bluuebunny/crossref_metadata_2025 huggingface dataset:
Design of a Photoelectron Microscope for Integrated Circuits and Other Areas of Surface Science
Authors: Rempfer G F , Griffith O H
Published: July 2020

In photoelectron microscopy (PEM; also known as PEEM) ultraviolet light is used to liberate electrons from the surface of a specimen through the photoelectric effect. The emitted electrons (photoelectrons) have low kinetic energies (∼1 eV) and are accelerated by an electric field between the specimen and an anode, before being imaged by a lens system. The lens system forms the image of the specimen in parallel mode, as in TEM, rather than in scanning mode, as in SEM. Image contrast arises from differences in work function and from electric or magnetic fields over the specimen surface. Micro-fields associated with surface topography are also a source of contrast. Because of the low emission energies of the electrons PEM is a highly surface-specific technique and also is relatively non-destructive.

Several versions of the photoelectron microscope have been designed and constructed jointly by the University of Oregon (U0) and Portland State University (PSU), and the instrument has evolved to an advanced state of development.
Digital Holographic Microscopy
Authors: Zhang Tong , Yamaguchi Ichirou , Morgan Hywel
Published: July 2020

We applied phase-shifting digital holography to microscopy in this paper. At first lensless microscopy is proposed, in which no optical adjustment is necessary. Then, the method is applied to relax the limitation of focal depth in traditional optical microscopy. A theory for image formation and experimental verification using a few specimens are described.

keywords: microscopy, digital holography, phase shifting

Due to the finite focal depth of an imaging lens, a limitation to normal optical microscopy-is that, only the 2-dimensional (2-D) information of an object can be obtained at one time. Besides, it is not convenient for quantitative analysis the observed image. Optical sectioning microscopy (OSM) and scanning confocal microscopy (SCM) which use opto-electronic detection have been proposed for quantitative analysis of a 3-D object. However, the former requires critical mechanical adjustment, while the latter uses timeconsuming mechanical 3-D scanning. Holographic microscopy can solve these problems because it can record 3-D information at one time. But, the chemical processing of holograms and the mechanical focusing at the reconstructed images cause more or less trouble. A 3-D imaging technique without use of photographic recording called optical scanning holography has recently been reported. However, there are also some trouble owing to the twin-image noise.
Digital Stereo Display and Projection Systems
Authors: Case Gary N , Mackenzie John M
Published: July 2020

For many years, we have been viewing stereo images created from a wide variety of microscopic sources. These images need to be easily viewed, shared and presented for stereo imagery to become more mainstream. We have been working for several years now to create affordable high resolution stereo display systems using the modern digital computers’ display capabilities. In the last couple of years, two methods have come into widespread use in our laboratory. One using shuttered goggles and the other using an active polarizing screen in front of the monitor. Both are synchronized with the display at refresh rates that allow for flicker free viewing. We use standard video display cards and shuttered glasses that cost under $300 (complete).

Unfortunately, the software and video cards turn over at such a fast rate that we have found our hardware “orphaned” several times.
Image Analysis: Converting from Manual to Automated Metrology
Authors: Cooper S , Saul E , Hunt J A
Published: July 2020

Many of the repetitive measurements required in the analysis of images in the semiconductor field can be replaced by partially or fully automated measurements. Automated measurements provide a number of benefits, including speed, repeatability, and removal of operator bias. The question of accuracy and precision in metrology applications is important to understand.

Recently we implemented a tool for automated analysis of resist bar images that was designed for use in acceptance testing-of lithographic stepper machines. The steppers are required to meet a defined set of criteria over the entire area of the wafer and through the focus range of the lens. Prior to the development of this package, the measurements were all done with a ruler and protractor on film.

The software identifies the boundary lines of the resist bar by locating a series of points along each edge and then fitting a line to those points. Edge identification is complicated by coating artifacts and by the variety in the images being analyzed. After identifying edge points along the base, side, and top of the resist bar, the software automatically generates information about the width, height, side wall angles, and various other measurements of the resist bar and stores the data in spread sheet format.
Imaging Cells in Liquid with a Near-Field Scanning Optical Microscope - Problems and Solutions
Authors: Gheber Levi A , Edidin Michael
Published: July 2020

Near Field Scanning Optical Microscopy (NSOM) is the newest member of the Scanning Probe Microscopy (SPM) family, which includes Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM). Like its predecessors, it uses a sharp tip and a very precise positioning system to scan the surface of samples. The important difference is that the NSOM tip is made of an optical fiber, with a sub-wavelength size aperture at its end, which is brought to near-field proximity to the sample. In the near-field regime, the resolution is only limited by the size of the light source (the aperture), and not by the wavelength of light, thus allowing NSOM to obtain images with subwavelength resolution. Applying NSOM to biological samples is very appealing, since biological research has developed a wide variety of fluorescent dyes which can be directed with high specificity to objects of interest. Moreover, biological research relies to a large extent on conventional fluorescence microscopy, which is diffraction limited. Application of NSOM to biological problems requires the ability to image rough and soft samples in liquid, a task that presents many challenges.

