bluuebunny/crossref_metadata_2025 huggingface dataset:
Is Low Accelerating Voltage Always the Best for Semiconductor Inspection and Metrology?
Authors: Postek M T , Vladár A E
Published: March 2018

Low accelerating voltage operation is an excellent mode of scanning electron microscopy and it is extensively used for measurements in semiconductor production. The beam penetration is small, and if properly applied, the specimen charging is kept at acceptable levels. But, is this always enough? Today, the scanning electron microscope (SEM) is being used in photomask metrology and imaging where charging is excessive. Charging is difficult to quantify and control as it varies greatly with instruments, operating conditions and sample. Therefore, it is also very difficult to model accurately. For accurate metrology charging must be overcome because the dynamic charging of the sample deflects the electron beam from its intended position and the intensity of the induced signal may vary uncontrollably. Deflection of the electron beam of even a few nanometers potentially results in a measurement error that is significant to modern semiconductor production.
Fluorescent Speckle Microscopy
Authors: Carmichael Stephen W , Lingle Wilma L
Published: March 2018

Whereas too much of a good thing can be bad, too little of a good tiling can be good. In this case, the accidental dilution of X-rhodamine tubulin injected into living cells resulted in a heterogeneous labeling of micro tubules, rather than visualization of continuous structures. This heterogeneous labeling rendered a speckled image, hence the term fluorescent speckle microscopy (FSM) was introduced. It turns out that FSM yields particularly useful information on dynamic events within living cells, for example the assembly and disassembly of microtubules.

There are two recent reviews of FSM by Clare Waterman-Storer and Gaudenz Danuser, one emphasizing the biologic applications of the technique, the other emphasizing the quantitative aspects. For their purposes, they defined a “speckle” as a diffraction-limited region of the image that is significantly brighter than its immediate environment. It can be calculated from the point-spread function (which is determined by the numerical aperture of the lens) that optimally a diffraction-limited region of about 250 nm can be imaged.
Digital Movies for Others to See
Authors: Sedgewick Jerry
Published: March 2018

In a departure from traditional articles on Photoshop, this article will provide a look at how to save movies for the purpose of distribution to Macintosh, Windows and Linux operating systems.

More than once, I have been asked to produce and help with the distribution of digital videos that rival those downloadable trailers for big Hollywood productions. I kindly inform the clients that I do not have a staff large enough to tweak each individual frame of their video, and that their expectation must go down a few notches. Upon seeing their baffled look, an explanation ensues, much like what follows:

First of all, digital video must be compressed in order to run seamlessly from a computer. Only the most robust computers can keep up with the number of frames flashing by per second (-30 frames for NTSC), and even these cannot play back for extended periods of time without the loss of some frames (what is called dropped frames in the digital editing world).
Use and Disposal of Uranyl Acetate in the Electron Microscope Laboratory: Glow in the Dark or Walk in the Park?
Authors: Tindall Randy
Published: March 2018

Let's face it—uranyl acetate is the perennial bad boy of chemicals, at least from the standpoint of disposal. A necessary evil in the electron microscopy laboratory, it presents a quandary for those trying to comply with hazardous materials regulalions, an effort complicated by its nominal radioactivity. Disposal costs of radioactive materials can be daunting. A past posting to the Microscopy Society of America listserver gave an estimate of US$7000 per liter and local follow up indicated that, although probably high, that may not be an unreasonable figure. In these times of diminished subsidies and expectations of increased cost recovery by academic service facilities like ours, this is of paramount concern.

The Electron Microscopy Core Facility at the University of Missouri-Columbia follows guidelines issued by our campus Environmental Health and Safety (EHS) office regarding hazardous materials and their disposal.
Confocal Microscopy for Diagnostic Cytology
Authors: Boon Mathilde E , Kok Lambrecht P
Published: March 2018

Laser scanning confocal microscopy has been used to analyze cytological specimens for research purposes. However, for many pathologists, its application in diagnostic cytology still is terra incognita. In our laboratory, confocal microscopy has been used for seven years, mainly to solve problems in cervical cytology, in breast aspirates, and for endometrium cytology.

In contrast to histology where we can cut new sections from the block, the cytologic sample is often limited to one smear. Accordingly, when we wish to expand our diagnostic possibilities, we have to extract the desired additional information from that single smear. This is possible by first de-staining the original smear and subsequently re-staining it. In this article, we describe how we restain old slides with the fluorescent dyes Ethidinm Bromide and Eosin, a DNA/protein stain, resulting in staining patterns comparable to the diagnostic Papanicolaou and Giemsa methods in that mere is an optimal distinction between nucleus and cytoplasm.
Rapid Cross-Section TEM Specimen Preparation of III-V Materials
Authors: Beanland R
Published: March 2018

Cross-section transmission electron microscope (TEM) specimen preparation of Ill-V materials using conventional methods can be a painful and time-consuming activity, with a day or more from receipt of a sample to examination in the TEM being the norm. This article describes the cross-section TEM specimen preparation technique used at Bookham Caswell. The usual time from start to finish is <1 hour. Up to 10 samples can be prepared at once, depending upon sample type. Most of the tools used are widely available and inexpensive, making the technique ideal for use in institutions with limited resources.
The Life and Death of a Tungsten Hairpin Filament
Authors: Chapman Steve
Published: March 2018

Many words have been written about filament saturation and filament life, not all with years of experience behind them] Sometimes when visiting laboratories one has the feeling that the life of the filament is far more important than the quality of the results, so let us try to demystify filament life?

