bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase Measurement in Electron Microscopy Using the Transport of Intensity Equation
Authors: Ishizuka Kazuo , Allman Brendan
Published: March 2018

Although many samples in electron microscopy are phase objects, as in the case of optical microscopy, we cannot directly measure phase modulation by microscopy. Dennis Gabor proposed a technique called in-line holography to record both amplitude and phase information at the rather early stage of electron microscopy (in 1947). With the development of highly coherent field emission electron sources, another type of holography, off-axis holography, became available for electrons. However, holography cannot be applied to general cases, since there is a requirement for a vacuum region where the reference wave passes through.
Making Anaglyphs in Photoshop
Authors: Sedgewick Jerry
Published: March 2018

In order to achieve a three dimensional appearance to a pair of two dimensional images, two off-axis images can be produced and colorized. These can be overlayed slightly apart and then viewed through glasses with two differently colored sides, one color for the left eye and another for the right eye in combinations containing red, green or blue colors. These off-axis and colorized images are referred to as anaglyphs.

Off-axis images can be achieved through the use of a tilting stage on a microscope, by physically changing the position of a camera in relation to a still object, or through changing the axis of an optical stack of sections, such as what is created by confocal/CT scans. Some images lend themselves more to a 3D look both by virtue of inherent three dimensionality limited by the resolution of the imaging system.
Virtual Electron Microscopy for Undergraduate/Graduate Classes
Authors: Humphrey Elaine
Published: March 2018

How do you give 1150 undergraduates in an introductory cell biology course (Biology 200) access to electron microscopes? Students often ask if they can see the electron microscopes. They often ask if they will have an opportunity to learn EM. As part of the course material, students are expected to recognize the images produced by different EM techniques, know the advantages and disadvantages of these techniques and interpret 2-dimensional micrographs in 3 dimensions. This is akin to teaching the theory of baking bread without ever smelling it in the oven. A pilot project was designed to address getting round the impossibility of bringing 1150+ students into the BioImaging Facility. This initiative gave selected undergraduates a hands-on experience of SEM in the BioImaging Facility and allowed the rest of their classmates to share that experience.
Behavior of Particle Size Distributions, Means and BET Values in Ideal and Non-Ideal Morphology Systems in a TEM
Authors: Beauregard Paul
Published: March 2018

Historically, one of the first jobs performed by the new instrument called a “transmission electron microscope” (TEM) was the determination of particle size and distribution. Particle size is still important in controlling the properties of sub-micron and nanoparticle products. The width and average particle size can determine the light scattering properties of a pigment like TiO2, for example. The cost of installing a TEM facility and TEM sample preparation issues makes TEM analysis expensive and labor intensive. So decades ago, nitrogen BET (Brunauer, Emmett, and Teller) surface area determinations were used on sub-micron powders and pigments to minimize these costs. The BET measurement of the exposed external surface area of a material is made by detecting and measuring the amount of nitrogen given off after absorption, assuming one atomic layer of nitrogen is adsorbed.
The Year That Was! Microscopy in Southern Africa
Authors: Harmsen Luc
Published: March 2018

If we look back at the year 2003 it becomes quite clear that the South African microscopy year was quite unique. …Of course, I can't speak for all the companies and laboratories in the country, but from my personal view it was definitely interesting.

FEI definitely had a great year in that they managed to sell 5 systems. Then LEO added to that another 3, and we have heard that there is also a Cameca probe being bought. This may not sound like a lot, but if you take into consideration that in the last few years an average of 4-5 EM columns per year would register as a banner year. Given that we have seen our currency improve over the past 2 years, it does imply that buying imported equipment is getting cheaper. However as this effect has also negatively affected exports, we can't say that we have become richer, and so could afford more systems.
Sub-Ångstrom Resolution with a Mid-Voltage TEM
Authors: O'Keefe Michael A , Hetherington Crispin JD , Chris Nelson E
Published: March 2018

The One-Ångstrom Microscope (OÅM) project was established at the NCEM to produce images at sub-Ångstrom resolution (O'Keefe, 1993). The project was implemented using a Philips CM300FEG/UT with hardware modifications designed to correct objective lens three-fold astigmatism and extend information transfer to 0.8 Ångstrom (O'Keefe et al., 2001a). A Gatan image filter (GIFTM) was used to bring the image magnification to more than three million times at the CCD camera to provide adequate real-space sampling.