We have developed a NSOM which is capable of imaging cells in liquid with a resolution of ∼150 nm. This has enabled us to image the organization of fluorescently labeled proteins on the membrane of cells in liquid, and demonstrate for the first time that the proteins are clustered.
Production and Analysis of High Resolution Polymer Replicas of Fibrillar Collagen
Authors: Sims P , Todd B , Eppell S , Li T , Park K , Albrecht R
Published: July 2020

Adherent cells generally construct the immediate substrate upon which they reside. This may occur via synthesis and secretion of new materials and/or by rearrangement and modification of existing substrate. The response of adherent cell types to an existing substrate can be influenced by a number of factors which include both the chemical and physical nature of the substrate. Cell adhesion, proliferation, differentiation and death can all be substrate dependent. Much effort has been directed toward chemical modification of substrates to regulate one or more of the parameters noted above. A significant, but somewhat smaller, degree of attention has been paid to the effects of the topography and microtopography on the cell response to substrate materials. Studies to date strongly suggest the topography is a significant factor in cell-substrate interactions. As noted above, it is most probable that both the chemistry and the structure of a substrate simultaneously influence the cellular response. However we wished to determine, particularly for artificial substrates, the role which microtopography can play in cell-substrate interactions.
High Resolution Microanalysis of Particles from the Human Lung
Authors: Bell David C , Rainey Lenore C , Vander Sande John B
Published: July 2020

Every time we take a breath, we are inhaling the results of twentieth century combustion technology. Combustion processes generally produce a multitude of soot and other sub micron sized particulates. The human lungs, via the process of cilia movement expel most of these particles; others are broken down with the aid of macrophage agents. These macrophages absorb particles and incorporate the constitute elements into our bodies. These elements maybe expelled, or they may remain in the body and accumulate over time, as is the case with certain heavy metals. Limited prior research on ‘single-particle’ interaction with lung or bronchial tissue has been conducted. Related research has focused on the statistical significance of soot inhalation on the lung tissue of rodents and primates [1]. Using the methods of single particle examination, founded by previous research into single particle source allocation [2], the examination particles of from human lung and bronchial tissues was performed.

Research on the particle characterization shown here is based on the application of an innovative method developed at MIT, which utilized high resolution transmission electron microscopy (HRTEM), scanning transmission electron microscopy (STEM) coupled with electron energy loss spectroscopy (EELS) and energy dispersive X-ray analysis (EDX).
Characterization of Pacemaker Electrode Surfaces Utilizing Scanning Electron Microscopy (SEM) and In-Vitro Electrochemical Analysis
Authors: Brabec Scott , Schindeldecker Bill , Brennen Ken , Okerstrom Sue , Pham Ky
Published: July 2020

The efficiency of implantable electrodes for cardiac pacing depends on the ratio of the conductive surface area to the geometric area of the interface with excitable tissue. New models of heart pacers require reduction of post-pulse polarization, i.e. the potential left on the electrode / tissue interface after a pacemaker pulse. Increasing the conductive surface area is an effective method to this end. Microscopy provides an important tool in elucidating the role of surface structure in electrode performance.

Three different surface textures were characterized on a 90% platinum(Pt)/10% iridium (Ir) polished electrode substrate of roughly 5 mm2 geometric surface area. These consisted of the polished substrate itself, a thin film of textured platinum in the 1-3 micron size range, and a sub-micron platinum black coating. Sample surface effects were characterized via scanning electron microscopy (SEM), in-vitro electrical impedance spectrum analysis, and polarization after-potential measurements.
The Potential of Cryo-Electron Tomography as Assessed by Reconstruction of Sea Urchin Axonemes
Authors: McEwen BF , Marko M , Hsieh C-E , Frank J
Published: July 2020

