Maskless pattern generation using probe-forming electron beam systems has been exploited to great advantage for several decades in lithographic processes of both mask making and direct write applications used in the production of integrated circuits (ICs). The key limitation of these e-beam lithography systems has been and still is throughput. More efficient exposure techniques using shaped beams to project a multitude of pixels simultaneously have improved productivity but were unable to keep pace with Moore's Law and the steady increase of pattern densities. The recent development of massively parallel pixel projection has opened new prospects for electron beam lithography. The early proof-of-concept demonstrations of these techniques are the main subject of the paper.

The low voltage scanning electron microscope (SEM) is widely used in many industrial and research applications due to its ability to image surface details and to minimize charging and beam damage effects on sensitive samples. However, fundamental limitations in beam performance have existed, most notably in the chromatic aberration effects, which become larger as the beam voltage is reduced. The introduction of the extreme high resolution (XHR) SEM has demonstrated that sub-nanometer resolution can be achieved at low beam voltages, revealing fine surface detail. This system uses a source monochromator to reduce the effects of chromatic aberrations, resulting in a more tightly focused electron beam. Beam deceleration is available to provide a further improvement in imaging at low voltages and to give additional flexibility in optimizing the image contrast. While the monochromator is a necessary enabler of the improved imaging performance, further system elements, such as scanning, detectors, stage and environmental controls - that go into completing the SEM - are also key to the usability and throughput when it comes to practical day-to-day performance.

Nanotechnology is pushing imaging and measurement instrument technology to high levels of required performance. As this continues, new barriers confronting innovation in this field are encountered. Particle beam instrument resolution remains one of these barriers. A new tool for imaging and metrology for nanotechnology is the scanning Helium Ion Microscope (HIM). The HIM is a new approach to imaging and metrology for nanotechnology which may be able to push this barrier lower. As a new methodology, it is just beginning to show promise and the number of potentially advantageous applications for nanotechnology and nanometrology has yet to be fully exploited. This presentation will discuss some of the progress made at NIST in collaboration with the manufacturing community in understanding the imaging and metrology for this new technology.

Helium ion beam microscopy (HIM) is a new high resolution imaging technique. The use of Helium ions instead of electrons enables none destructive imaging combined with contrasts quite similar to that from Gallium ion beam imaging. The use of very low probe currents and the comfortable charge compensation using low energy electrons offer imaging of none conductive samples without conductive coating. An ongoing microelectronic sample with Gold/Aluminum interconnects and polymer electronic devices were chosen to evaluate HIM in comparison to scanning electron microscopy (SEM). The aim was to look for key applications of HIM in material analysis. Main focus was on complementary contrast mechanisms and imaging of none conductive samples.

This work examined the Flow Rate, Average Flow Rate, Volume Flow Rate, and Viscosity of Epon 812, Dodecynyl Succinic Anhydride (DDSA), Nadic Methyl Anhydride (NMA), and 2,4,6- Tri(dimethylaminomethyl)phenol (DMP-30), the components traditionally offered in "kit" form for preparing epoxy embedding media utilized in biological electron microscopy. Other components not used in kit formulas, including Nonenyl Succinic Anhydride (NSA), the catalysts Benzyldimethylamine (BDMA) and Dimethylaminoethanol (DMAE), and several Epon 812-like epoxy resins (Embed 812, Pelco Medcast, LX 112, Polybed 812, Scipoxy 812, Eponate 12) also were similarly tested and compared with regard to physical and handling character to the kit ingredients. NSA, BDMA, and DMAE were significantly less viscous than the DDSA and DMP-30 counterparts from the kit formula. Embedding media were prepared utilizing Embed 812 and LX 112 in combination with different anhydrides and catalysts. Substituting less viscous NSA (in place of DDSA) and BDMA (in place of DMP-30) produced embedding media that were appreciably more fluid and less viscous. A "novel" mix of Embed 812/NSA/NMA/BDMA, for example, possessed a viscosity of 22.5 centipoises (cP) 60 minutes after initial mixing, as compared to a viscosity of 30.0 cP when the media was catalyzed with the more traditional DMP-30 catalyst. All novel combinations not using the original kit ingredients showed improvement in flow rate and fluidity, some more than others. This information suggests that microscopists are not bound to kit formulas, as they easily can prepare embedding media with specific characteristics to suit specific needs simply by altering the basic components that go into the makeup of the embedding medium.

The recent advent of focused ion beam (FIB) technology in combination with the more familiar scanning electron microscope (SEM) is bringing new insights to the characterization of a range of bulk materials. Furthermore, the FIB SEM can be augmented by a cryo-preparation/transfer system, enabling both frozen and frozen-hydrated soft materials to be FIB-milled at low temperature. This provides an opportunity to perform in situ site-specific crosssectioning, and hence study the interior of a bulk material in two and three dimensions, and serves as an alternative to the freeze-fracturing techniques associated with conventional cryo-SEM. For soft materials in particular, the quality of FIB SEM results is dependent on correct preparation of the specimen's top surface, which is rather challenging for specimens at low temperature. We therefore demonstrate methods for 'cold deposition' of a protective, planarising surface layer on a cryo-prepared sample, enabling high-quality cross-sectioning and investigation of structures at the nano-scale.

Focused Ion Beams (FIBs) provide a cross-sectioning tool for submicron dissection of cells and subcellular structures. In combination with Scanning Electron Microscope (SEM), FIB provides complementary morphological information, that can be further completed by EDX (Energy Dispersive X-ray Spectroscopy). This study focus onto intracellular microstructures, particularly onto metal granules (typically Zn, Cu and Fe) and on the possibility of sectioning digestive gland cells of the terrestrial isopod P. scaber making the granules available for a compositional analysis with EDX. Qualitative and quantitative analysis of metal granules size, amount and distribution are performed. Information is made available of the cellular storing pattern and, indirectly, metal metabolism. The extension to human level is of utmost interest since some pathologies of relevance are metal related. Apart from the common metal-overload-diseases (hereditary hemochromatosis, Wilson's and Menkes disease) it has been demonstrated that metal in excess can influence carcinogenesis in liver, kidney and breast. Therefore protocols will be established for the observation of mammal cells to improve our knowledge about the intracellular metal amount and distribution both in healthy cells and in those affected by primary or secondary metal overload or depletion.