The life of a tungsten hairpin filament very much depends upon its use and the applications present in a particular laboratory. Medturn to low magnification applications in the transmission electron microscope (TEM) may only require low emission currents (10 to 15 micro amps) with a long filament life resulting. High resolution studies require higher operating currents (20 to 45 micro amps) but, by demanding more emission current the life of the filament will suffer. Whilst a long filament life is good for the laboratory, it is not worthy of boasting, for those who have a short filament life may well be using the instrument near to the limit of its performance, a far more relevant boast!
Another Way to Implement Diffraction Contrast in SEM
Authors: Tao Xiaodong , Eades Alwyn
Published: March 2018

SEM users are familiar with two forms of contrast in SEM images: topographic contrast and atomic number contrast. We can now add a third form of contrast. Contrast can arise due to the different orientation of grains in the sample. However, in normal operation this con trast is very weak, since in the SEM the beam includes a range of incident angles. This has the effect of averaging out diffraction contrast from the different orientations of the grains. This contrast is generally much stronger when the trast is very weak, since in the SEM the beam includes a range of incident angles. This has the effect of averaging out diffraction contrast from the different orientations of the grains. This contrast is generally much stronger when the incident beam is an ion beam rather than an electron beam — contrast between the grains is strong in ion-beam images but not in normal SEM images.
Choosing a Cantilever for In Situ Atomic Force Microscopy
Authors: Woodward John T
Published: March 2018

What is the best cantilever for intermittent contact mode (often called Tapping Mode™) atomic force microscope (AFM) imaging under water? This is a question I hear often and one that recently generated some interesting discussion on an AFM newsgroup (more on the newsgroup below). The ability of the AFM to image samples En physiologically relevant environments has made it a popular technique in the biological sciences. However, because scanning the AFM tip in contact mode easily perturbs many biological samples, it was the advent of intermittent contact modes that lead to AFM's widespread use in biology.
Use of a New Imaging Technique to Document Deformations Recorded in the Environmental Scanning Electron Microscope
Authors: O'Neil Edward F , Jennings Hamlin M , Thomas Jeffrey J
Published: March 2018

The introduction of the environmental scanning electron microscope (ESEM) greatly Improved the functionality of scanning electron microscope (SEM) technology by allowing objects to be imaged in water-vapor under low vacuum, and without the need for coating to facilitate electrical conductivity. The low vacuum environment allows Imaging of delicate structures, such as needlelike crystals, without fear of damaging the object in the process of viewing it.
The Same Thing That Makes a Carrot Orange Also Makes a Molecular Wire
Authors: Carmichael Stephen W
Published: March 2018

The atomic force microscope (AFM) has been used in many ways to extract information from biologic specimens. Now Gerry Leatherman, Edgar Durantini, Devens Gust, Tom Moore, Anna Moore, Simon Stone, Ziniu Zhou, Peter Rez, YangZhang Liu, and Stuart Lindsay have used conduction atomic force microscopy (CAFM) to demonstrate that a molecule of carotene can function as a molecular wire.

Leatherman et al., synthesized carotene and then examined the molecules with the scanning tunneling microscope (STM), They could image spots that averaged 2 nm in diameter, although the size of the spots varied widely due to the geometry of the probe tip. Interestingly, the number of spots increased with the concentration of carotene, but the average size of the spots did not change. This made it clear that they were able to image carotene molecules with the STM, and that the carotene did not form large aggregates.
Choosing Your Tools©
Authors: Brinson Bruce E
Published: March 2018

The increasing availability of the user-friendly black box can present an increased probability of characterization errors. The uninformed choice (e.g., any SEM will see this) or dependence on one instrument can also lead to erroneous results. Failure to understand the limits and advantages of a particular instrument and bias through assumption or desired result can be equally deceptive.

In the example presented here, the objectives are to determine primary particle size and to identify the impurities in a specimen. We will look at the data produced by several instruments and discuss the ways each technology can support or mislead the analysts.

Images were acquired optically by secondary electron (SE), x-ray dot maps and back scattering electrons (BSE). The elemental analysis techniques used were Energy Dispersive Spectrometry (EDS), Wavelength Dispersive Spectrometry (WDS) and Powder x-ray diffractometry (PXD).

Field emission SEM (FESEM) and optical images are used.
Crystal Orientation Mapping of Copper Interconnects using EBSD
Authors: Dicks Keith
Published: March 2018

The semiconductor industry is committed to introducing copper interconnects in place of aluminum for the latest generation of semiconductor devices. With its greater current carrying capacity, the use of copper should enable further reductions in device geometry, power consumption and heat generation, and lead to higher performance and longer battery life in portable devices.