Recent improvements in digital image acquisition, and automated TEM, have lowered the electron dose requirement for collecting a tomographic tilt series to the point where cryo-electron tomography is now feasible. Theoretical calculations indicate that resolution values of 2-5 nm can be obtained for fully hydrated specimens 100-500 nm thick. This resolution is sufficient for identifying individual macromolecules, and macromolecular assemblies, within-organelles and small cells. However, initial applications to cells and organelles fell short of theoretical resolution limits, indicating further development is necessary before cryo-electron tomography reaches its full potential for characterizing macromolecules in situ. Axonemes are ideal test specimens for cryo-tomography because of their appropriate size (diameter of 250 nm), ease of isolation, and unique construction from regular arrays of well-characterized components. The latter include microtubules, radial spokes, central sheath components, outer dynein arm, inner dynein arms, and the microtubule subunit lattice. These components are convenient benchmarks for evaluating the actual detection limit of tomographic reconstructions within a resolution range of 3-30 nm. Furthermore, axoneme components are sufficiently well ordered that computed diffraction patterns contain layer-lines that are useful for judging the quality of individual 2D images.
A Versatile High-Vacuum Cryo-Transfer for Cryo-FESEM, Cryo-SPM and other Imaging Techniques
Authors: Ritter Martin , Henry Didier , Wiesner Stefan , Pfeiffer Stephan , Wepf Roger
Published: July 2020

A structure preservation of biological and organic samples, close to native state, can only be reached by cryo immobilization techniques. Cryo immobilization allows not only to preserve the high structural integrity but also to arrest dynamic processes in the μs- ms range.

After freezing the sample and preparing the surface of interest, it is important to prevent the sample from ice crystal damage, removal of structural water, condensation of water or other contaminants until imaging. Therefore, ideally the samples are kept below the recrystallisation temperature of water (< 147K) during the transfer from the preparation environment into the imaging chamber.

For the transfer of frozen samples several concepts have been followed in the,past: a) the specimen after manipulation/preparation were submersed in liquid nitrogen and transferred to the cold stage of the microscope or b) a preparation chamber was permanently attached to the microscope column allowing the direct transfers between the preparation chamber and the cold stage in the microscope. These concepts allow either a high grade of flexibility combined with a high risk of contamination or to prevent contamination but combined with inflexibility. In addition the later also does not allow using the microscope during the specimen preparation procedure, nor transferring the specimen to an other imaging device.
Use of Freeze Substitution With Ultrarapid and Controlled-Rate Freezing to Determine Quality of Cryopreservation
Authors: Hastings Cindy , Lightfoot Fred , Taylor Michael , Brockbank Kelvin
Published: July 2020

The field of cryobiology is expanding and incorporating conventional methods used in electron microscopy into protocols heretofore unexplored; two of those methods is that of ultrarapid freezing and freeze substitution. When tissues such as heart valves and/or vascular tissue is frozen for clinical applications it is imperative to know the quality of the freezing process.

Ultrarapid Freezing: Various methods of ultrarapid freezing have been employed routinely by those utilizing electron microscopy. In particular, questions involving immunocytochemistry and x-ray microanalysis may indeed require the use of one or more of these methods.. Metal mirror fixation (slamming) is the preferred method of cryopreservation for most subsequent protocols. It is also the preferred, and probably the only, method to check the accuracy of freeze substitution protocols. The metal mirror method utilizes a copper block, which is brought into contact with the tissue, at a predetermined speed, via a pneumatically operated system. The chilled metal block, devoid of scratches or imperfections, ensures maximal preservation of tissue to a depth of 15μm, which can serve as a control, for both fixation and the substitution process.
Freeze Substitution for EM-Studies Time Measuring.
Authors: R.K. Migunova , A.F Todrin , A.S. Kaprelyants
Published: July 2020

We conducted physical and mathematical modelling of the process of substituion of ice with organic solvent. The result was a relationship between the time of freeze-substitution and the sample thickness. The model is based on the solution of mass exchange problem between a porous plate (frozen tissue) filled with a solid matter (ice) and an organic solvent (alcohol, acetone) that is contained in an apparatus with an active fluid circulation. The model takes in account porousness of tissue, dissolving of ice with solvent that produces a new medium, dissolving of that medium and its substitutions with solvent after solvent concentration exceeds evtectic concentration for the given temperature. Our results (fig. 1) agree with previously published results [1].