Micromachining techniques are proposed to mass-manufacture innovative silicon oxide micropipettes and conventional boron-silicate pipettes with highly customized tips to address increasingly demanding cell handling procedures. Cell handling has become a crucial procedure in cell biology, especially in nuclear transfer, DNA injection, and in assisted reproductive techniques. Most pipette manufacturing procedures involve tedious artisanal methods prone to failure and with limited functionality. We expect high tip customization to have a large impact in current and future cell manipulation, paving the way for augmented functionality. Although proper biocompatibility assessments remain to be explored, initial pierced embryos are seen to continue their division procedure up to at least 24 hours. The continued cellular division is a good sign of biocompatibility. These results suggest that residual chemical agents or gallium ions injected during milling could be harmless to life development. We conclude that we have produced a novel technique combining microfabrication and Focus Ion Beam processes with great potential for industrial applications.

New developments in X-ray instrumentation and analysis have facilitated the development and improvement of various scanning X-ray microscopy techniques. In this contribution, we offer an overview of recent scanning hard X-ray microscopy measurements performed at the Swiss Light Source. We discuss scanning transmission X-ray microscopy in its transmission, phase contrast, and dark-field imaging modalities. We demonstrate how small-angle X-ray scattering analysis techniques can be used to yield additional information. If the illumination is coherent, coherent diffraction imaging techniques can be brought to bear. We discuss how, from scanning microscopy measurements, detailed measurements of the X-ray scattering distributions can be used to extract high-resolution images. These microscopy techniques with their respective imaging power can easily be combined to multimodal X-ray microscopy.

Milliprobe x-ray fluorescence (mXRF) with x-ray spectrum imaging (XSI) enables elemental mapping over centimeter lateral distances with a resolution of 40-150 μm. While highly complementary to classic elemental mapping scanning electron microscopy/energy dispersive x-ray spectrometry (SEM/EDS), mXRF has several advantages: (1) The lack of electron bremsstrahlung in the XRF spectrum, except for the elastic scattering of the primary continuum, means that the inherent detection sensitivity of mXRF is better. (2) The broad continuum excitation of mXRF enables sensitive access to secondary x-rays with photon energies in the range 10 keV to 40 keV, which are either not efficiently excited or are completely inaccessible with SEM. (3) The range of penetration of x-rays (with minimal sideways scatter) is typically 10 to 100 times the range of an electron beam, enabling deeper probing into the specimen or even viewing the specimen through protective covering such as glass or plastic. (4) The vacuum requirements of mXRF are much less than even environmental SEM, and primary excitation and secondary detection can occur through an atmospheric gas path if required. The scale of mXRF-XSI mapping is particularly useful for attacking problems that require following compositional structures over a wide spatial scale, the classic "micro-to-macro" spatial scale challenge.

Compositional measurements of microscopic particles by scanning electron microscopy/energy dispersive x-ray spectrometry (SEM/EDS) typically assume particle homogeneity so that a representative EDS spectrum can be collected by continuously bracket scanning the particle (overscanning). Particles are often found to be complex structures comprised of smaller entities that are elementally different, knowledge of which is inevitably lost with particle overscanning. Heterogeneity can be directly visualized with x-ray spectrum image mapping performed in a high resolution thermal field emission gun SEM combined with the silicon drift detector (SDD)-EDS. SDD-EDS is capable of x-ray collection with output count rates in excess of 1 MHz, enabling spectrum image mapping with useful pixel density (128x128 or more), intensity range (8 - 16 bits), and compositional sensitivity (detection to approximately 1 weight percent) with a total time of 3 - 30 s when a high beam current (e.g., 50 nA) is utilized. Spectrum image datacubes can range from 100 Mbyte to several gigabytes. NIST Lispix contains extensive image processing tools to extract spectral and image information from such large datacubes. In addition to particle chemical heterogeneity, spectrum image mapping can directly reveal the effects of geometric factors (size, shape, curvature) that modify x-ray generation and emission from particles and which must be considered in particle quantification calculations.

Pure and doped polyvinylidene fluoride (PVDF) films were prepared by casting. Films with various concentrations of transition metal halides TMHs (AlCl₃, ZnCl₂, and CoCl₂) were prepared. The microstructure and physical properties of these films were studied by IR analysis. The two factors affecting the interaction between the PVDF and MHs are (i) the dopant weight fraction (H_c) (0.05% -30%) by weght, and (ii) precasting time (t_pc) which is the time during which the PVDF pellets are maintained solved with the halides added before casting. From the IR quantitative analysis, it is evident that the addition of the three MH to the undoped PVDF film makes β-phase as the dominant crystalline structures in the doped films without the need for mechanical drawing treatment. The precasting time plays a role for new crystalline structures to appear which becomes strong for CoCl₂ doping, moderate in ZnCl₂ doping and weak in AlCl₃ doping. This phase is maximum for the relatively low doping levels < 5%. The stability of these structures in the samples doped with CoCl₂ is high compared to the doping with ZnCl₂ and AlCl₃. This result is extremely important hence the β-phase is that one which is electrically active compared with the other two phases and it is needed in all the samples used in the useful applications of the PVF₂ films. Remembering, that β-phase is obtained in the crystallization from melt samples by the uneasy mechanical stress and elevated temperature, it becomes evident the importance of the present result.