Grain orientations, crystal log raphic ‘texture’ trends and boundary types are crucial in determining properties such as electrical resistively, strength and corrosion resistance. These parameters may be implicated both in the performance of the device and in failure mechanisms, which are of great importance in determining reliability.

Our work demonstrates that high-resolution electron backscatter diffraction (EBSD) is now both possible and routinely achievable using both hot and cold field emission gun scanning electron microscopes (FEGSEMs). Our results demonstrate that EBSD can be successfully used to map grain orientation and to reveal and classify grain boundaries for this application.
Height Measurement of Interplanetary Dust Particles by Scanning Transmission Electron Microscopy (STEM)
Authors: Joswiak David J
Published: March 2018

Interplanetary dust particles (IDPs) comprise an important source of extraterrestrial materials available for study of our solar system and originate from either the asteroid belt or from short period comets. IDPs from cometary sources are particularly important as they constitute the only physical samples of comets available; all known meteorites are derived from the asteroids, the Moon or Mars. By measuring the densities of IDPs and using an appropriate atmospheric entry heating model, it is possible to determine whether an individual IDP has been derived from an asteraidal or cometary source region. Calculating the density of an IDP requires knowledge of both its mass and volume, which can be determined by using a combination of secondary and transmission electron microscopy techniques. We have developed methods to measure both of these parameters and thus routinely measure densities for individual IDPS in the size range of 5-15 μm.
Immunohistochemistry and Hematoxylin and Eosin on the Same Slide
Authors: Brooks Amos A
Published: March 2018

Small tissues are always difficult to work with. The limited size often leads to many technical problems. In spite of the care normally given to a specimen, the unthinkable can happen. A block may be cut through, slides may be broken, and sections may fall off. When this type of tragedy occurs, the creativity and technical abilities of a histology technician are often tested.

An example of this occurred when a technician was cutting a prostate biopsy. Two slides were taken, with two sections on each. The slides were taken, with two sections on each. The slides were stained with hematoxylin and eosin (H&E), and coverslipped with a Tissue Tek automatic coverslipper. This coverslipper uses a plastic film, which causes the film to adhere to the slide with xylene. The slides were then given to the pathologist who then requested an immunohistochemical (IHC) test to be performed. In the process, the specimen was exhausted.
How To Get Some Action (In Photoshop)
Authors: Sedgewick Jerry
Published: March 2018

All of us who work with images understand the enormous amount of time post-processing can require. Before you know it, a few hours can easily slip by even for the most seasoned among us. When so many other things need to be done, it can be frustrating to spend hours on image preparation.

Fortunately, some more frequent steps taken in Photoshop can be automated, and these can be assigned to one of the Function keys (F keys) at the top of the keyboard, or a droplet can be made (Photoshop 6x & 7x only).
Macro Imaging with Digital Cameras
Authors: Burnett Bryan , Blaauw Steven
Published: March 2018

Advances in charged couple device (CCD) design, low cast processor power, cheap memory and dropping prices of digital cameras over the last few years have made the CCD digital camera art attractive alternative to the film camera for many imaging applications. This is especially true in macro imaging where it appears likely that digital cameras will replace film cameras (curiously, Long (2001), says otherwise). As will be described here, a digital camera equipped with a quality macro-zoom lens, generates images with a depth of field (e.g., Fig. 1) that greatly surpass images produced by a film camera with a comparable lens system.
Rapid Preparation of a Polymer Fiber and a Free-Standing Polymer Film for Cross-Sectional Microtomy
Authors: Taubert Andreas , Ferris James H , Winey Karen I
Published: March 2018

Polymer films and fibers can readily be imaged in a “top-view“ mode by depositing the sample directly on a transmission electron microscopy (TEM) support grid. Sample preparation of thin polymer films and fibers for cross-sectional views in the electron microscope, however, is a major challenge. Owing to their small dimensions, the films or fibers cannot be mounted alone, because they will not remain immobilized In the microtome, even at low temperatures. Brittle polymers complicate sample preparation even further because they tend to break due to the mechanical stresses exerted on them by the microtome sample holder and the knife. Such sections often jump off the knife-edge or the sample film, thus preventing a systematic collection of thin sections. Similar arguments apply for cross-sectional atomic force microscopy (AFM).
Techniques for Combinatorial and High-Throughput Microscopy Part 1: Gradient Specimen Fabrication for Poiymer Thin Film Research
Authors: Sehgal A , Karim A , Stafford C , Fasolka M
Published: March 2018

Combinatorial and high-throughput (C&HT) approaches accelerate research by addressing multiple experimental parameters in a parallel or otherwise highly efficient fashion. First used by the pharmaceutical industry for product discovery, the C&HT paradigm is being extended to the study of complex materials systems that require measurements of properties and phenomena over a huge number of conditions. As with traditional materials science, microscopy and imaging of morphology are essential for C&HT materials research.
Preparation Of Soil Samples For Light And Transmission Electron Microsopy
Authors: Vrdoljak Gordon
Published: March 2018