We also conducted experiments using original device [2] that implements freeze-substitution method. The device is made of the desk apparatus, comprising cryogenic and electron parts. Cryochamber ( Fig. 2 ) contains cylindrical unit (1), central channel (2) and located around it cylindrical vessels (3), connected by means of upper (4) and lower (5) channels with the central one. Perforated containers (6) with frozen bioobjects are adjusted on metal rack (7). The flux inducer (8) provides the circulation of substituting medium. The cold-conduct or (9) is submerged into a liquid nitrogen. Temperature control is performed be the thermogauge (10). The range of operating temperatures stabilization is from +20° to −120° C.
Automatic Recording of Low Dose Images with Slow-Scan CCD Cameras
Authors: Zellmann E , Probst W , Seybold V , Hiller S A
Published: July 2020

Recording of images under low dose conditions is a rather critical operation including many intermediate steps. Moreover, there is not a single fully agreed protocol concerning low dose operation but several competing ones potentially including individual variations. In any case, however, there should be a very well defined sequence of steps which must not allow for any mistake. Therefore tailored individual automation is the only way to obtain reliable results with maximum throughput at the same time.

Such automation requires the functional integration of a fully remote controllable digital TEM, a Slow-Scan camera with a high sensitivity, and an acquisition/remote control software providing macro capabilities dedicated to different logical cameras. These requirements are met by LEO (EF)TEMs, by the ProScan SSCCD 14 bit camera, and the analySIS software 3.0.

Three different logical cameras have to be configured in the SIS image processing software. The first camera is defined for searching for suitable specimen areas. It operates with 5 MHz and 8 bit image depth providing 1k×1k field size and resolution. The exposure time should be as short as possible, e.g. 50 ms for each frame. Via a preprocessing macro image brightness and EM magnification are set to the appropriate values, the desired shutter is activated, and the viewing screen is lifted for the exposure. A second postprocessing macro blanks the beam and does additional settings when the camera is stopped.
Imaging Dynamic Events in Living Tissue using Water Immersion Objectives
Authors: Brenner M
Published: July 2020

The use of water immersion objectives can provide a significant benefit to research involving the study of dynamic events in living tissue. Optical and quantitative imaging techniques like Confocal, Differential Interference Contrast (DIC), and Epi-fluorescence microscopy are dependent on the ability of high magnification, high numerical aperture immersion objectives to provide a high resolution image. The need to image the specimen not only at the surface next to the cover glass, but deeper down to 200 microns or more, can be greatly assisted by the use of highly corrected water immersion objectives. With oil immersion objectives, the deeper into the specimen and aqueous media one images, the more spherical aberration occurs.

Spherical aberration is the main optical problem in imaging living cells with oil immersion, and spherical aberration increases proportionately with depth into the aqueous media and cellular material. The destructive effect of spherical aberration is seen as a loss of intensity and contrast, inability to collect and resolve small spatial frequencies, and reduced accuracy of reproduction in an optical system, all in direct proportion to the distance from the cover glass. It is possible to compensate mathematically for these distortions and extend resolution with deconvolution or image restoration software. These methods require accurate measurement of the point-spread function; if resolution varies with depth, then measurements must be taken at different depths, which complicates the process.
Optimization and the Evaluation of Confocal System Performance to Quantify Fluorescence
Authors: Zucker Robert M , Price Owen T
Published: July 2020

The laser scanning confocal microscope (LSCM) has enormous potential in many fields of biology. Quality assurance is essential for the equipment to function correctly and realize its fullest potential. Currently the assessment of a confocal microscope’s performance is subjective primarily by evaluating the system with a specific test slide provided by the user. To achieve better performance, it is necessary to run a series of validation tests. These tests that were applied to measure field illumination, lens, clarity, dichroic functioning, objective spectral alignment, axial resolution, laser power and stability, photomultiplier tube (PMT) functioning, and system noise. Although th?se tests were derived on a Leica TCS-SP system and Leica TCD4D system, they should be applicable to other manufacturers’ systems.

For optimal images, it is important to control the variables of laser power, PMT tube voltage, averaging, and bleaching. The relationship between PMT voltage, laser power, and averaging was determined using a Spherotech Rainbow 10 μm diameter test bead. It was demonstrated that the noise (coefficient of variation of bead intensity, CV) in an image greatly increases as the PMT increases. The system variables that affected the precision and accuracy of the confocal system will be discussed.
Quenching or Misalignment? Confocal Microscopy Onset of the Mitochondrial Permeability Transition in Cultured Hepatocytes
Authors: Qian Ting , Trost Lawrence C , Lemasters John J
Published: July 2020