Phase-contrast imaging in the HRTEM produces images with peaks at atom positions by extracting the spatial distribution of the relative phase from the electron wave. Usually, the electron wave is imaged by direct interference of diffracted beams at optimum focus (Scherzer, 1949).
Viability and Versatility of the Yeast Cell
Authors: Henry-Stanley Michelle J , Wells Carol L
Published: March 2018

Yeasts are single-celled eukaryotic microorganisms (generally about 5 to 10 microns in diameter) that divide by a budding process and are classified with the fungi. Yeast cells are ubiquitous in our environment and can be found on plants and in soil and water. Yeasts have considerable importance Ln industrial and agricultural settings, Saccharomyces cerevisiae (Figure 1) is also known as “bakers yeast” or “brewers yeast.” Specific strains of yeast are used to make pastries, bread, beer, ale, wine, distilled spirits, and industrial alcohol. In the paper industry, Candida utilis is used to break down die sugars from processed wood pulp. Yeast cells are also nutritious. In some societies, “cloudy” beer (containing yeast cells) provides essential B vitamins, minerals, and amino acids.
Secondary Melt Ornamentation on Westwater, Utah Microspherules: Evidence of an Extraterrestrial Provenance
Authors: Fandrich J W , Dujay Richard C
Published: March 2018

Microspherules (MS) ranging in size from 50μm to 425μm were discovered at Westwater, Utah in 1999. Examination of these particles using a scanning electron microscope identifies certain physical features that support an extraterrestrial (ET) provenance for these objects. Energy dispersive spectrometry (EDS) was used to determine the qualitative chemistry of these particles.
Inspecting Surfaces With a Sharp Stick: Scanning Probe Microscopy - Past, Present, and Future
Authors: West Paul
Published: March 2018

With the growing emphasis on nanotechnology, scanning probe microscopy (SPM) is emerging from the surface science laboratories and becoming a mainstream inspection and metrology tool along side optical and SEM microscopes. Scanning probe instrumentation and applications evolved dramatically during the past quarter-century (Table I). By 1998 SPM-related papers were being published at the rateof nearly 5000 per year Here we review the history of scanning probe microscopy, describe its current role as a critical enabler in nanotechnology, discuss why it has become a routine laboratory tool, and present a view of future directions for this advanced technology.
Setting White, Black and Gamma on Continuous Tone Grayscale and Color Images using Photoshop
Authors: Sedgewick Jerry
Published: March 2018

The contrast range of many digital images needs to be reset before outputting images to various ends. This is especially important when printing to paper because ink absorbs into papers through a capillary action, most in the cheapest papers (such as newspaper) and least in high gloss paper from slick magazines. The capillary phenomenon is known as dot gain In the printing world, since images are redefined as small dots when printing to publications, The result of dot gain is seen most in a loss of details or muddiness in the shadow or the black region of images.
Nutlet Morphology and the Use of SEMs to Determine Characteristics for Identification of Species in the Genus Cryptantha Lehm. ex G. Don Section Oreocarya (E. Greene) Payson
Authors: Murphy Willow B , Kelley Walter A , Dujay Richard C
Published: March 2018

The genus Cryptantha Lehm ex G. Don section Oreocarya (E. Greene) Payson of the family Boraginaceae presents some problems to botanists, both professional and amateur, in the keying and identification of species. The genus contains approximately 150 species, the section about 60 perennial and biennial herbs located generally in western North America. Identification has presented some taxonomic difficulty due to the variability and lack of distinctive vegetative characters. Botanists have turned to the nutlet (fruit) and flower morphology to aid in identification for precise specific differentiation. In the past, 10X magnification and a decent botanical illustrator were required to provide the illustrations necessary to assist in this identification. We are in the process of collecting micrographs of nutlets (dorsal, sagital, and ventral views) and developing a webpage containing these micrographs along with descriptions of their morphological variations.
Media-Agarose Slides: A Mounting Method for Observation of Living Cells
Authors: Merla Alia
Published: March 2018

Often during light and fluorescent microscopy, one wishes to observe samples of cells (e.g., yeast) grown in liquid culture. This is sometimes difficult as the cells can move around under the coverslip and a wet mount can dry out, killing the cells. Protocols in which the cells are resuspended in molten agarose results in cells that are in many different focal planes and often stressed by the high temperature of the molten agarose. This protocol describes a method of depositing small pads of growth media containing agarose onto slides, which eliminates these problems. Choice of growth media depends upon both the growth requirements of the cells and the requirements of the microscopy method. For example, yeast synthetic media works better for fluorescent microscopy than yeast rich media, because the yeast extract in rich media is autofluorescent in the green (fluorescein/GFP) channel.
Coating Effects On BSE Imaging
Authors: White Woody
Published: March 2018

Poorly conducting specimens can be examined without coating by using a variable pressure SEM. However, many labs may only have a high vacuum SEM, or for other reasons, choose a high vacuum mode. In order to examine insulating specimens in a high vacuum SEM (using operating conditions conducive to BSE and X-ray analysis) specimens must be coated with a conductive thin film.

The perspective of this article is from a materials point of view, but the principles remain the same for biological examinations requiring similar information.