Soil structure influences water supply to plant roots, aeration, water infiltration rates, suitability of soil medium for seed germination and growth, growth of plant roots, drainage, evaporation, mechanical strength, and workability (Dexter 1988). Adequate description of soil structure for cultivation, engineering, or remediation is typically done by light microscopy and transmission electron microscopy. Literature exists in numerous sources for preparation of soils for microscopy, but often preparation steps are left out due to the shortening of Methods Sections in journal articles to conserve print space. I present here, protocols I've used for preparation of tropical soils (Oxisols) for microscopy.
Cleaning a Cold Cathode Gauge Tube
Authors: Mills Owen P
Published: March 2018

This topic appeared on the MSA list server In December 2002 and January 2003. While cleaning my gauge tube recently, I documented the steps. In this article I describe cleaning a Varian 524-2 gauge tube. There are a number of other cold cathode and penning style gauge tubes on the market, Edwards CP-25 (old penning), CVC GPH-001A(old cold cathode), Pfeiffer IRK 250 (new cold cathode) to name a few. The cleaning steps are very similar. Consult the instructions that came with the gauge tube or visit the vendor's websites.
Correlative Electron Tomography and Elemental Microanalysis in Biology: A Preview
Authors: Marko Michael , Wagenknecht Terence , Mannella Carmen
Published: March 2018

The combination of electron tomography (three-dimensional reconstruction of an individual object from a series of EM tilt images) and elemental mapping (based on element-specific differences in interaction between the primary electron beam and the specimen) is at the very beginning of its application. This article reviews the first published results and considers some requirements and potential uses in biology, employing two examples from our own exploratory work.
An Innovative Method for Imaging and Chemical Analysis of Wet Samples in Scanning Electron Microscopes
Authors: Ruach-Nir Irit
Published: March 2018

Electron microscopy (EM) of fully wet samples is a valuable tool for studies in the material, medical and biological sciences. In order to appreciate the natural structures of tissues or materials they should be examined in their native wet state, as opposed to a dry form that incorporates artifacts of sample processing. Viewing and analyzing wet samples at high resolution has undergone a significant improvement only recently due to the innovative WETSEMTM technology developed by QuantomiX.
EBSD Sample Preparation: Techniques, Tips, and Tricks
Authors: Nowell Matthew M , Witt Ronald A , True Brian W
Published: March 2018

Automated analysis of Electron Backscatter Diffraction (EBSD) patterns for orientation imaging and phase identification in materials and earth sciences has become a widely accepted microstructural analysis tool. To briefly review, EBSD is a scanning electron microscope (SEM) based technique where the sample is tilted approximately 70 degrees and the electron beam is positioned in an analytical spot-mode within a selected grain. An EBSD pattern is formed due to the diffraction of the electron beam by select crystallographic planes within the material. The EBSD pattern is representative of both the phase and crystallographic orientation of the selected area. The pattern is imaged by a phosphor screen and recorded with a digital CCD camera and then analyzed.
Energy-Filtered Electron Holography
Authors: Herring Rodney A
Published: March 2018

When electrons pass through a material, they can pass through without losing energy such as elastically scattered electrons or they can lose or gain energy by inelastically scattering with the material's electrons. The elastically scattered electrons have been used in the simulations of lattice images, which are used to help determine the atomic structure of materials. Inelastically scattered electrons were ignored in the simulations because it was believed that they did not have the required coherence to interfere with themselves and they contributed only to the background intensity. Recently though, a great deal of interest has been generated in knowing whether the inelastically scattered electrons can also contribute to the lattice images in order to help explain the Stobbs factor [1], where the contrast in the lattice images is often three or more times less than the theory predicts. The Stobbs factor makes it difficult, if not impossible, to establish quantitative values to high-resolution lattice images.
Eucentric Stage for Recording Stereo Pair Photomacrographs
Authors: Clarke Ted
Published: March 2018

The stereo microscope is the most commonly used microscope. This applies to both the materials science and biological science fields. My past career as a materials engineer depended critically upon the three dimensional imaging of the stereo microscope for studying metal parts and fracture surfaces. Unfortunately our clients had no readily available way to view stereo pairs, so stereo pair recording was not done. Likewise, our electron microscopes were not equipped with costly eucentric stages, which make stereo pair recording easily done.
Microscopy in Costa Rica
Authors: Eades Alwyn
Published: March 2018

Costa Rica is amazing. You probably know that Costa Rica has no armed forces and that fully a quarter of its area is given over to national parks and other protected areas. You probably don't know that Costa Rica is also amazing for its electron microscopy. Almost anonymously Costa Rica has a center for electron microscopy on a par with centers in the United States or Europe.

