Making the practically impossible “Merely difficult”—Cryogenic FIB lift‐out for “Damage free” soft matter imaging

The preparation of thinned lamellae from bulk samples for transmission electron microscopy (TEM) analysis has been possible in the focussed ion beam scanning electron microscope (FIB‐SEM) for over 20 years via the in situ lift‐out method. Lift‐out offers a fast and site specific preparation method for TEM analysis, typically in the field of materials science. More recently it has been applied to a low‐water content biological sample (Rubino 2012). This work presents the successful lift‐out of high‐water content lamellae, under cryogenic conditions (cryo‐FIB lift‐out) and using a nanomanipulator retaining its full range of motion, which are advances on the work previously done by Rubino (2012). Strategies are explored for maintaining cryogenic conditions, grid attachment using cryo‐condensation of water and protection of the lamella when transferring to the TEM. Microsc. Res. Tech. 79:298–303, 2016. © 2016 Wiley Periodicals, Inc.     

Beam characteristics for various sizes of annular aperture on scanning electron microscope.

Using an analogy between light optics and electron optics, we have calculated beam characteristics such as the beam profile and the optical transfer function for several sizes of annular and circular apertures on a scanning electron microscope (SEM). It has been found that an annular aperture improves the image quality with regard to several kinds of image resolution and the depth of focus at the price of good low-frequency (nu) contrast. In contrast with conventional circular-aperture SEM images, a combination of a low-nu-pass filtered, circular-aperture SEM image with a high-nu-pass filtered, annular-aperture SEM image has the potential to enhance the image quality in terms of both the image resolution and the depth of focus.     

‘Collapsing rings’ on Schottky electron emitters

The electron beam in systems that use a Schottky emitter as the electron source can display periodic fluctuations when the emitter is operated at an extraction voltage that gives a relatively low field strength at the tip. In the past, these fluctuations have been associated with the so-called “collapsing rings” without much further information. In this paper, the tip’s geometry changes associated with these beam instabilities are investigated in more detail by recording the evolution of the emission pattern of a Schottky emitter showing ‘collapsing rings’ for different operating conditions. Scanning electron microscope (SEM) images of different Schottky emitters have been used to support the interpretation.     

The relationship between accelerating voltage and electron detection modes to linewidth measurement in an SEM

The basic premise underlying the use of the scanning electron microscope (SEM) for linewidth metrology in semiconductor research and production applications is that the video image acquired, displayed, analyzed, and ultimately measured accurately reflects the structure of interest. However, it has been clearly demonstrated that image distortions can be caused by the detected secondary electrons not originating at the point of impact of the primary electron beam and by the type and location of the secondary electron detector. These effects and their contributions to the actual image or linewidth measurement have not been fully evaluated. Effects due to uncertainties in the actual location of electron origination do not affect pitch (line center-to-center or similar-edge-location-to-similar-edge-location spacing) measurements as long as the lines have the same edge geometries and similar profiles of their images in the SEM. However, in linewidth measurement applications, the effects of edge location uncertainty are additive and thus give twice the edge detection error to the measured width. The basic intent of this work is to demonstrate the magnitude of the errors introduced by beam/specimen interactions and the mode of signal detection at a variety of beam acceleration voltages and to discuss their relationship to precise and accurate metrology.     

Imaging of specimens at optimized low and very low energies in scanning electron microscopes.

The modern trend towards low electron energies in scanning electron microscopy (SEM), characterised by lowering the acceleration voltages in low-voltage SEM (LVSEM) or by utilising a retarding-field optical element in low-energy SEM (LESEM), makes the energy range where new contrasts appear accessible. This range is further extended by a scanning low-energy electron microscope (SLEEM) fitted with a cathode lens that achieves nearly constant spatial resolution throughout the energy scale. This enables one to optimise freely the electron beam energy according to the given task. At low energies, there exist classes of image contrast that make particular specimen data visible most effectively or even exclusively within certain energy intervals or at certain energy values. Some contrasts are well understood and can presently be utilised for practical surface examinations, but others have not yet been reliably explained and therefore supplementary experiments are needed.     

Optical detection system for probing cantilever deflections parallel to a sample surface

To date, commercial atomic force microscopes have been optimized for measurements of forces perpendicular to the sample surface. In many applications, sensitive parallel force measurements are desirable. These can be obtained by positioning the cantilever with its long axis perpendicular to the sample: the so-called pendulum geometry. We present a compact optical beam deflection system which solves the geometrical constraint problems involved in focusing a light beam onto a cantilever in the pendulum geometry. We demonstrate the performance of the system on measurements of forces imparted by a muscle myofibril, which is in-plane to a high-magnification objective of an optical microscope.     

Scanning electron microscopy of two-dimensional magnetic stray fields

Methods used in metrology of two-dimensional magnetic microfields and based on direct interaction of the electron beam of scanning electron microscope (SEM) with the studied fields are described. An analytical expression for calculating the value of the field is presented. The errors and applicability of the methods have been estimated. The concepts discussed are illustrated by the experimental results of the measurements of some types of statistical and dynamic stray fields.     