Pure and doped polyvinylidene fluoride (PVDF) films were prepared by casting. Films with various concentrations of transition metal halides TMHs (AlCl₃, ZnCl₂, and CoCl₂) were prepared. The microstructure and physical properties of these films were studied by X-ray diffraction (XRD). The two factors affecting the interaction between the PVDF and MHs are (i) the dopant weight fraction (Hc) (0.05% -30%) by weight, and (ii) precasting time (tpc) which is the time during which the PVDF pellets are maintained solved with the halides added before casting. From the qualitative analysis of XRD, the addition of MHs to the undoped PVDF film enhances the appearance of the important β-phase without the need for mechanical drawing treatment. The precasting time plays a role for new crystalline structures to appear which becomes strong for CoCl₂ doping, moderate in ZnCl₂ doping and weak in AlCl₃ doping. Annealing and Corona poling have a negative effect on the new crystalline structures hence their peaks in X-ray diffraction are reduced. The stability of these structures in the samples doped with CoCl₂ is high compared to the doping with ZnCl₂ and AlCl₃.

For decades, high resolution scanning electron microscopes (SEM) have strived to offer improved performance in the high and low energy regimes. High energies have always been attractive, because they lead to sub-nanometer resolution without complex electron optics, especially when using a scanning transmission electron microscopy (STEM) mode in the SEM. Lower energies have caught the attention of microscopists, due to their increased surface sensitivity, minimized charging effects or reduced depth of radiation damage. While going to very low beam landing energies was demonstrated more than 20 years ago, keeping a nanometric spot-size below 1 keV proved to be a technological challenge. Only a few years ago did the first commercial SEM succeed in delivering sub-nanometer resolution at 1 kV, but with some restrictions. Recently, the introduction of the extreme high resolution (XHR) SEM has demonstrated subnanometer resolution in the entire 1 to 30 kV range, thanks to a monochromatized Schottky electron source that reduces the effects of chromatic aberrations at lower energies. Of at least equal interest is the fact that the same XHR SEM can take advantage of its optics, modularity, platform stability and cleanliness developments to explore new avenues, such as high resolution imaging at very low beam energies or up to 30 kV STEM-in-SEM. For the first time, complementary information from the very surface and internal structure at the true nanometer level is obtained in the same SEM.

Patterned Si surfaces, p- and n-type doped, were examined for different secondary electron yield (contrast between ptype and n-type regions) under the electron beam of a scanning electron microscope. The contrast as a function of primary beam energy was studied for samples with a thick oxide layer and with the layer removed using an HF solution. It was found that the contrast between p- and n- type areas reversed on the samples with a thick oxide layer as the primary beam energy was increased. However, after the oxide layer was removed, the contrast reversal was no longer apparent. In addition, it was also found that regions on a patterned Si sample could reverse in contrast when the scan speed of the electron beam was changed. The various competing theories describing the dopant contrast effect of doped semiconductors are discussed and compared to the results reported here and elsewhere in the literature. It is concluded that oxygen at sub-monolayer coverage through to thick films plays an important role in the dopant contrast effect. However, adventitious carbon is equally important where a metal-oxide-semiconductor structure could exist with the presence of these two materials. Results from the literature using other techniques such as photoemission and field emission are also considered and it is found that these studies give results which are inconsistent with several of the current theories which attempt to explain the dopant contrast effect.

The secondary electron and backscattered electron coefficients have been measured as a function of primary beam energy for as-inserted and cleaned pure element samples. Clearly, the effect of cleaning samples makes a significant effect on both these key measurements needed for understanding the electron transport measurements in scannng electron microscopy and a number of other technologies. The results from the cleaned samples suggest that the currently accepted theory for secondary electron emission (SEE) of Baroody does not take account of an important physical effect. We propose that the SEE in transition metals is mainly controlled by the inelastic mean free path (IMFP) of the secondary electrons. In combination with current theories on the transport of hot electrons in transition metals, where sensitivity to the density of empty d states is important, the apparent correlation of the work function with SEE can be explained. The effect of errors in the electron elastic scattering cross-section and the electron stopping power on the estimates of backscattered electron coefficient, η, are explored for the case of Cu. It is found that percentage errors in one parameter (e.g. stopping power) cause very similar changes in η as equal but opposite percentage errors in the other parameter (e.g. elastic scattering cross-section).

Miniature electron beam columns have the advantage of high resolution (<10 nm) at low beam energies (0.5 - 2 kV), making them well-suited for probing the surface structure of a wide variety of nano-scale materials. Because miniature columns have the further benefits of small form factor and low cost of manufacturing, they are uniquely suited for high resolution tabletop Scanning Electron Microscopy (SEM). Miniature columns are also good candidates for use in multiple beam lithography and high throughput mask writing systems. A miniature electrostatic column has been developed for the Novelx mySEM tabletop SEM using monolithically processed bonded stacks of silicon and glass, mounted to a ceramic substrate. It has been shown previously that this type of design can be used to produce a highly manufacturable and reliable column. This column design has demonstrated high resolution imaging and lithography capabilities. The column includes a condenser lens integrated into the source silicon stack, which provides variable beam current density at the limiting aperture in order to vary the probe current. This paper presents results from the condenser lens in the mySEM column, demonstrating continuously variable beam current over a wide range. Simulations presented in this paper show that this column is capable of >2 nA beam current using the condenser lens, with only a slight increase in beam size at high beam current. Measurements are in good agreement with the simulations, demonstrating > 7 nA beam current with the condenser turned on.

In order to provide the uniformity of measurements at the nanoscale, seven national standards have been developed in the Russian Federation. Of these seven standards, three standards specify the procedures of fabrication and certification of linear measures with the linewidth lying in the nanometer range. The other four standards specify the procedures of verification and calibration of customer's atomic force microscopes and scanning electron microscopes, intended to perform measurements of linear dimensions of relief nanostructures. For an atomic force microscope, the following four parameters can be deduced: scale factor for the video signal, effective radius of the cantilever tip, scale factor for the vertical axis of the microscope, relative deflection of the microscope's Z-scanner from the orthogonality to the plane of a sample surface. For a scanning electron microscope, the following two parameters can be deduced: scale factor for the video signal and the effective diameter of the electron beam. The standards came into force in 2008.