El Centro de Investigation en Estructuras Microscopicas, CIEMIC (The Center for Microstructural Research) has its own purpose-built building with an infrastructure that would make most of us envious.
Microscopy and Microbes at Plum Island: Protecting America's Livestock
Authors: Burrage Thomas G
Published: March 2018

Plum Island Animal Disease Center, located on a small island off the coast of Long Island's North Fork, has been clouded in mystery and misinformation for years. Often the topic of conspiracy theorists, this secret place has generated many myths—from aliens to anthrax and pink eels to secret submarines. But the truth of the center's mission is far less colorful yet far more crucial to the state of the nation's agriculture.

In June 2003, operational responsibility for the Plum Island Animal Disease Center (PIADC) transferred from the United States Department of Agriculture (USDA) to the United States Department of Homeland Security (DHS), and PIADC became the core of the DHS effort to protect US livestock from foreign animal disease agents.
50th Anniversary Celebrations of Atomic-Resolution Imaging
Authors: Kelly Thomas F , Melmed Allan J
Published: March 2018

October 2005 marks 50 years since the first images achieving atomic resolution were obtained by Erwin Miiller and Kanwar Bahadur at the Pennsylvania State University using field ion microscopy. An image from that seminal work is shown in Figure 1. Two separate meetings were held this year to commemorate this important event in the history of microscopy; the 50th Anniversary of Atomic Resolution Microscopy, held June 15-17, 2005 at Penn State and the Golden Anniversary of Atomic Resolution Imaging, a symposium at Microscopy and Microanalysis 2005 in Honolulu held July 31 to August 4, 2005. These celebrations were timed to coincide also with the World Year of Physics 2005 http://www. wyp2005.org/.
The Microscopy Society Of America's Project MICRO: The Vermont Experience
Authors: Taatjes Douglas J , Schwarz Janet
Published: March 2018

Project MICRO (Microscopy in Curriculum - Research Outreach) is an initiative by the Microscopy Society of America (MSA) to connect scientists with middle school teachers in an effort to introduce young students to the scientific method. Through a collaboration with the Lawrence Hall of Science (LHS) at the University of California, Berkeley, a teacher's manual was produced as part of the LHS GEMS (Great Explorations in Math and Science) series. This manual, entitled “Microscopic Explorations”, can be used by scientists and middle school teachers alike to prepare a Project MICRO “Festival” to be presented in the classroom. Detailed information concerning Project MICRO in general, and the Microscopic Explorations manual can be obtained from the Project MICRO web page from MSA (http://www.msa.microscopy.com/ProjectMicro/PMHomePage.html).
Telepresence Confocal Microscopy
Authors: Youngblom J H , Wilkinson J , Youngblom JJ
Published: March 2018

The advent of the Internet has allowed the development of remote access capabilities to a growing variety of microscopy systems. The Materials MicroCharacterization Collaboratory, for example, has developed an impressive facility that provides remote access to a number of highly sophisticated microscopy and microanalysis instruments, While certain types of microscopes, such as scanning electron microscopes, transmission electron microscopes, scanning probe microscopes, and others have already been established for telepresence microscopy, no one has yet reported on the development of similar capabilities for the confocal laser scanning microscope.
LR White Flat Embedding Of Cells Grown On Coverslips
Authors: Howard Tamara
Published: March 2018

We do this two different ways, using polyethylene (PE) molds or aluminum weigh boats, depending on the sample, You can either heat- or UV-cure these molds-just change the type of “lid” on the embedding mold.

The PE molds are used when the coverslip can be made to fit in the mold. We use JB-4-type molds, available from your favorite microscopy supplier. The key to using these molds is that they have to be pre-treated...fill the inner cup of the mold with LR White, cover with an aluminum JB-4 chuck (try to have enough resin in the mold to come up around the base of the chuck), and polymerize in a 60° C oven for a day or two, until the resin is hard.
Removing Halftone Patterns From Scanned Images
Authors: Russ John , Russ Chris
Published: March 2018

Using illustrations from published technical articles is part of many lectures and presentation citations, and given the availability of flat bed scanners, should be easy to accomplish. The problem that remains is removing halftone patterns and other periodic noise that result from printing and scanning technology. Practically all magazines and newspapers are printed using a regular halftone pattern that uses an array of dots varied in size to produce the visual illusion of continuous gray scale. Color images are usually printed with three, four or even more such patterns using different colored inks and different pattern orientations. Scanning such images into a computer can introduce a further pattern due to the moire interference between the printed pattern and the spacing of the sensors in the scanner. Such patterns are also characteristic of images obtained from single-chip video cameras because of the color filters present in front of the light sensors on the chip.
Microwave Immunohistochemistry on Mohs Cryostat Sections
Authors: Miller Diane G
Published: March 2018

With the advent of Mohs surgery in the dermatology clinics, making sure the margins are clear of any cancer is of the utmost importance to both patient and dermatopathologist. While the patient is still in the surgery suite, immunohistochemstry staining can be done in the laboratory microwave (Pelco microwave with water load cooler, wattage controller and temperature probe). The immunohistochemistry procedure takes only 25-30 minutes as compared to the more conventional methods that take 2 hours or more.