Back Scattered Electron (BSE) image contrast is primarily a function of the average atomic number of an imaged area. This is particularly true for polished specimens where there is no topography to contribute to contrast. The BSE coefficient is the ratio of back scattered electrons to incident (beam) electrons.
Expiration Dating Of Chemicals And Reagents
Authors: Kline Rande
Published: March 2018

With today's requirements for chemicals and reagents in research and clinical laboratory environments, questions often arise concerning expiration dating. Why are not all chemicals and reagents expiration dated by the manufacturer when they are made? How should expiration of chemicals and reagents not dated by the manufacturer be determined? How would a laboratory establish expiration of solutions and reagents prepared in-house?

To assure good manufacturing practices, many manufacturers comply with guidelines set for them by agencies such as the International Organization for Standardization (ISO) and are inspected by ISO auditors. Some manufacturers are required to comply with the Food and Drug Administration (FDA.) These manufacturers are known as GMP facilities (good manufacturing practices) and are assessed by FDA inspectors. Like regulatory agencies who govern clinical sites, ISO and FDA require manufacturers to establish uniform quality systems for product testing and certification.
Imaging of Big and Messy Biological Structures Using Electron Tomography
Authors: Sosinsky Gina E , Martone Maryann E
Published: March 2018

Any student of cell biology cannot help but be impressed with the exquisite organization of cellular and subcellular compartments, where Individual proteins and even nucleic acids are targeted to specific domains and held together in macromolecular assemblies to achieve orderly and efficient interactions. Perhaps nowhere is the specialization of cellular compartments more apparent than in the nervous system, where the cell contains multiple, well-defined functional domains, each with its own unique biochemical character.
Converting Right-Left Stereo Pairs into Colored Pairs for Electronic Presentation
Authors: Tengowski Mark W
Published: March 2018

Since Microsoft PowerPoint has become the dominant scientific meeting presentation format, my collection of right-left stereo slide pairs has basically been rendered obsolete. Yet, contained within these striking images are valuable scientific data. This communication describes an easy method whereby analog slide data (i.e. scanning electron micrograph RL pairs, Figs. 1 & 2) are transformed into an electronic format such that the need for projector polarizers is replaced by inexpensive red-green or red-blue 3-D glasses.
An Aberration Corrected (S)TEM Microscope for Nanoresearch
Authors: Kujawa S , Freitag B , Hubert D
Published: March 2018

The continued focus on improving materials, combined with the fact that it is now commonly understood that material properties are affected by characteristics at the atomic level, give rise to the need to characterize and image at the best resolutions possible. The (Scanning) Transmission Electron Microscope ((S)TEM) has the capability to image structures with atomic resolution and provides, at the same time, information on the chemical composition, bonding and electronic structure of the material. The nanoresearcher's continued need for the ultimate resolution has accelerated the development of next generation electron optics and technology.
Getting the Green Light to Measure Small Forces
Authors: Carmichael Stephen W
Published: March 2018

Green fluorescent protein (GFP) and related molecules have been important signal molecules for cell biologists because they emit a bright signal to mark the position of tagged structures as well as changes in calcium and proton concentrations. Related molecules even emit in different colors. Now Hendrik Dietz and Matthias Rief from the Technical University of Munich have given us a detailed look at the "energy landscape" of GFP with the promise that it may also be useful indicator for forces.2 In this context, energy landscape refers to a three-dimensional map of the energy levels of the molecule as it is transitioned from its native state to an unfolded state.
Integrating Light and TEM Information with F-TEM images
Authors: Sims PA , Lockwood CA , Hardin JD
Published: March 2018

Fluorescent fusion proteins are widely used to visualize the localization of proteins in worms, fish, flies and tissue culture cells. We have used two different methods that use high pressure freezing (HPF) combined with correlative light microscopy (LM) and TEM. The first method uses fluorescence from live organisms immobilized in agarose followed by HPF and standard freeze substitution in dry solvent with osmium. This pre-embedding method optimizes ultrastructural preservation. A second, post-embedding method preserves fluorescence and immunoreactivity from embedded and polymerized thin sections. Here we describe post-embedding fluorescent integrated TEM images (F-TEM).
A Novel FIB Method for Preparing Three Dimensional TEM Specimens
Authors: Wang Nathan
Published: March 2018

Presently, there is an increasing demand for multi-view TEM observation of semiconductor device failure locations where the feature size of interest is smaller than the specimen thickness, and where the thin section has other features that may have high Z materials that obscure the site of interest in the TEM. Therefore, different viewing angles are necessary — such as a cross-section view of a defect-containing planar thin specimen. A major obstacle for the preparation of three dimensional specimens of this nature is specimen preparation. Great care must be taken when preparing a thin specimen from an already thin existing TEM specimen.
ASTM Standards in Microscopy
Authors: Friel John J
Published: March 2018

ASTM International publishes many standards specifically about microscopes and using microscopy. The value of these standards falls into three distinct categories. First, they can be a quick tutorial on how to perform some operation. E 1508, "Standard Guide to Quantitative Analysis by EDS" is only eight pages long and falls into this category. Second, they can be used to standardize a test and reporting method. The methods described in E 1382, "Standard Test Methods for Determining Grain Size by Semiautomatic and Automatic Image Analysis" are examples of procedures that have been agreed upon for many years.
Very Cool Clathrin
Authors: Carmichael Stephen W
Published: March 2018