Two types of test objects for automated measurements of critical dimensions with scanning electron microscopes (SEMs) are described. The first type can be used for SEM calibration along two coordinates in a wide range of magnifications (to perform dimensional measurements in the range from 10 nm to 100 μm without making recalibration), including determination of the electron beam diameter. The second type is recommended for embedding into the integrated circuits (ICs) to monitor the focusing of the SEM electron beam in the course of dimensional measurements of IC elements. Measurement and monitoring of the SEM magnification and electron beam diameter is necessary to measure the linewidth (the sizes of the upper and lower bases of the IC trapezoidal relief elements) in the nanometer range.

Models for simulating Scanning Probe Microscopy (SPM) may serve as a reference point for validating experimental data and practice. Generally, simulations use a microscopic model of the sample-probe interaction based on a first-principles approach, or a geometric model of macroscopic distortions due to the probe geometry. Examples of the latter include use of neural networks, the Legendre Transform, and dilation/erosion transforms from mathematical morphology. Dilation and the Legendre Transform fall within a general family of functional transforms, which distort a function by imposing a convex solution. In earlier work, the authors proposed a generalized approach to modeling SPM using a hidden Markov model, wherein both the sample-probe interaction and probe geometry may be taken into account. We present a discussion of the hidden Markov model and its relationship to these convex functional transforms for simulating and restoring SPM images.

Today the vast majority of the scanning electron microscopes (SEMs) are incapable of taking repeatable and accurate images at high magnifications. Geometric distortions are common, so are drift, vibration, and problems related to disturbing electro-magnetic fields, contamination. These issues tend to degrade the quality of the image. Hence, in many cases it is not the focusing ability of the electron optical column, but these factors that limit the achievable resolution, repeatability and accuracy. This is a significant issue for nanometer-scale measurements, because the errors are many times greater than the measured distances. However, there are new image acquisition techniques that could improve the accuracy and repeatability of such measurements. One of these techniques is being developed at National Institute of Standards and Technology (NIST). This technique is based on cross-correlation combined with frequency filtering. Because the power this technique strongly depends on many conditions, like shape and periodicity of the sample features, noise, frequencies of the vibrations, etc., it needs to be properly evaluated and limits of usability of the technique should be specified. For this, a statistically significant number of SEM images varying in all of these parameters is necessary and the parameters must be known. Unfortunately, this is impossible to achieve using an SEM, because most of these parameters are random and the instrument parameters are unknown. A possible solution is to use the artificial SEM images, which can simulate these effects repeatably and deterministically. It is not an issue to generate a large number of such images in a reasonable time. The artificial image generator was developed at NIST originally for the evaluation of resolution-calculation techniques, but it is also very usable for assessment of new imaging techniques too. The new version of the generator is being developed at NIST. It allows for modeling different types of samples and several new effects, which also allow for taking into account the different characteristics of different SEMs. This paper describes, how the artificial images are built and how they may be used to improve the new SEM imaging techniques.

A proprietary metrological scanning probe microscope (SPM) with an interferometer, developed by the Institute of Process Measurement and Sensor Technology at the Ilmenau University of Technology (IPMS), is used as a stationary probe system in the nanomeasuring and nanopositioning machine (NPMM). Due to the movements of the NPMM, the total microscope measuring range is 25mm × 25mm × 5mm with a positioning resolution of less than 0.1nm. Examples for specimens are step height standards and one-dimensional gratings. The repeatability has been determined at less than 0.5nm for measurements on calibrated step height standards and less than 0.2nm for the gratings. The measurement results of these samples are always directly related to the corresponding measurement uncertainty, which can be calculated using an uncertainty budget. A new traceable method has been developed using a vectorial modular model. With this approach, it is possible to quickly insert new sub-models and to individually analyze their effects on the total measurement uncertainty. The analysis of these effects with regard to their uncertainties is done by Monte Carlo Simulation (MCS), because some models have partially or fully nonlinear character of which one example is the interferometer model of the metrological SPM. The complete development and analysis of these models is presented for one specific measurement task. The measurement results and the corresponding measurement uncertainty were obtained by Monte Carlo Simulation. Comparisons with the GUM have shown that the proposed procedure is a good alternative to achieve reasonable measurement results with uncertainty estimation.

Calibration of scanning electron microscopy (SEM) for accurate measurement of critical dimensions (CDs) poses a significant problem. Measured CDs depend on the specific SEM setup, as well as the materials and shape of the sample. In addition to systematic errors, charging of the wafer plays an important role in SEM and defect inspection tools. Charging introduces a dynamic component into the linewidth measurement error. CD-SEM calibration can be significantly improved when measurements are complemented by accurate simulations. An advanced Monte-Carlo software, CHARIOT, with an emphasis on low-voltage electrons was developed to simulate image formation in SEM, energy deposition in electron-beam lithography, and charging of a target. Scattering of an electron beam in a microstructure, generation of secondary electrons, and characteristics of the detector, as well as the material and 3D shape of the features, are considered. Physical and mathematical models are described to comply with the accuracy required by modern technology, especially with low-voltage electrons. Examples of CD-SEM simulation in the presence of charging are presented.

The Nanometer-Coordinate-Measuring-Machine(NCMM) has been developed for comparatively fast large area scans with high resolution for measuring critical dimensions. The system combines a metrological atomic force microscope (AFM) with a precise positioning system. The sample is moved under the probe system via the positioning system achieving a scan range of 25 x 25 x 5 mm with a resolution of 0.1 nm. A concept for critical dimension measurement using a-prioriknowledge is implemented. A-priori-knowledge is generated through measurements with a white light interferometer and the use of CAD data. Dimensional markup language (DML) is used as a transfer and target format for a-priori-knowledge and measurement data. Using a-priori-knowledge and template matching algorithms combined with the optical microscope of the NCMM, the region of interest can be identified automatically. In the next step an automatic measurement of the part coordinate system and the measurement elements with the AFM sensor of the NCMM is performed. Automatic measurement involves intelligent measurement strategies, which are adapted to specific geometries of the measurement features to reduce measurement time and uncertainty.