INTRODUCTION: The mitochondrial permeability transition (MPT) has been implicated in mediating both necrotic and apoptotic cell death. Opening of the permeability transition pore in the mitochondrial inner membrane causes the MPT. Previously, our laboratory developed a method to detect the MPT in cultured hepatocytes by visualizing redistribution of calcein fluorescence from the cytosol into the mitochondria after permeability transition pore opening, using confocal microscopy. (1). However, a recent paper suggests that unstained mitochondria are unlikely to be detected against bright cytosolic signal, because fluorescence spreading magnifies the size of emitting objects while reducing that of dark spot (2). Rather, it was suggested that dark mitochondrial voids in green calcein images were the result of fluorescent quenching by concurrent use of a red-fluorescing mitochondrial dye, tetramethylrhodamine (TMRM). The AIMS of the present study were to determine 1) whether small voids can be visualized in the absence of any potential quenching agent; 2) whether quenching can produce dark mitochondrial voids and 3) the importance of alignment in visualizing mitochondria as negative contrast by confocal microscopy. METHODS: Overnight cultured rat hepatocytes were used. To load calcein exclusively into cytosolic space, hepatocytes were incubated with 1 μM calcein AM for 15 min at 37°C. To load calcein into both cytosol and mitochondria, cells were incubated with 1 μM calcein AM for 1 hour at 4°C.
Video-Confocal Microscopy: An “Electronic-Pinhole” Method using Narrow-Field Illumination and Wide-Field Image Detection.
Authors: Benedetti Pier A , Evangelista Valter , Guidarini Dante , Vestri Stefano
Published: July 2020

State of the art in optical microscopy

Optical microscopy is still and increasingly one of the most valuable tools in biological investigation. In particular, confocal microscopies are capable of achieving best performances in the study of three-dimensional fluorescent and reflecting specimens. Nevertheless, current techniques adopted in confocal microscopy present some drawbacks and limitations that stimulate to devise and set-up further techniques, suited to a wider range of applications.

Advantages of confocal microscopes mainly correspond to an improved spatial resolution, especially in the axial direction. Depending on the narrow-field scanning approach used, there are two main forms of confocal microscopes: single-point (SP) and multi-point (MP) ones. Unfortunately, SP confocal microscopes require the use of lasers as illumination sources with consequent high costs and scarce spectral flexibility. Moreover, specimen photo-damage due to relatively high instantaneous irradiation doses involved, can often limit their investigative capabilities. On the other hand, proposed MP confocal microscopes still rely on the revolving-disk approach and exhibit a relatively low luminous efficiency, substantial constructional complexity, and limited contrast in the study of thick fluorescent objects.
Colocalization of mRNAs by Fluorescence in Situ Hybridization
Authors: Baskin D G , Breininger J F
Published: July 2020

Fluorescence in situ hybridization (FISH), long the method of choice for chromosomal cytogenetics, is becoming recognized as a powerful method for correlative histochemical detection of multiple messenger ribonucleic acid (mRNA) in cells. The technique is based upon the principle of the binding of a labeled strand of DNA (an oligonucleotide probe) or RNA (a riboprobe) to complementary strands of mRNA. In the traditional in situ hybridization method, nucleic acid probes are labeled with radioactive isotopes and the hybrids are localized by autoradiography. More recently, labeling of the probes with digoxigenin, biotin, or fluorescein has allowed nonisotopic detection of probe-mRNA hybrids with immunoenzymatic techniques that are based on peroxidase or alkaline phosphatase histochemistry, in which the results are observed as brightfield stains. FISH is similar to conventional nonisotopic in situ hybridization except that the labeled nucleic acid probes are detected by fluorescence microscopy.

The FISH technique has advantages for performing correlative histochemistry. The results of a FISH protocol can be observed within a few hours of completing the hybridization protocol instead of waiting for days as is the case with isotopic methods. FISH permits cellular resolution and facilitates multiple labeling, where in situ hybridization probes can be resolved at the level of individual cells (Hahn et al. 1998).
Correlative LM/TEM Studies are Essential in Evaluating the Effectiveness of Liposome Mediated Delivery of the Cystic Fibrosis Transmembrane Regulator (CFTR) as a Corrective Therapy in a CFTR Knockout Mouse that Develops Lung Disease
Authors: Ackerley CA , Tilups A , Bear CE , Becker LE
Published: July 2020

An animal model for Cystic Fibrosis (CF) has been developed to further comprehend the pathogenesis of the disease as well as the development of therapeutic interventions. This is a mouse line from a phenotype of an inbred congenic strain of CFTR-knockout mouse that spontaneously develops lung disease. Pathology of the mice include severe impairment of the mucociliary transport, postbronchiolar over inflation of alveoli and parenchymal interstitial thickening with evidence of fibrosis and inflammatory cell invasion.