Clathrin-coated vesicles are the shuttle containers within cells. The vesicles carry lipids and proteins between membrane-bound compartments. Clathrin forms a cage-like structure around the membrane-bound vesicle that is pinched off from the plasma membrane (in endocytosis) or a membranous component of the cytoplasm. Clathrin recruits cargo that is within a vesicle through intermediary proteins known as adaptors that help select membrane-anchored protein and form an interface between the clathrin cage and the membrane bilayer.
Metallography for the European Copper Age: Research on the Axe-Blade of the Glacier- Mummy from the Ötztaler Alps in Tyrol
Authors: Sperl Gerhard O
Published: March 2018

The beginning of metallurgy in the Old World is characterized by hammering native metals such as gold, copper and meteoric iron. Owing to the need of annealing the metal, for softening it after cold working, pyrometallurgy, the use of fire for producing metals from ores, could have been found by trial and error. Parallel to the rise of metallurgy is the use of a campfire (low temperature: max. 800°C) for baking clay-objects, which also seems to be an additional origin of metallurgy. The very first piece of molten copper-ore, dating back to the 7th millennium BC, was found in Catal Hoyiik, Turkey, together with hammered native copper and beads made of galena (PbS), initially mistakenly thought to be metallic lead.
LN2 Handling—Fact and Fiction
Authors: Compton Bob
Published: March 2018

“I will be getting my first SEM/EDS shortly, and to obtain the required operating permit from my safety group, I need a written procedure for handling LN2. Specifically I need a written procedure for filling the 3 L dewar on the EDS detector from a 50 L dewar mounted on a cart. I plan to make the transfer by using a lab source of LN2 to pressurize the 50 L dewar and have obtained all the valves and fittings required to do this from another SEM lab. I would appreciate copies of the procedure.”

This question recently posed to the Microscopy List Server unleashed a flurry of comments, not only on the safety of various LN2 procedures but on the safety of other materials as well. Also discussed was the necessity of formal LN2 handling procedures.
Philadelphia College of Osteopathic Medicine Examines Embryonic Cells to Find the Origin of Muscles With Image Analysis Software
Authors: DeSandies Kisha D , Madden Gabriella
Published: March 2018

Tens of thousands of Americans suffer from muscular dystrophy (MD), a disease that gradually deteriorates a person's skeletal muscle. While there is no effective cure, scientists know MD is caused by a genetic defect and are searching for treatments that will stop or retard the deterioration of muscle. In June, American and British researchers announced the success of a long-term treatment that repairs the genetic defect in Duchenne muscular dystrophy, the most common childhood form of MD.

Last fall, these researchers injected a short strand of nucleic acid into the shin muscle of a six-week old golden retriever - which had a genetic defect that leads to Duchenne MD in dogs - in order to maintain normal levels of dystrophin, the muscle protein missing in Duchenne MD. Their goal was for the nucleic acid to trigger the dog's system to correct the genetic defect. Eleven months later, the injected muscle continues to show normal levels of dystrophin.
Advantages of Adobe Photoshop Elements 2.0* over the full version of Photoshop*
Authors: Sedgewick Jerry
Published: March 2018

The high cost of Photoshop and its increasing complexity with each new version can leave the user longing for a version that is a fraction of the price, but still contains the basic features. That longing may be met with Adobe's Photoshop Elements.

Certainly its price is attractive at $99.00 on some websites and about $50.00 on others (including sites for those in Education), For the shopper who wishes to do a little extra research, a web search may reveal that Elements is included with a scanner or ink jet printer at well under $99.00, making the purchase a 2-for-1.

Elements is a pared down version of Photoshop, but it is still a viable solution for almost all image processing needs. Simply because it is pared down, it becomes very attractive for occasional users who do not desire to re-learn the full version of Photoshop, let alone yet another upgrade and yet more frustrating minutea to discover.
Integrated Electron and X-Ray Induced Microbeam XRF in the SEM
Authors: Cross Brian J , Witherspoon Kenny C
Published: March 2018

Energy-Dispersive X-Ray Spectroscopy (ED-XRS or EDS) is a powerful and easy-to-use technique for the elemental analysis of a wide variety of materials. Most commonly, this technique is called X-Ray Fluorescence (XRF), which classically uses x-ray photon sources to excite the sample. A Scanning Electron Microscope (SEM), of course, uses electrons as the excitation source for microbeam x-ray spectroscopy together with sample imaging using characteristic x-rays and/or secondary electrons. These two XRS techniques are used independently, although often the same sample is analysed by both, to provide complementary information.