The microwave needs to be checked for hot spots using the Pelco #36140 microwave bulb array. Make sure the area in which you are going to put your slides has no hot spots, indicated by no illuminating bulbs. Using a Sigma Diagnostics #H6644 Humid Chamber, place a paper towel in the bottom of the chamber and fill with 1/4 inch of water. This is in addition to the water loads used to eliminate hot spots.
The Structural and Chemical Analyser – A New Analytical Technique for SEM
Authors: Brooker AD , Prusnick T , Jarvis RM , Goodacre R , Bennett R , Dawe CJ , Leak DJ , Hill MR , Lainchbury MJ
Published: March 2018

Renishaw's structural and chemical analyser (SCA) provides new analytical techniques for scanning electron microscopes (SEM). For many years energy dispersive X-ray analysis (EDS) has been the default in-SEM analytical technique, and whilst it can provide fast and accurate data from any samples, it can only provide elemental information about the sample being analysed. For materials applications, assumptions about stoichiometry may enable accurate characterisation, but in most Instances “chemistry by guesswork” does not provide an acceptable solution. Furthermore, EDS is rather insensitive to light elements - atomic number 11 (sodium) and below, which severely limits its applicability to organic chemistry and life sciences.
Three-dimensional Volume Reconstructions Using Focused Ion Beam Serial Sectioning
Authors: Dunn DN , Hull R
Published: March 2018

Traditionally, focused ion beam (FIB) microscopy systems have been used as instruments to repair integrated circuits, isolate defects or prepare mateiials for characterization with other techniques. Recently however, FIB microscopy has been used extensively as a standalone characterization technique. Typically, FIB microscopy can be used to probe site specific areas of a sample through secondary electron and ion imaging, energy dispersive x-ray analysis (EDX), or secondary ion mass spectroscopy (SIMS). These analyses will often combine two-dimensional elemental mapping or sputter depth profiles with imaging to investigate spatial variations of chemical and imaging signals. Inherently, these investigations are confined to two dimensions but often, complex materials problems are more easily understood if chemical and geometric information can be collected.
Advanced Confocal Microscopy An Essential Technique for Microfluidics Development
Authors: Lundy Terence
Published: March 2018

Many believe that microfluidics has the potential to do for chemistry and biology what the integrated circuit has done for electronics — integrating tremendously complex chemical and biological processes into simple easy-to-use devices that will eventually pervade our lives. While microfluidics has made great progress in the last decade — addressing many of the fundamental questions related to manipulating nanoliter volumes of chemicals and solutions — it still faces some very basic challenges as it moves out of the laboratory and into use. Perhaps most basic is the need for fast, accurate characterization of the size and shape of the microfluidic devices themselves. Conventional imaging and measurement techniques have proven adequate for initial development, but are unable to provide the speed and accuracy needed to support the continued development of microfluidic technologies.
The Staining of Polymers II
Authors: Smith Ronald W , Bryg Victoria
Published: March 2018

E.H. Andrews [1] was the first to produce transmission electron micrographs that differentiated between crystalline and amorphous forms in long chain polymers. Working with thin cast films of cis-polyisoprene {natural rubber (NR)}; he examined them in the unstrained and strained states. Staining the unstrained thin films with osmium tetroxide vapors, he showed that spherulitic crystals were in light contrast whereas the darker contrasted matrix was the amorphous continuum (Figure 1).
Microscopes Reveal Prehistoric High Technology
Authors: Carmichael Stephen W
Published: March 2018

Since the beginning of human civilization, people have apparently been fascinated with the reflection of light from surfaces. The creation of shiny surfaces extends from 4,000 B.C., when Neolithic farmers in ancient China polished jade objects, to the present day where modern polishing techniques are key to creating flat surfaces in the fabrication of electronic microcomponents. The question we are posing here is how does one determine when high technology was introduced in the process of polishing? Recently, Peter Lu, Nan Yao, Jenny So, George Harlow, Jianfang Lu, Genfu Wang, and Paul Chaikin offered an answer to this question. Interestingly, microscopes were used to provide the answer.
The Value of S/TEM: Matching Solutions, Applications, and Economics
Authors: Hubert Dominique
Published: March 2018

A new generation of scanning/transmission electron microscopes (S/TEM) offers a broad range of capabilities and dramatic improvements in usability, challenging the conventional perception of these techniques as needlessly difficult and expensive. Improvements in usability address both the operation of the instrument and the interpretation of results, while the cost of the analysis must be evaluated relative to the value of the information it provides—a value that has appreciated significantly with the burgeoning growth of the nanotechnology sector. A million dollar instrument is a bargain if it solves a multi-million dollar problem. Moreover, the broad range of capabilities available in current generation instruments makes careful matching of the cost of the capability with the potential value of the solution the key to sound economic choices. This article reviews the factors that typically drive TEM costs and provides examples of valuable matches in a number of applications.
Overcoming Severe XEDS Peak Overlaps with the AEM
Authors: Walck Scott D
Published: March 2018