Although protein replacement therapy using liposomes containing purified reconstituted functional CFTR has been done on the nasal epithelium of CFTR-knockout mice the strain did not develop lung disease nor were the liposomes applied beyond the nasal cavity. Assessment of the treatments was done by measuring the transepithelial potential difference (PD), immunofluorescent labeling of the CFTR and immunogold labeling of the segments of nasal epithelium which were found to have a correction in their PD readings and CFTR present by immunofluorescence. It was clear after these studies that some form of correlative microscopy technique was needed to be devised in order to improve the efficiency of detection.
Changes in F-actin Organization Induced by Hard Metal Particle Exposure in Rat Pulmonary Epithelial Cells as Observed by Laser Scanning Confocal Microscopy
Authors: Antonini J , Starks K , Millecchia L , Roberts J , Rao K
Published: July 2020

Hard metal is an alloy of tungsten carbide and cobalt along with other components such as chromium carbide, molybdenum carbide, tantalum carbide, and nickel. Chronic exposure to hard metal particles by inhalation causes alveolitis leading to interstitial fibrosis, the pathogenesis of which is still undefined. The initial inflammatory response includes a change in epithelial cell permeability barrier function (1) which has been shown to be regulated by the state of assembly and organization of the actin cytoskeletal network (2, 3). Therefore, the objective of this study was to evaluate the effect hard metal particles have on F-actin organization of rat lung epithelial cells in an in vitro culture system.

Rat lung epithelial cells (L2: ATCC, CCL-149) were grown to confluence on glass coverslips and exposed to various concentrations of hard metal particles for 24 hours. The effect on F-actin organization was visualized by confocal microscopy following Bodipy-Phallacidin staining, while changes in cell morphology were assessed by phase contrast microscopy. Hard metal particles of cobalt, tungsten carbide, and tungsten carbide/cobalt (6 % cobalt) were tested at concentrations of 1, 3, and 5 μg/ml. There was a dose-dependent change in the F-actin organization in the cells. The actin microfilaments lost their uniform distribution and aggregated into homogeneous masses of F-actin staining. Significant change in F-actin state was observed even at a 1 μg/ml concentration of tungsten carbide/cobalt particles. This is consistent with previous observations that pathological effects of tungsten carbide/cobalt particles are more pronounced compared to either metal alone. Phase contrast microscopy revealed no significant change in the cell morphology at this short incubation time.
Retinoic Acid Synthesizing Astrocytes within the Developing Spinal Cord: Correlative Microscopic Study
Authors: Rosa-Molinar E , Frolich V , Vaughn R , McCaffery P J , Fritzsch B
Published: July 2020

The Western Mosquitofish, Gambusia affinis affnis, a sexually dimorphic internal fertilizing species of the family Poeciliidae, shows distinct sexual dimorphism in the anal fin and its axial and appendicular support.

The development of the regional vertebral differences (i.e. precaudal [vertebra bearing ribs] and caudal [vertebra bearing hemal spines]) in female and male G. a. affinis is generated through a development program of first posterionzing more anterior vertebra (formation of hemal spines more anteriorly) then antenonzing more posterior vertebra (loss of hemal spines and formation of parapophysis with pleural ribs). This developmental program is reminiscent of effects obtained by disturbing developmental programs by exogenous treatment with retinoic acid.

Recently we generated data suggesting that all-trans retinoic acid (tRA), a non-steroidal lipophilic molecule may play a role in the regulation and patterning of sexual dimorphic phenotype(s), an area considered the domain of steroid hormones.

We wanted to identify the specific neural cell type(s) synthesizing endogenous tRA within the developing spinal cord which might coordinate sexually dimorphic patterning of spinal cord levels in G. a. affinis. To achieve this, we employed retrograde transport studies using 3,000 molecular weight (MW) dextran amine and biocytin combined with immunogold and/or lmmunohistochemistry to identify the specific neural type(s) which express tRA synthesizing enzymefs] (i.e. retinaldehyde dehydrogenase [RALDH]).
Up-Conversion and Two-Photon Excitation Fluorescence Properties of Phloxine B.
Authors: Malak Henryk
Published: July 2020

A dye, Phloxine B, a common food coloring and one of the active components of a photoreactive insecticide was recently approved by Food and Drug Administration as D&C Red #28 for use in drugs and cosmetics. Phloxine B is also one of the most widely use stain in fluorescence microscopy. However, in spite of the widespread interest in multi-photon spectroscopy and imaging, no information is available on the electronic transitions properties of Phloxine B with red edge fluorescence excitation and with multi-photon excitation.