The Gray Institute ‘open’ high‐content,fluorescence lifetime microscopes

We describe a microscopy design methodology and details of microscopes built to this ‘open’ design approach. These demonstrate the first implementation of time‐domain fluorescence microscopy in a flexible automated platform with the ability to ease the transition of this and other advanced microscopy techniques from development to use in routine biology applications. This approach allows easy expansion and modification of the platform capabilities, as it moves away from the use of a commercial, monolithic, microscope body to small, commercial off‐the‐shelf and custom made modular components. Drawings and diagrams of our microscopes have been made available under an open license for noncommercial use at http://users.ox.ac.uk/~atdgroup . Several automated high‐content fluorescence microscope implementations have been constructed with this design framework and optimized for specific applications with multiwell plates and tissue microarrays. In particular, three platforms incorporate time‐domain FLIM via time‐correlated single photon counting in an automated fashion. We also present data from experiments performed on these platforms highlighting their automated wide‐field and laser scanning capabilities designed for high‐content microscopy. Devices using these designs also form radiation‐beam ‘end‐stations’ at Oxford and Surrey Universities, showing the versatility and extendibility of this approach.     

Scanning Optical Microscopy via a Scanning Electron Microscope

We have described in detail the principles of operation of a commercially available SEM-based scanning optical microscope. The stage consists of a cathodoluminescent material which converts the scanning electron beam into a scanning optical beam and an optical system to focus the light beam onto the specimen. The resolution attainable with such a stage is discussed as well as the efficiency of electron-to-photon conversion. Simplified formulae are given and a set of curves plotted which can be used to determine the appropriate tradeoff between resolution and optical power. Examples of the imaging ability in the optical beam induced current mode from various semiconductor devices are presented.     

A precise stage arrangement for correlative microscopy for specimens mounted on glass slides,stubs or EM grids

The instrumentation necessary for precise and fast correlation of images derived from a light optical microscope (LM) and a scanning electron microscope (SEM) operated in the reflective mode, is described. The specimens can be mounted on standard microscope slides (25 × 75 mm), SEM-stubs (12 mm ø), or on transmission EM grids (3 mm ø). The instrumentation consists of two parts: an attachable precision stage for an LM, and an attachable slide carrier for the stage of an SEM. By taking into account the vernier readings of the stages of both microscopes (LM and SEM), identical particles in a specimen can be found instantaneously under either microscope. Therefore it is concluded that the use of this instrumentation in correlative microscopy (LM → SEM → LM) is time saving, and especially recommended on fragile biological specimens, which may deteriorate rapidly under the electron beam of an SEM.     

Application of the low-loss scanning electron microscope image to integrated circuit technology part II--chemically-mechanically planarized samples.

Chemical-mechanical planarization (CMP) is a process that gives a flat surface on a silicon wafer by removing material from above a chosen level. This flat surface must then be reviewed (typically using a laser) and inspected for scratches and other topographic defects. This inspection has been done using both the atomic force microscope (AFM) and the scanning electron microscope (SEM), each of which has its own advantages and disadvantages. In this study, the low-loss electron (LLE) method in the SEM was applied to CMP samples at close to a right angle to the beam. The LLEs show shallower topographic defects more clearly than it is possible with the secondary electron (SE) imaging method. These images were then calibrated and compared with those obtained using the AFM, showing the value of both methods. It is believed that the next step is to examine such samples at a right angle to the beam in the SEM using the magnetically filtered LLE imaging method.     

An automated image alignment system for the scanning electron microscope

We have developed an automated image alignment system for the scanning electron microscope (SEM). This system enables specific locations on a sample to be located and automatically aligned with submicron accuracy. The system comprises a sample stage motorization and control unit together with dedicated imaging electronics and image processing software. The standard SEM sample stage is motorized in the X and Y axes with stepping motors which are fitted with rotary optical encoders. The imaging electronics are interfaced to beam deflection electronics of the SEM and provide the image data for the image processing software. The system initially moves the motorized sample stage to the area of interest and acquires an image. This image is compared with a reference image to determine the required adjustments to the stage position or beam deflection. This procedure is repeated until the area imaged by the SEM matches the reference image. A hierarchical image correlation technique is used to achieve submicron alignment accuracy in a few seconds. The ability to control the SEM beam deflection enables the images to be aligned with an accuracy far exceeding the positioning ability of the SEM stage. The alignment system may be used on a variety of samples without the need for registration or alignment marks since the features in the SEM image are used for alignment. This system has been used for the automatic inspection of devices on semiconductor wafers, and has also enabled the SEM to be used for direct write self-aligned electron beam lithography.     

Objective comparison of scanning ion and scanning electron microscope images

Common and different aspects of scanning electron microscope (SEM) and scanning ion microscope (SIM) images are discussed from a viewpoint of interaction between ion or electron beams and specimens. The SIM images [mostly using 30 keV Ga focused ion beam (FIB)] are sensitive to the sample surface as well as to low-voltage SEM images. Reasons for the SIM images as follows: (1) no backscattered-electron excitation; (2) low yields of backscattered ions; and (3) short ion ranges of 20–40nm, being of the same order of escape depth of secondary electrons (SE) [=(3–5) times the SE mean free path]. Beam charging, channeling, contamination, and surface sputtering are also commented upon.     