In our contribution we present a technique for characterizing the piezo-driven sensor head of a scanning probe microscope (SPM) using a laser vibrometer. The experimental setup, the signal processing and some experimental results are described. Furthermore a mechanical model for the sensor head is proposed and verified by the experimental results. Finally a guidance for an online or offline correction of the position of the sensor head is presented.

The Kelvin Probe Force Microscopy (KPFM) is a method to detect the surface potential of micro- and nanostructured samples using a common Scanning Probe Microscope (SPM). The electrostatic force has a very long range compared to other surface forces. By using SPM systems the KPFM measurements are performed in the noncontact region at surface distances greater than 10 nm. In contrast to topography measurement, the measured data is blurred. The KPFM signal can be described as a convolution of an effective surface potential and a microscope intrinsic point spread function, which allows the restoration of the measured data by deconvolution. This paper deals with methods to deconvolute the measured KPFM data with the objective to increase the lateral resolution. An analytical and a practical way of obtaining the point spread function of the microscope was compared. In contrast to other papers a modern DoF-restricted deconvolution algorithm is applied to the measured data. The new method was demonstrated on a nanoscale test stripe pattern for lateral resolution and calibration of length scales (BAM-L200) made by German Federal Istitute for Materials Research and Testing.

In this article, an approach based on the recently-developed inversion-based iterative control (IIC) to cancel the cross-axis coupling effect of piezoelectric tube scanners (piezoscanners) in tapping mode atomic force microscope (AFM) imaging is proposed. Cross-axis coupling effect generally exists in piezoscanners used for 3D (x-y-z axes) nanopositioning in applications such as AFM, where the vertical z-axis movement can be generated by the lateral x-y axes scanning. Such x/y-to-z cross-coupling becomes pronounced when the scanning is at large range and/or at high-speed. In AFM applications, the coupling-caused position errors, when is large, can generate various adverse effects, including large imaging and topography distortions, and damage of the cantilever probe and/or the sample. This paper utilizes the IIC technique to obtain the control input to precisely track the couplingcaused x/y-to-z displacement (with sign-flipped). Then the obtained input is augmented as a feedforward control to the existing feedback control in tapping-mode imaging, resulting in the cancellation of the coupling effect. The proposed approach is illustrated through the exemplary applications of the nanoasperity measurement on harddisk drive. Experimental results show that the x/y-to-z coupling effect in large-range (20 μm) tapping-mode imaging at high scan rates (12.2 Hz to 24.4 Hz) can be effectively removed.

The logic and memory semiconductor device technology strives to follow the aggressive ITRS roadmap. The ITRS calls for increased 3D metrology to meet the demand for tighter process control at 45nm and 32nm nodes. In particular, gate engineering has advanced to a level where conventional metrology by CD-SEM and optical scatterometry (OCD) faces fundamental limitations without involvement of 3D atomic force microscope (3D-AFM or CD-AFM). This paper reports recent progress in 3D-AFM to address the metrology need to control gate dimension in MOSFET transistor formation. 3D-AFM metrology measures the gate electrode at post-etch with the lowest measurement uncertainty for critical gate geometry, including linewidth, sidewall profile, sidewall angle (SWA), line width roughness (LWR), and line edge roughness (LER). 3D-AFM enables accurate gate profile control in three types of metrology applications: reference metrology to validate CD-SEM and OCD, inline depth or 3D monitoring, or replacing TEM for 3D characterization for engineering analysis.

SEM has greatly increased our knowledge of the microstructure of seeds. Mature seed coats are rather thick walled and stable in a vacuum: this allows quick preparation for SEM examination, without the need of complicated dehydration techniques. The low level of technical expenditure required, in combination with the high structural diversity exhibited and the intuitive ability to understand the "three dimensional", often aesthetically appealing micro-structures visualized, has turned seed-coat studies into a favorite tool of many taxonomists. We used dry mature seeds of 26 species of 4 Leguminous genera, Acacia, Albizia, Cassia and Dalbergia to standardize a procedure for identifying the seeds through SEM on the seed surface and seed sections. We cut transverse and longitudinal sections of the seeds and observed the sections from different regions of seeds: midseed, near the hilum and two distal ends. Light microscopy showed the color, texture, pleurograms, fissures and hilum at lower magnification. The anatomical study with SEM on the seed sections revealed the size, shape, and number of tiers and cellular organization of the epidermis, hypodermis, endosperm and internal structural details. We found the ornamentation pattern of the seeds including undulations, reticulations and rugae that were species specific. Species of Dalbergia (assamica, latifolia and sissoo), Albizia (odoratissima and procera), Acaia (arabica and catechu) and Cassia (glauca, siamia and spectabilis) are difficult to distinguish externally, but SEM studies provided enough characteristic features to distinguish from the other. This technique could be valuable in identifying seeds of important plant species for conservation and trading.

Fresh-cut produce has a huge following in today's supermarkets. The trend follows the need to decrease preparation time as well as the desire to follow the current health guidelines for consumption of more whole "heart-healthy" foods. Additionally, consumers are able to enjoy a variety of fresh produce regardless of the local season because produce is now shipped world-wide. However, most fruits decompose rapidly once their natural packaging has been disrupted by cutting. In addition, some intact fruits have limited shelf-life which, in turn, limits shipping and storage. Therefore, a basic understanding of how produce microstructure relates to texture and how microstructure changes as quality deteriorates is needed to ensure the best quality in the both the fresh-cut and the fresh produce markets. Similarities between different types of produce include desiccation intolerance which produces wrinkling of the outer layers, cracking of the cuticle and increased susceptibility to pathogen invasion. Specific examples of fresh produce and their corresponding ripening and storage issues, and degradation are shown in scanning electron micrographs.

