STXM) at beamline XIA at the National Synchrotron Light Source (NSLS)2. We extract component thickness maps of each polymer in a sample region. This allows for quantitative investigation of the dynamics of polymer phase separation and should allow the determination of important parameters affecting the domain formation in these thin films.

Rubber toughening was first developed about seventy years ago as a method of increasing the impact resistance of brittle glassy polymers. Since that time, a wide variety of techniques have been developed to produce discrete dispersions of rubber within a glassy polymer matrix. We are exploring a new route to rubber-toughen polymer through the non-equilibrium process of mechanical alloying (high-energy ball milling). Here we have blended poly(methyl methacrylate) (PMMA) with polyisoprene (PI) (see the insets in Fig. 1 for chemical structures) and have characterized the resultant blends with Scanning Transmission X-ray Microscopy (STXM) in order to obtain chemical, as well as morphological, information about the blends.

The STXM technique utilizes tuneable soft x-rays that are focused with a diffraction zoneplate to a microprobe of 45 nm full width at half maximum. Thin sections of the sample are placed at the microprobe and the transmitted photon intensity is measured.

The intimate blending of two incompatible polymeric materials constitutes one of the most important fields of study in polymer science. A vast arsenal of techniques, ranging from copolymerization to melt-blending, has been assembled to mix intrinsically immiscible polymers and optimize the physical properties of the resultant system. For some systems, however, even established techniques cannot produce useable blends. To overcome this limitation, non-equilibrium mechanical alloying (high-energy ball milling of two or more dissimilar powders) has been employed to produce blends of poly(ethylene terephthalate) (PET) and Vectra (73/27 oxybenzoate/oxynaphthoate). Characterization of these blends by TEM is confounded by the absence of a preferential staining agent, in which case Scanning Transmission X-ray Microscopy (STXM) provides a valuable alternative in examining the morphology and chemistry of these blends.

The x-ray microscope utilizes a diffraction element called a zone plate to focus soft x-rays into a microprobe. A thin section of sample is placed in this microprobe, and the transmitted photon intensity measured.

An O-N-O layer within a DRAM is designed to be an insulator between a single-crystal silicon substrate and a poly-crystalline silicon gate. The nominal structure of the layer is SiO2-Si3N4-SiO2. A small amount of phosphorus is doped in the poly-crystalline silicon gate to improve the electrical characteristics of the device. Transmission Electron Microscopy (TEM) was first used to view the micro structure in the layer. A bright field image gave clear contrast in the O-N-O layer, however, TEM images do not provide direct information about the elemental distribution. In this study X-ray elemental mapping was thus used to show this distribution.

The specimen was cut from a 16M-DRAM device and thinned using the cross-section ion milling method. The O-N-O layer was examined using a JEM-201 OF FE-(S)TEM equipped with a JEOL UTW-EDS detector. Fig. 1 shows a high resolution TEM image of the O-N-O layer.

A dielectric O-N-O multi-layer is formed between a single-crystal silicon substrate and a polycrystalline silicon gate to insulate them. The nominal structure of the layer is SiO2-Si3N4-SiO2. In this study, in order to visualize the elemental distribution, energy filtered mapping technique was used and also the relative concentration was calculated from the intensities of the elemental maps to characterize the structure quantitatively.

The specimen was cut from a 16M-DRAM device and made suitable for TEM observation by the cross-section ion milling method. The O-N-O layer was examined using a JEM-2010F FE-TEM equipped with a post column energy filter(GIF 200). Fig. 1 shows energy filtered elemental maps of Si, O and N with an elastic image of the O-N-O layer obtained by the three-window method.

As a map is produced after a background intensity extrapolated from two pre-edge images is subtracted from a post-edge image, each pixel of these maps provides energy loss intensity of an element.

An O-N-O layer within a DRAM is designed to be an insulator between a single-crystal silicon substrate and a poly-crystalline silicon gate. The nominal structure of the layer is SiO2-Si3N4-SiO2. It has been reported that oxygen and nitrogen diffuse into each other layer while preserving the three layered structure. In order to measure the elemental distribution quantitatively, EELS was used for Si and light elements O and N in this study.

The specimen was cut from a 16M-DRAM device and prepared for TEM observation by the crosssection ion milling method. The O-N-O layer was analyzed using a JEM-201 OF FE-(S)TEM equipped with a scanning imaging device including a High-Angle Annular Dark Field(HAADF) detector which is capable of collecting electrons scattered 50 to 110 mrad, and also a PEELS(GATAN model 676). Fig. 1 shows a HAADF image of the O-N-O layer.