The advantages of both techniques have been reviewed several times [e.g. 1,2], SEM-EDS being more suited to imaging and microbeam quantitative compositional analysis and maps, and XRF more suited to accurate quantitative analysis, especially for trace elements, while analyzing a much larger area.
Microscopical Evaluation of Glass Delamination in Pharmaceutical Vials: A Look at Three Different Vial Manufacturers
Authors: Diebold Kristie
Published: March 2018

Delamination is defined as separation into constituent thin layers (lamellae). Glass delamination, which can have several causes, could then be defined as a separation of surface glass, as from a vial, into thin layers resulting in a flaky appearance. Pitting could be related to, or even be the initial stages of glass delamination; data will be provided that supports a relationship. The presence of glass delamination is a serious concern to the pharmaceutical industry, not only because suspended glass flakes in any pharmaceutical product poses an obvious health hazard, but because of regulatory quality control standards, and medical profession requirements.

There are, of course, many ways of addressing the problem of glass delamination in pharmaceutical vials, including looking into the relationship of chemical nature of vial contents to degree of delamination and conditions of vial manufacture. Specialized lighting and imaging methods are other research approaches.
Chromatic Aberration in Digital Photomicrographs from Microscopes Requiring Compensating Eyepieces
Authors: Clarke Ted
Published: March 2018

Chromatic aberrations are defects in an imaging system caused by the fact that different wavelengths or colors of light are refracted by different amounts. There are two types of chromatic aberration: longitudinal and lateral. Longitudinal Chromatic Aberration arises when a lens fails to focus various colors sharply in the same plane. If white light is used, the resulting image will be unsharp due to the different focal points of its component colors. Some colors will be in focus (and therefore sharp) and other colors will be out of focus. Lateral Chromatic Aberration results in a lateral shift of the different color components of an image as a single lens with a fixed refractive index will disperse each color by different amounts. This results in color stripes at slightly different magnifications, much like a rainbow, around hard edges and a general softening or decrease in resolution in all areas.
Why Flies Walk with Wet Feet
Authors: Carmichael Stephen W
Published: March 2018

Many animals can walk up vertical surfaces or even along the ceiling. And many studies have examined how flies accomplish this impressive task. Whereas these studies have revealed the anatomy of the contact area and there have been many assumptions about the fly attachment mechanism, the main elements that contribute to the attachment force were unknown. However, Mattias Langer, J. Peter Ruppersberg, and Stanislav Gorb have successfully demonstrated that the fluid secreted from the fly's feet is a critical factor in attaching the fly to the ceiling.
Temperature Monitoring of an EM Environment
Authors: Fellmann Denis , Bañez Rachel , Carragher Bridget , Potter Clinton S
Published: March 2018

The ultimate performance of high resolution electron microscopes depends on a variety of factors including the stability of the temperature surrounding the instrument. Variations in temperature can cause drift of the specimen, the microscope electronics, and the mechanical tolerances in components such as microscope lenses and scan coils. For high resolution work or for image reconstruction that requires acquisition of multiple exposures over extended periods of time, it is critical that the temperature variations be strictly controlled. A typical manufacturer's specification for temperature stability of a room housing a transmission electron microscope (TEM) is a tolerance of <0.5°C/hour with fluctuations of <0.05°C/minute. While modern instrumentation rooms are carefully designed to maintain stable environmental conditions (O'Keefe etal, 2004), it seems unlikely that these stringent specifications are routinely met in practice when the microscope is in operation and personnel are present in the room.
The Influence of Microspectroscopy on Evaluating and Analyzing Forensic Evidence
Authors: Reffner John A , Leary Pauline E
Published: March 2018

The evaluation and analysis of evidence using infrared, Raman, and SEM/EDX microprobe methods has advanced forensic science. Forensic science deals with the interaction of science with the law. This interaction requires that certain standards are met before scientific evidence is admitted in either a civil or criminal case. While the Court's burden of proof is different for a civil case than for a criminal case, the standards of evidence admissibility are not. Forensic scientists must defend their methods and conclusions in Court, regardless of whether they are a trace analyst identifying fibers, a drug analyst determining if the white powder found on a suspected criminal was cocaine, or a pharmaceutical researcher discovering new solid-state forms of a drug. Today, microspectroscopy is a primary technology used within all forensic science disciplines to increase the value of evidence. Modern microbeam methods are extending observations, enhancing documentation, providing additional information, aiding deductions, and testing hypotheses. This increase in value of scientific evidence is pivotal in adjudicating both civil and criminal litigation.
The Day Atomic Resolution Microscopy Happened
Authors: Melmed Allan J
Published: March 2018

Microscopists only dreamed about seeing atoms until 1951. The concept of atoms is believed to have originated in the writings of Democritus in 430 B.C., actually seeing images of atoms in a microscope became a fact only in 1951 with the publication by Erwin W. Müller, announcing his field ion microscope, FIM. Typical of the great scientist that he was, Müller immediately thought of ways to improve his new microscope and his goal then became achieving atomic resolution microscopy as he thought of it - the ability to not only see individual atoms, but to resolve the adjacent atoms comprising the atomic lattice of a crystal surface.
Microscope Instrumentation and Optics: A Researcher's "Road Test"
Authors: Smith Robert F
Published: March 2018