Oats have long been recognized as having superior quality among cereals with respect to protein and lipid composition as well as soluble dietary fibre (β-glucan). The microstructure and chemistry of oats influence oat quality, and thus are determinants of the end products derived from oats. Light and scanning electron microscopies have been used to elucidate microstructure and nutrient distribution in oats. The influence of variation in these parameters on oat quality can be demonstrated, from milling through to oat products for consumption. Milling quality is determined in part by hull architecture. SEM examination of oat hulls can help predict ease of dehulling, which affects the efficiency and economics of oat milling. In addition to protein and lipid, β-glucan is an important nutritional component of oats. Fluorescence microscopy can reveal both the relative amount and distribution of β-glucan in oat kernels. Consumption of oats or oat products containing β-glucan has been shown to have beneficial effects on carbohydrate and lipid metabolism. These health benefits have generated a demand for new and palatable ways to incorporate oats into the diet as consumer demand increases. To help meet this need, we have been investigating the use of micronized naked oats as a whole grain to be cooked and consumed as a rice alternative. Different varieties of naked oats had dramatically different acceptance levels from a sensory panel. SEM of the pericarp, light microscopy of the endosperm, and analyses of starch properties of the different varieties revealed differences that corresponded with sensory data.

The advent of novel techniques using the Transmission and Scanning Electron Microscopes improved observation on various biological specimens to characterize them. We studied some biological specimens using Transmission and Scanning Electron Microscopes. We followed negative staining technique with Phosphotungstic acid using bacterial culture of Bacillus subtilis. Negative staining is very convenient technique to view the structural morphology of different samples including bacteria, phage viruses and filaments in a cell. We could observe the bacterial cell wall and flagellum very well when trapped the negative stained biofilm from bacterial culture on a TEM grid. We cut ultra thin sections from the fixed root tips of Pisum sativum (Garden pea). Root tips were pre fixed with osmium tetroxide and post fixed with uranium acetate and placed in the BEEM capsule for block making. The ultrathin sections on the grid under TEM showed the granular chromatin in the nucleus. The protein bodies and large vacuoles with the storage materials were conspicuous. We followed fixation, critical point drying and sputter coating with gold to view the tissues with SEM after placing on stubs. SEM view of the leaf surface of a dangerous weed Tragia hispida showed the surface trichomes. These trichomes when break on touching releases poisonous content causing skin irritation. The cultured tissue from in vitro culture of Albizia lebbeck, a tree revealed the regenerative structures including leaf buds and stomata on the tissue surface. SEM and TEM allow investigating the minute details characteristic morphological features that can be used for classroom teaching.

We describe a new type of scanning electron microscope which works by directly imaging the electron field-emission sites on a nanotip. Electrons are extracted from the nanotip through a nanoscale aperture, accelerated in a high electric field and focussed to a spot using a microscale einzel lens. If the whole microscope (accelerating section and lens) and the focal length are both restricted in size to below 10 microns, then computer simulations show that the effects of aberration are extremely small and it is possible to have a system with approximately unit magnification, at electron energies as low as 300 eV. Thus a typical emission site of 1 nm diameter will produce an image of the same size and an atomic emission site with give a resolution of 0.1-0.2 nm (1-2 Å), and because the beam is not allowed to expand beyond 100nm in diameter the depth of field is large and the contribution to the beam spot size from chromatic aberrations is less than 0.02 nm (0.2 Å) for 500 eV electrons. Since it is now entirely possible to make stable atomic sized emitters (nanopyramids) it is expected that this instrument will have atomic resolution. Furthermore the brightness of the beam is determined only by the field-emission and can be up to a million times larger than in a typical (high-energy) electron microscope. The construction of this microscope, based on using a nanotip electron source which is mounted on a nanopositioner so that it can be positioned at the correct point adjacent to the microscope, entrance aperture, is described. In this geometry the scanning is achieved by moving the sample using piezos. Two methods for the construction of the microscope column are reviewed and the results of preliminary tests are described. The advantages of this low energy, bright-beam, electron microscope with atomic resolution are described. It can be used in either scanning mode or diffraction mode. The major advantage over existing microscopes is that because it works at very low energies the elastic backscattering is sensitive to the atomic species and so these can be identified directly without any energy discrimination on the detector. Furthermore it is also possible to use the microscope to do low energy electron diffraction which, because the scattering cross-section is large, can be carried out on single molecules. If these are biological samples such as DNA, proteins and viruses then the low energy means that the radiation damage is minimised. Some possibilities for mounting these samples, which can reduce radiation damage, are discussed. Finally we show a system for producing holograms of single protein molecules.

While electron microscopes and AFMs are capable of high resolution imaging to molecular levels, there is an ongoing problem in integrating these results into the larger scale structure and functions of tissue and organs within a complex organism. Imaging biological samples with optical microscopy is predominantly done with histology and immunohistochemistry, which can take up to a several weeks to prepare, are artifact prone and only available as individual 2D images. At the nano resolution scale, the higher resolution electron microscopy and AFM are used, but again these require destructive sample preparation and data are in 2D. To bridge this gap, we describe a rapid non invasive hierarchical bioimaging technique using a novel lab based x-ray computed tomography to characterize complex biological organism in multiscale- from whole organ (mesoscale) to calcified and soft tissue (microscale), to subcellular structures, nanomaterials and cellular-scaffold interaction (nanoscale). While MicroCT (micro x-ray computed tomography) is gaining in popularity for non invasive bones and tissue imaging, contrast and resolution are still vastly inadequate compared to histology. In this study we will present multiscale results from a novel microCT and nanoCT (nano x-ray tomography system). The novel MicroCT can image large specimen and tissue sample at histology resolution of submicron voxel resolution, often without contrast agents, while the nanoCT using x-ray optics similar to those used in synchrotron radiation facilities, has 20nm voxel resolution, suitable for studying cellular, subcellular morphology and nanomaterials. Multiscale examples involving both calcified and soft tissue will be illustrated, which include imaging a rat tibia to the individual channels of osteocyte canaliculli and lacunae and an unstained whole murine lung to its alveoli. The role of the novel CT will also be discussed as a possible means for rapid virtual histology using a biopsy of a human regenerated bone sample done without contrast agents and that of other soft tissues with contrast agents. Comparison between histology, SEM and MRI will be given.