In the present report we described the steady state and time-resolved fluorescent properties of Phloxine B with up-convert photon excitation and with two-photon excitation. We examined the electronic transitions properties of Phloxine B when was excited by femtosecond pulses from a mode-locked titanium sapphire laser or a mode-locked optical parametric oscillator laser. Phloxine B under excitation wavelengths above 775 nm was found to display two-photon excitation fluorescence with a spectrum maximum at 580 nm, which emission is consistent with one-photon excitation fluorescence spectrum. At the red edge excitation of Phloxine B, from 590 nm to 650 nm, we observed one-photon excitation fluorescence indicating that Phloxine B behaves like upconverting dye with one-photon excitation process.
Pattern Analysis Meets Cell Biology
Authors: Murphy Robert F , Boland Michael V
Published: July 2020

The widespread availability of automated fluorescence microscope systems has led to an explosion in the acquisition of digital images by biologists. This has created a need for computer applications that automate the analysis of these images and an opportunity to develop new approaches to classical problems. An example is the determination of the subcellular location of a protein from immunofluorescence images (or, more recently, images of GFP fluorescence). Current practice is to compare such images to mental images that a cell biologist has developed over time, and to reach a tentative conclusion about the structure (i.e., organelle) that a protein is found in. Since this determination is subjective, it often must be followed up by double labeling with a marker protein from the suspected structure.

As an initial exploration of the feasibility of automating the determination of subcellular location, we developed a system that is able to classify the localization patterns characteristic of five cellular molecules (proteins and DNA) in Chinese Hamster Ovary (CHO) cells. Images were acquired on an epifluorescence microscope after the cells had been fixed, permeabilized, and labeled with appropriate fluorescent reagents (usually antibodies conjugated to fluorescent dyes). The labels used were directed against a Golgi protein, a lysosomal protein, a nuclear protein, a cytoskeletal protein, and DNA.
Three Dimensional Light Microscopy: Imaging & Corrections for Quantitative Analysis
Authors: Turner JN , hain W , Szarowski DH , Lasek S , Kam L , Can A , Al-Kofahi K , Roysam B
Published: July 2020

There are several forms of three-dimensional (3-D) light microscopy but all utilize the principle of optical section recording, i.e. the 3-D image is a sequence of two-dimensional (2-D) images called optical sections. The optical sections are particular focal planes formed within the thick specimen and usually correspond to the conventional image projections recorded in a light microscope, referred to as x,y projections. The optical sections are recorded for a sequence of focus- or z-positions. This “stack” of 2-D images is the data set for the 3-D image. If quantitative analysis is to be performed on the 3-D images, the choice of the z-dimension increment between 2-D images is especially important, and its value may be more or less critical depending on the analysis algorithm used. A reasonable starting value for this dimension is the depth-of-field of the objective lens, but the actual value may have to be smaller to optimize the image analysis or larger to decrease the influence of photobleaching. The most photostable dyes should be selected and the specimen should be mounted in index-of-refraction matching media with an antioxidant.

The image resolution in all three-dimensions is determined by the 3-D point-spread-function (psf), and as a rough rule of thumb the z-resolution is degraded by a factor of 3 relative to the x,y resolution. To achieve or at least approach isotropic resolution the 3-D image can be deconvolved. Figure 1 shows the 2-D maximum value projection of a 3-D image of a cultured glial cell dual labeled for actin and vinculin before and after deconvolution. The actin fibers and vinculin focal contacts are more clearly resolved after deconvolution. Although a single cultured cell might traditionally be considered a thin object, it is really a thick object if the desired spatial resolution is less than the thickness of the cell. It is desirable to image as deep into a thick object as possible to maximize the tissue volume sampled. However, it has been shown that the image signal decreases with depth into the specimen. Figure 2 demonstrates this effect in a 3-D image of the nuclei of the rat hippocampus that have been labeled with the fluorescent Schiffs reagent acriflavine. In the x,y projection, it is not clear why some nuclei are dimmer than others, but the x,z projection shows that the dimmer ones tend to be deeper in the section. It has been shown that this depth dependent signal attenuation follows the form of an exponential function.
Electron Beam Induced Phase Transformations from Metakaolinite to Mullite
Authors: Kim Y J , Lee S
Published: July 2020

The kaolinite-mullite reaction series by heating has been controversial since the proposal of a new transformation mechanism. Recently energy-filtered electron diffraction data obtained from the specimen furnace-heated at 920 °C indicated that the spinel-type phase coexisted topotactically with metakaolinite. On the other hand, mullite first appeared from the specimen furnace-heated at 940 °C without showing any crystallographic relationship to the parent metakaolinite.