Microscopists in today's laboratories face multiple challenges. Many must examine specimens according to legal, governmental or grant-specific requirements. Protocol requires that the specimens be documented by conventional photography, electronic imaging techniques, or both. Because of the thousands of specimens examined, operators work under conditions of potential physical strain for hours at a time. And the material they examine requires that they have expertise with large and complex imaging instruments.
A Tiny Toolbox
Authors: Carmichael Stephen W
Published: March 2018

A recent article by Matthias Rief, Filipp Oesterhelt. Berthold Heymann, and Hermann Gaub concluded with this sentence: "Single molecule force spectroscopy by AFM has proven to be a powerful addition to the nanoscopic piconewton toolbox," Everything about that conclusion is tiny. Clearly, the atomic force microscope (AFM) has given us a tool to examine structure at or near the atomic level. Earlier work from Gaub's laboratory, reviewed in this column, demonstrated that the AFM could directly measure the binding force between single molecules of biotin and avidin. This established that the AFM could be used as a tool to measure forces, not just observe structure. Their most recent experiments has added to this tiny toolbox.
Optical Sectioning in Fluorescence Microscopy by Confocal and 2-Photon Molecular Excitation Techniques
Authors: Cannell MB , Soeller C
Published: March 2018

Fluorescence microscopy has proved to be an invaluable tool for biomedical science since it is possible to visualise small quantities of labeled materials (such as intracellular ions and proteins) in both fixed and living cells, However, the conventional wide field fluorescence microscope suffers from the disadvantage that objects outside the focal plane also fluoresce (in response to the excitation light) and this leads to a marked loss of contrast for objects in the focal plane, This is especially a problem when the fluorescent probe is distributed throughout the thickness of the cell and the cell is thicker than about 1 µm. The confocal microscope overcomes this problem by illuminating the preparation with a point source of excitation light and limiting the collection of light with a pinhole that is confocal with the illumination source.
A Simple Technique For The Removal Of Plant Cell Protoplasm To Facilitate Scanning : Electron Microscopy Of Fungal Haustoria And Plant Cell Wall Features
Authors: Richardson B A , Mims C W
Published: March 2018

Several years ago Honegger (1985) described a simple technique for removing plant cell protoplasm in order to reveal details of interfaces between plant cells and fungal structures. This technique involves the use of Ariel a commercially available washing powder (Proctor and Gamble) containing a Bacillus substilis derived protease. We since have used this technique with excellent results to examine not only the morphology of fungal haustoria inside leaf cells of various host plants but also features of the inner surface of the host cell wall with scanning electron microscopy (SEM). Here we describe the procedure we have used to prepare samples for study and provide examples of the types of images we have obtained from our samples.
Improving the Resolution of Sputter-coated Films
Authors: Mager Mary
Published: March 2018

After an inquiry from the Microscopy Listserver, I went back to my 1980 copy of Scanning Electron Microscopy, volume I. Several authors had investigated the structure of thin metal films by depositing the films onto carbon-film-covered TEM grids and imaging the films at high magnification. There were several proposals for new devices that have since become standards for high-resolution coaters, but the Listserver inquiry was for a fine conducting film suitabie for high-resolution SEM from an existing sputter coater.There were several factors studied that influenced the fine structure of the films. The first was the materials sputtered: for a given set of conditions of voltage, current and time, platinum gave the finest film, 60% gold-40% palladium (Au/Pd) the next finest and pure gold the least fine.
Parallel Monte Carlo Simulation Using Desktop Computers
Authors: Scott John Henry J , Myklebust Robert L , Newbury Dale E
Published: March 2018

Monte Carlo simulation of electron scattering in solids has proven valuable to electron microscopists for many years. The electron trajectories, x-ray generation volumes, and scattered electron signals produced by these simulations are used in quantitative x-ray microanalysis, image interpretation, experimental design, and hypothesis testing. Unfortunately, these simulations are often computationally expensive, especially when used to simulate an image or survey a multidimensional region of parameter space.

Here we present techniques for performing Monte Carlo simulations in parallel on a cluster of existing desktop computers. The simulation of multiple, independent electron trajectories in a sample and the collateral calculation of detected xray and electron signals fall into a class of computational problems termed “embarrassingly parallel”, since no information needs to be exchanged between parallel threads of execution during the calculation.
Precise SEM Cross Section Polishing via Argon Beam Milling
Authors: Erdman N , Campbell R , Asahina S
Published: March 2018

SEM observation of a specimen cross section can provide important information for research and development as well as failure analysis. In most cases, surface observation alone cannot provide information concerning the cross sectional structure of granular materials, layered materials, fibrous materials, and powders. Preparing highly-polished cross sections of these materials is both a science and an art.