Biologically compatible quantum dot (QD) nanoparticles are hybrid inorganic-organic materials with increasing popularity as fluorescent probes for studying biological specimens. QDs have several advantageous optical features compared to fluorescent dyes and they are electron-dense, allowing for correlated fluorescence and electron microscopic imaging. Despite these features, widespread use of QDs as biological probes has generally been limited by the complex chemistry required for their synthesis and the conjugation. In this work, we show that easily prepared quantum dot (QD) probes provide excellent contrast for fluorescent confocal and environmental scanning electron microscopy (ESEM) analysis of pure microbial cultures and microbial communities. Two conjugation strategies were employed in order to specifically target the QDs to bacterial cell surfaces. The first was biotinylation of the bacteria followed by labeling with commercially available QDs incorporating the high-affinity partner for biotin (QD-streptavidin). Second, we designed a novel QD probe for Gram negative bacteria: QD-polymyxin B (PMB), which binds to lipopolysaccharide (LPS) in the Gram negative cell wall. Pure cultures of Gram positive and Gram negative strains were used to illustrate that QDs impart electron density and irradiation stability to the cells, and so no other preparation apart from QD labeling is required. The techniques were then extended to a set of recently characterized microbial communities of perennial cold springs in the Canadian High Arctic, which live in close association with unusual sulfur crystals. Using correlated confocal and and ESEM, we were able to image these organisms in living samples and illustrate their relationship to the minerals.

Optical methods, including confocal microscopes, are widely used for measurements of surface topography. The knowledge of surface morphology and roughness parameters is crucial for many applications, i.e. in industrial and automotive environment, in tribology, wear and functionality prediction. However, confocal microscopy has a limited field of view. A time consuming stitching process is required for extending to long profile lines measurement. Therefore, in this paper we present a novel concept of a Confocal Line Scanning Sensor (CLSS) to cover theoretically infinite profile lengths. The new technique is proposed with no moving parts required for axial scanning, and it has a simpler setup than those of Chromatic Confocal Sensor (CCS). The idea is to produce a stack of focal points on an inclined plane covering a certain axial measurement range. Therefore, by scanning the stack of focal points in lateral direction we can realize a long profile line. By doing that we expect to achieve shorter scanning time, while providing high lateral and axial resolution by using a true confocal principle. A long profile line of a few ten millimeters with a lateral resolution in sub-micrometer range and an axial resolution in tens of nanometers can be expected. Moreover, this concept is easily extensible to an areal measurement. Among other key components, a new design of the pinhole mask has been developed. We design it to produce an inclined focal line with optimum optical parameters. Optimization of the pinhole design fulfills two objectives; minimizing its size by allowing optimal reflected-light intensity, and minimizing crosstalk between nearby pinholes. Further detail of the pinhole design is beyond a scope of this paper. In this paper an overview of the new concept is presented, accompanied by validation of first experimental results.

Scanning confocal microscopy is a widely recognized technique due to its applicability to the imaging of 3D geometries. Image formation in this technique is often analyzed using the Fresnel approximation. However, such an approximation is not sufficient when object dimensions are comparable to the operating wavelength and, most of all, when the target is composed of different semi-transparent materials. Yet, this is very typical for modern integrated circuits where we work with subwavelength features. In such a case target needs to be modeled using full-wave Maxwell theory. However, most of electromagnetic modeling methods (like well established FDTD method) become computationally impractical when the modeled scenario has dimensions measured in hundreds or even thousands of wavelengths like in the far-field microscopy. Therefore, in this paper we propose a hybrid approach that takes advantage of both FDTD and Fresnel approximation methods. The first method will be applied to the modeling of close vicinity of the target. The advantage of that is versatility in definition of arbitrarily shaped geometries as well as wideband approach of the FDTD method. Subsequently, results provided by the FDTD solver will be transferred to the procedure based on the scalar Fresnel approximation used to process the final image pixel by pixel. We will show that the presented method allows imaging of 3D shape of targets proving unique advantage of using FDTD method to the modeling of scanning confocal microscopy.

The major thrust of modern day fluorescence laser-scanning microscopy have been towards achieving better and better depth resolution embodied by the invention and subsequent development of confocal and multi-photon microscopic techniques. However, each method bears its own limitations: in having sufficient background fluorescence and photodamage resulting from out-of-focus illumination for the former, while low multi-photon absorption cross-sections of common fluorophores for the latter. Here we show how the intelligent choice of single-photon ultrashort pulsed illumination can circumvent all these shortcomings by exemplifying the tiny spatial stretch of an ultrashort pulse. Besides achieving a novel way of optical sectioning, this new method offers improved signal-to-noise ratio as well as reduced photo-damage which are crucial for live cell imaging under prolonged exposure to light.

Resonant Energy Transfer (RET) from an optically excited molecule to a non-excited molecule residing nearby has been used to detect molecular interactions in living cells. Information such as the number of proteins forming a molecular complex has been obtained so far for a handful of proteins, but only after exposing the samples sequentially to at least two different excitation wavelengths. Changes in the molecular makeup of a cellular region occurring during this lengthy process of measurement has limited the applicability of RET to determination of cellular averages. We developed a method for imaging protein complex distribution in living cells with sub-cellular spatial resolution, which relies on a spectrally-resolved two-photon microscope. The use of diffractive optics in a non-descanned configuration allows acquisition of a full set of spectrally-resolved images after only one complete scan of the excitation beam. This presentation will briefly describe our basic experimental setup and a simple theory of RET in oligomeric complexes, and it will review our recent results on determination of the geometry and size of oligomeric complexes of several proteins in yeast as well as in mammalian cells. This method basically transforms RET into a method for performing veritable structural determinations of protein complexes in vivo.