Electron energy-loss spectroscopy (EELS) in the transmission electron microscope (TEM) is a powerful technique that analyzes the inelastic scattering distribution of the fast TEM electrons after they have lost energy within the sample. The resultant energy-losses are characteristic of elemental, chemical, and dielectric properties and are typically measured in one of two ways. Parallel-detection EELS spectrometers (PEELS) acquire spectral data over a large range of energy-loss simultaneously for rapid acquisition of spectral data at a single point. In contrast, the energy filtering TEM (EFTEM) acquires only a single energy band at once, but does so for thousands or even millions of image pixels simultaneously.

Spectrum-imaging concerns the acquisition of spectroscopic data of sufficient detail for rigorous analysis at each pixel in a digital image. (Fig. 1) A STEM EELS spectrum image “data cube” can be acquired by stepping a focused electron probe to each pixel and filling the spectrum image one spectrum at a time.

In Near Edge X-ray Absorption Fine Structure (NEXAFS) microscopy, excitations of core electrons into unoccupied molecular orbitals or electronic states provide sensitivity to a wide variety of chemical functionalities in molecules and solids. This sensitivity complements infrared (IR) spectroscopy, although the NEXAFS spectra are not quite as specific and “rich” as IR spectra. The sensitivity of NEXAFS to distinguish chemical bonds and electronic structures covers a wide variety of samples: from metals to inorganics and organics. (There is a tendency in the community to use the term NEXAFS for soft x-ray spectroscopy of organic materials, while for inorganic materials or at higher energies X-ray Absorption Near Edge Spectroscopy (XANES) is utilized, even though the fundamental physics is the same.) The sensitivity of NEXAFS is particularly high to distinguish saturated from unsaturated bonds. NEXAFS can also detect conjugation in a molecule, as well as chemical shifts due to heteroatoms.

Conventional Fourier transform infrared (FTIR) spectroscopy is a workhorse analytical technique providing quantitative and qualitative information on a vast array of materials, ranging from semiconductors to biological samples. By coupling a Michelson interferometer and infrared (IR) microscope to an IR sensitive focal-plane array detector, we have added an imaging component to the existing analytical capabilities of FTIR spectroscopy. This additional capability makes it possible to image the distribution of subtle structural and compositional changes reflected in the infrared spectrum within heterogeneous materials. As such, it is an extremely powerful analytical tool which is currently employed in a variety of basic research and industrial applications. Similarly the utility of FTIR spectroscopy for the investigation of biological materials can be greatly extended by recording spectroscopic imaging data sets instead of single FTIR spectra. This spectroscopic imaging capability, especially when combined with microscopy, ensures that information concerning spatial variations in the system’s biochemistry, as well as its cellular morphology, is maintained.

Over the several years that imaging energy filters have been available commercially, numerous and wide-ranging applications have demonstrated elemental mapping at resolutions approaching 1 nm. From reviewing applications of energy-filtered transmission electron microscopy (EFTEM) to interfacial segregation and concentration profiles in metals and ceramics, some guidelines and issues may be identified. The work has been performed with a Gatan imaging filter (GIF) interfaced to a Philips CM30 operated at 300 kV with a LaB6 cathode.

For quantitative elemental mapping by EFTEM a number of interrelated parameters [field of view, resolution, collection angle (β), slit width (Δ), incident beam divergence (α), illumination uniformity, probe current (i), drift rate, exposure time (T), and local specimen thickness (t)] have always to be properly selected and controlled. For example, the collection angle should be large enough for a strong signal, but not so large that the signal/background (S/B) and chromatic-aberration-limited spatial resolution (δ) are both degraded.

Compositional mapping with energy dispersive x-ray spectrometry (EDS) in the scanning electron microscope (SEM) is one of the most widely used techniques for elemental imaging of microstructures. SEM/EDS maps are usually qualitative presentations of elemental distributions in x-y space. In older implementations of mapping, the x-ray intensity from a single spectral region of interest (ROI) above a threshold was recorded on film as a white “dot” during analog scanning. With modem digital control and data recording, x-ray intensities from many ROIs can be recorded in parallel and true counts stored in memory. For display purposes a gray level or color value is used to represent the stored x-ray intensity at each pixel. For major constituents (arbitrarily, major >10%), it is generally possible to make valid, robust comparisons between regions within an individual compositional map, but comparisons may be problematic among several maps of different constituents within the same field of view, depending on the sophistication of the mapping software.