Typically, a cross section is prepared using mechanical means like conventional mechanical polishing methods or a microtome. The sample is first embedded in a holder or device, and then polished to achieve a flat cross section. In some cases, a staining procedure is used to highlight a specific component of the sample. Such methods can be lengthy procedures that require a great deal of skill, and can introduce artifacts into soft materials, deform the material around voids, or compress layers of soft and hard materials in composite samples. Mechanical polishing can miss fine details such as the presence of hairline cracks, and present a challenge to water-soluble phases.
A Comment on AFM vs. Replicas for High Resolution Imaging
Authors: Chernoff Don
Published: March 2018

High-resolution Pt/C shadowing and replication provided important insights into the size and shape of polymers beginning over 40 years ago. The first images of DNA molecules were made this way. However, in my opinion, this methodology has largely been supplanted by the use of Atomic Force Microscopy (AFM), both for direct height measurements (available in Pt/C replicas by measuring shadow lengths when the coating is deposited from one direction only) and for imaging molecular contours.

As an everyday example, consider that making a magnetic read and write head for a hard disk drive requires controlling the relative heights of several different regions to a tolerance of about 1 nm. AFM supports production by providing a rapid means of offline analysis, far faster and more precise than any replica method could be.
Insights on Diffraction
Authors: Eades Alwyn
Published: March 2018

This article presents ideas on some topics related to electron diffraction in the TEM. These are in regard to topics that I have come to think of as standard parts of what it means to do microscopy. However, they represent insights that not all users share (or even agree with, maybe).

Kikuchi lines are of great use in orienting a sample. Unfortunately, in modern microscopes, Kikuchi lines are not seen in selected-area diffraction (SAD). This is because immersion lenses send parallel electrons, from different parts of the sample (like the Kikuchi lines from a flat specimen), to different places in the diffraction pattern.
More on the Calibration of TEMs
Authors: McCaffrey J P , Beanland R
Published: March 2018

There was a recent thread on the microscopy listserver by John McCaffrey (who hosted the discussion on TEM calibration at the 2001 M&M facility managers session) and Richard Beanland, dealing with the calibration of TEMs. This discussion was prompted by a calibration question from John Basgen, who was looking for more precision and more long-lived calibration specimens. The discussion complements and extends the one of the M&M 2001 managers meeting on EM calibration (Microscopy Today, January/February 2002, issue #02-1), and we are running this separately from that meeting discussion. (MT-ed.)
Lateral Resolution in Scanning Force Microscopy
Authors: Todd Brian A , Eppell Steven J
Published: March 2018

Two particular questions keep coming up regarding Atomic Force Microscopy (AFM), and we would like to address them here.

The lateral resolution of AFM is a parameter that currently has no accepted definition. The definition used in our lab is “the minimum distance between objects at which you can distinguish distinct objects.” In other words, imagine a surface with two sharp features: upon bringing the two features closer and closer together, at some point only one object is seen. That distance is the resolution limit. It is useful to define “see” as having a dip in height greater than the noise level in the image.
The Staining of Polymers
Authors: Smith Ronald W
Published: March 2018

In the beginning there was water-the first stain that delineated structure within a polymeric system. The polymerwas natural rubber (NR) and the system was an air-dried film of natural latex. Grenquist reported in 1929 that a dried film of natural rubber latex soaked in hot water became milky (Figure 1) and, at that stage, the latex particles could be detected by the optical microscope (OM), The contrast mechanism was provided by the absorption of water by the naturally occurring proteinaceous layers present on the congealed NR particle surfaces. There was little demand for polymer staining until the age of electron microscopy.
Imaging of Shallow Surface Topography by the Low-Loss Electron (LLE) Method in the Scanning Electron Microscope
Authors: Wells Oliver C
Published: March 2018

The low-loss electron (LLE) method in the scanning electron microscope (SEM) was proposed by Dennis McMullan in 1953: “…the beam from the specimen could be restricted to the electrons which have lost only small amounts of energy and which have therefore travelled only short distances through the specimen.”

Subsequent studies showed that the LLE method gives different image contrasts from the more familiar secondary electron (SE) method: (i) it is less affected by specimen charging; (ii) has a shallower information depth for a given beam energy; (iii) shows less serious penetration effects at sharp edges; (iv) shows stronger channeling contrast; and (v) Is better for showing shallow surface topography.
Photoshop Distilled II
Authors: Carvaiho Tina (Weatherby)
Published: March 2018

Preparation of digital figure plates for publication is best done with software designed for that purpose, such as QuarkExpress or Adobe Page-Maker, However, if you have only Photoshop in your repertoire, you can tackle page layout in the following ways.