In the past decade, the development of new tools to better visualize microbes at the cellular scale has spurred a renaissance in the application of microscopy to the study of bacteria in their natural environment. This renewed interest in microscopy may be largely attributable to the advent of the confocal laser scanning microscope (CLSM) and to the discovery of the green fluorescent protein. This article provides information about the use of fluorescence microscopy combined with fluorescent labels such as GFP, DsRed, and DNA stains, with immunofluorescence, and with digital image analysis, to examine the behavior of bacteria and other microbes on plant surfaces. Some of the advantages and pitfalls of these methods will be described using practical examples derived from studies of the ecology of foodborne pathogens, namely Salmonella enterica and E. coli O157:H7, on fresh fruit and vegetables. Confocal microscopy has been a powerful approach to uncover some of the factors involved in the association of produce with epidemics caused by these human pathogens and their interaction with other microbes in their nonhost environment.

Direct imaging of charge transport is obtained in luminescent materials by combining the excitation capability and resolution of a scanning electron microscope (SEM) with high sensitivity optical imaging. A regular optical microscope (OM) or a near field scanning optical microscope (NSOM) is operated within the SEM to allow for characterization of semiconductor materials by imaging the spatial variation of luminescence associated with minority carrier recombination. The NSOM system uses a Nanonics MultiView 2000 that allows for independent scanning of both sample and collecting fiber. The technique builds upon traditional cathodoluminescence (CL), but differs in that spatial information from the luminescence is maintained, allowing for direct imaging of carrier transport. The approach will be introduced with results from double heterostructures of GaAs and the effect of radiation damage on minority carrier diffusion lengths. Then, its application to structures requiring near field imaging will be illustrated with results from measurements of carrier diffusion in GaN nanowires.

An optical profilometer has been used to obtain 3-dimensional data for use in two research projects concerning toolmark quantification and identification. In the first study quantitative comparisons between toolmarks made using data from the optical system proved superior to similar data obtained using a stylus profilometer. In the second study the ability of the instrument to obtain accurate data from two surfaces intersecting at a high angle (approximately 90 degrees) is demonstrated by obtaining measurements from the tip of a flat screwdriver. The data obtained was used to produce a computer generated "virtual tool," which was then employed to create "virtual tool marks." How these experiments were conducted and the results obtained will be presented and discussed.

The authentication and identification of the source of a printed document(s) can be important in forensic investigations involving a wide range of fraudulent materials, including counterfeit currency, travel and identity documents, business and personal checks, money orders, prescription labels, travelers checks, medical records, financial documents and threatening correspondence. The physical and chemical characterization of document materials - including paper, writing inks and printed media - is becoming increasingly relevant for law enforcement agencies, with the availability of a wide variety of sophisticated commercial printers and copiers which are capable of producing fraudulent documents of extremely high print quality, rendering these difficult to distinguish from genuine documents. This paper describes various applications and analytical methodologies using scanning electron miscoscopy/energy dispersive (x-ray) spectroscopy (SEM/EDS) and related technologies for the characterization of fraudulent documents, and illustrates how their morphological and chemical profiles can be compared to (1) authenticate and (2) link forensic documents with a common source(s) in their production history.

The victim was alleged to have been shot in the head with a .40 caliber pistol from several feet. The defendant claimed that the shot was on the order of inches. Examination in the scanning electron microscope of the hair from around the victim's wound showed no adherent gunshot residue (GSR). However, when the hair was pulled apart by the adhesive of a standard GSR sampler, GSR was found associated with the exposed inner surfaces of the cuticle and cortex fragments. The pistol was discharged close enough to the victim's head that some of the cuticular scales were lifted in the muzzle blast which allowed GSR to be inserted under those scales. Gunshot residue associated with the surface of the victim's hair had somehow been removed. The defendant's account of the shooting was verified by the presence of under-scale GSR.

In 2008, an unknown white powder was discovered spilled inside of a shipping container of whole kernel corn during an inspection by federal inspectors in the port of Baltimore, Maryland. The container was detained and quarantined while a sample of the powder was collected and sent to a federal laboratory where it was screened using chromatography for the presence of specific poisons and pesticides with negative results. Samples of the corn kernels and the white powder were forwarded to the Food and Drug Administration, Forensic Chemistry Center for further analysis. Stereoscopic Light Microscopy (SLM), Scanning Electron Microscopy/Energy Dispersive X-ray Spectrometry (SEM/EDX), and Polarized Light Microscopy/Infrared Spectroscopy (PLM-IR) were used in the analysis of the kernels and the unknown powder. Based on the unique particle analysis by SLM and SEM as well as the detection of the presence of aluminum and phosphorous by EDX, the unknown was determined to be consistent with reacted aluminum phosphide (AlP). While commonly known in the agricultural industry, aluminum phosphide is relatively unknown in the forensic community. A history of the use and acute toxicity of this compound along with some very unique SEM/EDX analysis characteristics of aluminum phosphide will be discussed.

Like many forensic science labs, the Belgian National Institute of Forensic Science (NICC) is involved in a Quality Assurance program aiming towards an ISO17025 Accreditation. Since last year, a project is underway in the GSR lab to validate the method used in the analysis of GSR samples acquired from the hands of suspects by SEM/EDX. The project is well underway, and is planned to lead to accreditation for this technique by the start of 2010. The presentation will discuss several aspects of the functioning of the lab that have to be addressed when preparing for this accreditation. Some of these issues and problems are so involved that separate sub-projects were defined in order to provide a manageable solution. The following topics will be treated in detail: definition of the scope of the accreditation, the validation of the SEM/EDX method with respect to : accuracy, precision, reproducibility and robustness, and the documentation of the Chain of Custody (CoC) of the samples and their storage. One specific sub-project that will be discussed is the study of contamination monitoring in different relevant locations of the lab. Finally, as we have recently acquired a new microscope, the technical criteria we used in the acquisition study will be presented with a focus on their relevance in a QA context. We feel this discussion is informative, both for labs that are pursuing a formal accreditation in the future, and those that work already in such a context and are in the process of acquiring new equipment.