An online optical system for inspecting tool condition in milling of H13 tool steel and IN 718 alloy

Tool wear adversely affects surface integrity due to higher cutting forces and temperatures. However, an accurate and efficient tool wear measurement is a challenging problem. The traditional direct tool wear measurement methods such as optical microscope and scanning electron microscope (SEM) leads to error of tool reassembly, tool orientation, and low accuracy, while the indirect measurement methods cause poor accuracy. In this paper, tool wear phenomena in milling of tool steel AISI H13 and superalloy Inconel 718 have been studied. A novel online optical system has been developed to integrate with a CNC machine to directly inspect and measure tool wear conditions in milling which minimizes the above-mentioned measurement errors in traditional methods. The evolutions of tool flank wear of PVD-coated inserts in end milling of the two materials were inspected to demonstrate the function of the optical measurement system. The tool wear evolution versus cutting time were obtained and examined. The characteristic images of fast tool wear in milling of Inconel 718 were captured using SEM and compared with the optical images to estimate flank wear. Three basic modes of tool wear—flank wear, nose wear, and crater wear—were compared and analyzed. A two-parameter method has been developed to evaluate both flank wear and nose wear with respect to cutting time in milling of Inconel 718. The advantages of the on-line optical tool inspection system were discussed.     

Method to deterministically study photonic nanostructures in different experimental instruments

We describe an experimental method to recover a single, deterministically fabricated nanostructure in various experimental instruments without the use of artificially fabricated markers, with the aim to study photonic structures. Therefore, a detailed map of the spatial surroundings of the nanostructure is made during the fabrication of the structure. These maps are made using a series of micrographs with successively decreasing magnifications. The graphs reveal intrinsic and characteristic geometric features that can subsequently be used in different setups to act as markers. As an illustration, we probe surface cavities with radii of 65 nm on a silica opal photonic crystal with various setups: a focused ion beam workstation; a scanning electron microscope (SEM); a wide field optical microscope and a confocal microscope. We use cross-correlation techniques to recover a small area imaged with the SEM in a large area photographed with the optical microscope, which provides a possible avenue to automatic searching. We show how both structural and optical reflectivity data can be obtained from one and the same nanostructure. Since our approach does not use artificial grids or markers, it is of particular interest for samples whose structure is not known a priori , like samples created solely by self-assembly. In addition, our method is not restricted to conducting samples.     

Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging

Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three-dimensional micro- and nanostructures. Although this technique can be routinely applied to conducting materials, FIB–SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block-face imaging using low-kV backscattered electrons. In addition, a new ion milling geometry is used that reduces the effects of intensity gradients that are inherent in conventional geometry used for FIB–SEM tomography.     

Monte Carlo Simulation of CD‐SEM Images for Linewidth and Critical Dimension Metrology

In semiconductor industry, strict critical dimension control by using a critical dimension scanning electron microscope (CD‐SEM) is an extremely urgent task in near‐term years. A Monte Carlo simulation model for study of CD‐SEM image has been established, which is based on using Mott's cross section for electron elastic scattering and the full Penn dielectric function formalism for electron inelastic scattering and the associated secondary electron (SE) production. In this work, a systematic calculation of CD‐SEM line‐scan profiles and 2D images of trapezoidal Si lines has been performed by taking into account different experimental factors including electron beam condition (primary energy, probe size), line geometry (width, height, foot/corner rounding, sidewall angle, and roughness), material properties, and SE signal detection. The influences of these factors to the critical dimension metrology are investigated, leading to build a future comprehensive model‐based library. SCANNING 35: 127‐139, 2013. © 2012 Wiley Periodicals, Inc.     

Image formation in scanned heterodyne microscope systems

Several new scanning microscopic techniques have recently been developed which rely on modulation of the optical beam to enhance the imaging performance. These systems have the common feature that the image is formed by detection or demodulation of an a.c. signal. Techniques have been developed which are sensitive to both intensity and phase information in the sample. We refer to such microscopes as heterodyne imaging systems (and by analogy single-frequency microscopes, operating at d.c. only, are referred to as homodyne) although as we will point out this term is only strictly applicable to the interferometric-based methods. Although the theory for the conventional homodyne scanning optical microscope is well developed there has been no systematic study of heterodyne microscopic techniques, which is the purpose of this paper. Several techniques are discussed and compared and the different ways of extracting amplitude and phase information are considered in detail with reference to experimental systems which have been demonstrated to have good imaging performance.     

The use of a novel image-shearing technique on a scanning electron microscope for comparative measurement of linewidths on photomasks

A technique for measuring the widths of lines on chromium-on-glass photomasks is described which combines the resolution of the scanning electron microscope (SEM) with the accuracy of a helium-neon laser interferometer, and the setting sensitivity of image-shearing methods. Electron microscope image intensity profiles and the choice of signal which yields the best measurement repeatability are discussed. The technique described here yields measurements which are relatively insensitive to defocus and beam diameter.