Of all the analytical techniques in electron microscopy, X-ray energy dispersive spectroscopy (XEDS) is perhaps the most useful. It provides a quick identification of the elements and even with semiquantitative methods; a reasonable composition can be obtained. However, in the scanning electron microscopy (SEM), there are materials systems in which severe peak overlaps of heavier elements L and M lines cannot be easily deconvolved with lighter elements' K lines. In addition, without a sufficient overvoltage in the SEM, even identification of the heavier elements can be difficult. In the analytical electron microscope (AEM), there is always sufficient overvoltage to excite all of the elements' K-lines. However, all of the K-lines might not be able to be detected with commercially available instruments. This is illustrated in Fig.l where the maximum energy of the detector system might be set to 10, 20, or 40 keV.
Rhodamine Fluorescence After 15-year Storage in Methyl Salicylate
Authors: Philip L. Hertzler
Published: March 2018

Fading of fluorochrome is a significant limitation to fluorescence microscopy. Several anti-fade agents, e.g. n-propyl gallate, are commonly used for glycerol-based mounting media (Longin et al., 1993; Ono et al., 2001). Samples mounted in glycerol must be kept at -20°C for long-term storage to prevent bacterial degradation. In contrast, fluorescent samples cleared and mounted in organic media can be stored indefinitely at room temperature.

Methyl salicylate or oil of wintergreen is an excellent clearing agent (refractive index = 1.53), which works well with a variety of fluorochromes. It has a pleasant aroma but is somewhat difficult to work with since it remains liquid after mounting. It was previously reported that shrimp embryos labeled with tubulin antibody and rhodamine-conjugated secondary antibody maintained their fluorescence after six months (Summers et al. 1993). These same samples, stained in November, 1990 and imaged by confocal microscopy for publication in Hertzler and Clark (1992), are still fluorescent after continuous storage in methyl salicylate at room temperature in the dark (Figure 1). The images of 62-cell stage shrimp embryos taken from these 1990 samples were collected with an Olympus Fluoview 300 laser scanning confocal microscope in January, 2006 in the Dept. of Biology, Central Michigan University.
When Point To Point Is Not Enough
Authors: Heckman Carol , Cayer Marilyn , Varghese Mita
Published: March 2018

As all microscopists are taught, resolution can be measured by knowing the closest distance between two points that can be discriminated in the image. There are some occasions when we want to measure something in a digital image, and the nominal resolution isn't sufficient for the task. One of these, extracting a contour from the image, was summarized in a previous research report. The principles would be valid for any filled area, such as a particle or structure from an X-ray dot map. The ground rule was set that the outermost pixel would be selected at every point on the contour. It was clear from the geometry shown in Fig. 1 that only values of 0°, 45°, 90°, 135° and 180° could be measured. Because of the stair step defect, tracing the sequence of pixel locations only gave offsets to 8 pixels, one directly below, two on the corners below, three pixels in the same location above, and one pixel on either side of the subject pixel. Therefore it was impossible to measure the true curvature of the contour.
What Happens When the Electron Beam Hits the Specimen
Authors: Tivol William
Published: March 2018

When an electron passes through matter, the energy of the electron is transferred to the material in the form of ionizations and excitations. Almost all of this transfer is the result of the Coulomb interaction of the beam electrons with the electrons in the specimen; that is, scattering of beam electrons by nuclei is not an important energy-transfer process.

On average, about 30 eV is transferred for each ion pair (the electron and the parent atom) produced, and most of the energy transferred goes into primary or secondary ionizations. In a typical energy-loss process, an electron can be removed from an inner shell (it can leave the atom with a velocity comparable to that of the incident electron).
Freeze Fracturing of Hot Samples
Authors: Rades Thomas
Published: March 2018

Liquid crystals are substances which combine properties of both liquids and solids. To investigate the structures and slructurai defects of liquid crystals, freeze fracturing of these gel-Like substances has been performed successfully for many years. In the TEM the structures of the different types of liquid crystals have been investigated and the results confirmed the earlier investigations using polarizing light microscopy. These TEM studies extended the knowledge of structures and structural defects of this fascinating state of matter. However, freeze fractures were only carried out on liquid crystals which form around or below room temperature.
Using Projection X-ray Microscopy to Observe the Solidification Phenomena in Metals
Authors: Kaukler William F
Published: March 2018

This article outlines how projection x-ray microscopy can be used to view the structures that develop within solidifying metals during the freezing process (in real-time and in situ), A description of x-ray microscopy methods will be given. The specialized furnace design and techniques required to permit observation of solidification phenomena will also be described. This forms the outline of the salient features of our X-ray Transmission Microscope (XTM) for solidification studies. This instrument is being developed for microgravity materials research at NASA's Space Science Laborataory in the Marshall Space Flight Center, Alabama. Finally, a sampling of radiomicrographs taken during alloy solidification will be presented.
Principal Components Analysis of Multispectral Image Data
Authors: Neal Brent , Russ John C
Published: March 2018