This first method is probably easiest, but results in the loss of a few pixels along the inside edges of each of the images.
Unzipping a Membrane
Authors: Carmichael Stephen W , Fernandez Julio M
Published: March 2018

The atomic force microscope (AFM) is well known for its outstanding spatial resolution, but it is becoming increasingly useful as the instrument for force spectroscopy. In the force spectroscopy mode, the AFM can measure tiny tension forces, in the piconewton (pN) range. Daniel Müller, Wolfgang Baurmeister, and Andreas Engel have used the AFM in both the imaging and force spectroscopy modes to pull proteins out of membranes in a controlled fashion.

Müller et al. used Deinococcus radiodurans, a bacterium best known for its high resistance to radiation (as its Genus name implies), as their test subject. They extracted a highly regular membrane from the bacterium, the hexagonally packed intermediate (HPl) layer. They mounted the HPI on mica, so that the hydrophilic outer surface of the HPI adsorbed strongly to the mica, exposing the hydrophobic inner surface to the silicon nitride AFM stylus.
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. The Al chuck can usually be removed by hand, but a flathead screwdriver used as a pry will help pop it off if you have trouble.
A Tip on Purifying Glutaraldehyde
Authors: Melsen Lawrence R
Published: March 2018

Being an old timer, I have been around since before the advent of vials of glutaraldehyde. In the 1970s, the Merck index described a method for purifying glutaraldehyde using activated charcoal.

Basically, glutaraldehyde begins to polymerize when stored at room temperature. Since best fixation is achieved with the monomeric form, this purification protocol was designed to remove the more complex forms. Storage under nitrogen in a sealed vial slows the polymerization, and lower concentrations degrade more slowly. Storing at 4°C is advised.
How Long Should A Diamond Knife Stay Sharp?
Authors: Garber Charles A , Mesa Bernard
Published: March 2018

The answer to this question is about as elusive as predicting which way the Dow Jones average will close tomorrow! But seriously, there are the “ten commandments” for a diamond knife to enjoy a long life, the most important ones being as fallows:

1) Because of the extreme sharpness of a diamond knife edge, it should not be touched with any solid object, even for cleaning, This is controversial since some manufacturers actually recommend that the edges be cleaned with sticks of varying types. We ourselves believe such treatment accelerates the wear of a diamond knife. 2) Don't let sections or the remains of sections or other debris dry down onto the knife edge. Keep the knife edge wet until it is ready for cleaning before being put to bed for the night. 3) Use a diamond knife cleaner sold by several firms (including ours) specifically for this purpose, Some typical laboratory ultrasonic cleaners can have enough power to be damaging to a knife. 4) Wash the knife edge one last time with distilled water, and then dry with some kind of "blast", such as from a clean duster, 5) Avoid conditions of chatter at all times. Reduce chatter by varying the clearance angle or slowing the cutting speed. Other common causes of chatter are insufficient tightening of the boat in the microtome, an insufficiently tightened block, or an incompletely cured block. 6) Final block trimming with a razor blade can ieave metal particles from the blade, which are of course damaging to the knife edge, This can be minimized by using a fresh razor blade each time, Then wash the end of the freshly cut block face with distilled water, followed by drying with a duster blast is the final step before the first cut with the knife, This is a final chance to wash away metal particles that could damage the knife edge.
DAB on Human Hepatocytes
Authors: Gilbert Charles S
Published: March 2018

Neutrophil myeioperoxidase standard 3,3'-Diaminobenzidine Tetrahydrochioride (DAB) procedure:

When staining neutrophils, a cell suspension would be preferred, but a finely minced buffy coat can be used, if you thick section and locate the stained cells before thin sectioning. Staining should be carried out as soon as possible after fixation, The myeioperoxidase (MPO) activity falls off such that we process samples within 2-3 weeks of fixation.

Note that fixation is for 1 hour. Enzyme activity may be destroyed if tixed for longer periods.
A Really Cool Image Database Environment:
Authors: Hilton Jim
Published: March 2018

Growing your own microscopy client/server image database requires addressing many issues. If you are going to grow your own, the following ideas will get you “out of the box.”

The ideal microscopy image database environment might appear like this: The core of the image database would be a very powerful, universally available, extensible, cost effective, flexible and serviceable database engine. The core engine would allow the user to perform fast and seamless SQL searches across a large database without reducing the available network bandwidth. It would be able to take advantage of current and future technologies (i.e., 64 bit processing, 1 GB networks, digital, video, multi media, etc.) The same core would be able to share information with other databases, applications and platforms. What an odd concept.
To Make a Leeuwenhoek Microscope Replica
Authors: Shinn Alan
Published: March 2018

For some time now I have been studying Antony Van Leeuwenhoek's microscopes and building prototypes, and have finally settled on a design that I would like to offer to the public.

They are made of thin beaten brass, about 47 mm tall by 25 mm wide, with the lens captured between two such sheets. The subject is placed on a metal pin, adjusted with screws, to change the focus and position. One of the adjusting screws protrudes down from the body and also serves as a handle. These microscopes look very much like the one in the museum at Utrecht (circa ∼ 1690). They have a lens made of a 2 mm diameter glass ball and have a power of about 180X, with surprisingly good resolution.