Principal components analysis of multivariate data sets is a standard statistical method that was developed in the early halt or the 20th century. It provides researchers with a method for transforming their source data axes into a set of orthogonal principal axes and ranks. The rank for each axis in the principal set represents the significance of that axis as defined by the variance in the data along that axis. Thus, the first principal axis is the one with the greatest amount of scatter in the data and consequently the greatest amount of contrast and information, while the last principal axis represents the least amount of information.
Directly Reading The Genome
Authors: Carmichael Stephen W , Maihle Nita J
Published: March 2018

The code for the human genome is essentially complete, opening an expansive book about our biologic composition. But the problem is to read this book. The font size is very small, What you are reading now is 9 point font, and genetic information would correspond to approximately 10-50 point font, six orders of magnitude smaller. There are methods to derive genetic information, but recently Adam Woolley, Chantal Guillemette, Chin-Li Cheung, David Housman, and Charles Lieber have developed an elegant way to directly read genetic information with a microscope. The microscope they used was an atomic force microscope (AFM) that employed a single-walled carbon nanotube as its probe.
Programmable Array Microscopes
Authors: Hanley Quentin , Heintzmann Rainer , Arndt-Jovin Donna , Jovin Thomas
Published: March 2018

The programmable array microscope (PAM) is a powerful tool combining the capabilities of nearly all previously described optical sectioning techniques in a single microscope. Not only can the user create optical sections of threedimensional objects, but the PAM's unique adaptive optical strategy allows a user to select the best sectioning method for a particular sample or experimental need. The key to the PAM is a spatial light modulator (SLM). This device, when placed in the image plane of a microscope, can be used to create optical sectioning, generate spatial encoding masks, and/or define regions of interest.
Resolution of Digital Photomicrographs from Scanned Film
Authors: Clarke Theodore M
Published: March 2018

Some may have read my previous articles in Microscopy Today and know that I am a strong proponent of digital imaging for photomicrographs and photomacrographs. I require that the digital images match the resolution and field size of traditional film images, with the 4X5 Polaroid film most commonly used in metallurgical laboratories where I worked before early retirement. My development of an affordable universal student microscope for home microscopy resulted in a need for color digital images that would meet my requirements at an affordable cost. This need was met by recording on 35 mm film with subsequent scanning of selected film images to a Kodak Master Photo CD. More recently I purchased a CanoScan FS2710 film scanner for digitizing my family's collection of slides and negatives for saving on CD's.
Double-Coating Of Three-Dimensional SEM Specimens
Authors: Jeffree Chris
Published: March 2018

Many of the SEM specimens which we are asked to deal with are highly three-dimensional non-conductive objects, such as silica beads, pollen grains and spores, small inorganic and organic crystals such as zeolites and drug preparations, starch grains, etc. These often cause severe charging problems which result in all of the familiar and tiresome charge-related image artefacts, such as flare, excessive contrast, specimen movement, and variable scan lines. Charging can also make it very difficult to obtain high-resolution images or do satisfactory analysis.
Cryostat Sectioning of Undecaicified Mouse Appendage Joints for use in Immunohistochemical Staining Procedures
Authors: Plummer Timothy B , Ziesmer Steven C
Published: March 2018

Immunohistochemical analysis was required to identity lymphoid cell subtypes in mouse appendage joint synovium tissue. Hamster and rat monoclonal antibodies directed toward lymphoid cell subtypes were found to be nonreactive in formalin-fixed decalcified appendages, and this necessitated the use of cryostat sections for immunohistochemical procedures. Initial attempts to section mouse joints using conventional cryostat techniques did not yield adequate sections, and it was clear that a different approach was necessary.
Casting Cells
Authors: Carmichael Stephen W
Published: March 2018

It's always refreshing to read about two old techniques being combined in a novel way to yield new information. Osamu Ohtani and Yuko Ohtani recently accomplished this. Louis Ranvier probably introduced the use of silver nitrate as a histology stain over a century ago. Whereas Ranvier is best known for describing nodes along the nerve fiber, he also demonstrated that silver nitrate stained endothelial cells, allowing their borders to be clearly visualized. In the 1970's, Takuro Murakami first used the scanning electron microscope (SEM) to examine corrosion casts of the vasculature. Much of what we currently know about the microarchitecture of blood vessels has been discovered using this technique.
Microscopy for Children: A Bibliography Update, April, 2001
Authors: Schooley Caroline
Published: March 2018

One of Murphy's Laws says that whenever something is published, it is immediately out of date. Project MICRO'S “Microscopy for Children: a Bibliography”, which was distributed with the December issue of this publication is no exception. I have been contacted recently about a few CD-ROMs that will interest many readers. Since the web version of the bibliography (URL below) will not be updated for a while, I am providing listings as follows.
Notes On Vacuum Techniques For Microscopists
Authors: Walck Scott D
Published: March 2018

This article was originally written to respond to a discussion on the Microscopy Listserver found on the Internet concerning backfilling an electron microscope with nitrogen versus laboratory air. It was expanded to include general vacuum comments, suggestions, and techniques that could be of possible value to the working microscopist.