Direct beam heating experiments of the 920 °C-heated specimens using an energy-filtering TEM supported the previous results and ideas. Fig. 1 displays topotactic coexistence of metakaolinite and the spinel-type phase (spots in hexagonal arrangement) before beam heating. Electron beam heating gave rise to a noticeable change in the diffraction pattern, that is, the formation of mullite rings and enhancement of the diffuse intensity around the direct beam shown in Fig. 2. Comparison of EDS data for the specimen before beam heating and after beam heating (Fig. 3) indicates considerable increase of Si and O peaks by beam heating.
Study of Olivine Morphology, Spinifex-Textured Komatiite, by Atomic Force Microscopy
Authors: Coutol Lucia F , Vallet Catherine E
Published: July 2020

Atomic Force Microscopy (AFM) imaging of a komatiite specimen from the Morro do Ferro Greenstone Belt, M.G, Brazil, has revealed thin (0.2 - 2.0 micron in width), randomly oriented olivine crystals (fig. 1a,b), of prismatic shape. The length of such crystals is highly variable, ranging from less than 1 μm to more than 10 μm. The randomly oriented crystals typically occur in three or more distinct directions, indicating that the spinifex texture is reproduced at the micron-scale.

In addition, typical skeletal crystals were observed (figure 1c,d) with width of the order of one micron. Figure 1d is the sketch representation of the skeletal crystal shown on figure 1c Figures 1c,d also show that skeletal crystals sometimes branch out thinner crystals, indicating that nucleation also occurs at the surface of previously crystallized minerals, besides generating isolated new crystal seeds.

It is worth noting that the olivine crystals are characterized as low topographic regions on the AFM images. What distinguishes them from polishing scratches is the fact that scratches typically do not mimic crystal shapes, especially skeletal, or fabrics which are readily seen on different scales, i.e., petrographic sections or hand-specimens.
The Hunt for Red Corrosion: A Study of Microbial Rock Corrosion in Caves
Authors: Spilde MN , Northup D E , Boston PJ
Published: July 2020

A number of cave systems host colorful deposits of what has been termed “corrosion residue” (CR), material that appears to be the breakdown product of bedrock minerals. The CR may be red, pink, orange, ocher, brown gray, or black and usually occurs in a variety of places within caves (ceilings, walls, tops and sides of boulders, etc.). Geologists have hypothesized that CR is the long-term result of upwelling corrosive air. However, discovery of evidence of microbial activity has led to a complimentary explanation that microbes could be active participants in the production of the corrosion residue. These deposits have been cursorily examined in several caves, including Jewel Cave (SD), Lechuguilla and Spider Caves (NM), and Cueva de Villa Luz (Tabasco, Mexico) using microscopy techniques of SEM and TEM, along with EDS and WDS analysis.

In all cases, the CR is a complex mixture of iron and/or manganese oxides, clays, quartz, and corroded bedrock material. In several cases, rare earth element (REE) phosphate minerals and other unusual minerals have been observed. The REE minerals are believed to result from recrystallization of apatite present in the bedrock; rare earth elements are commonly present in trace amounts within the apatite minerals. X-ray diffraction and bulk chemical analysis has shown that Fe3+ makes up the bulk if not all of the Fe-oxides in the CR while microprobe analysis of thin sections of the bedrock has found Fe2+ in dolomite as high as 700 ppm.

Within much of examined material, evidence of microbial life is present in the form of structures shaped like coccoid or filamentous bacteria. There is often a close association of presumptive bacteria with small dissolution pits in corroded fragments of bedrock minerals in the CR (Figure 1). In addition to the putative bacteria, the mineral residue often hosts small star-shaped minerals containing Fe-oxide (Figure 2).
Electron Microprobe Dating of Uraninite from the Mcallister Pegmatite, Alabama
Authors: Bersch Michael G
Published: July 2020

In the early days of geochronology, attempts were made to date U-Th bearing rocks and minerals using the Pb-U-Th chemical method, but fundamental assumptions that all the lead was of radiogenic origin, and the system was closed, often were invalid. With the advent of the U-Th-Pb isotopic and common-lead methods of dating rocks and minerals, the chemical method was deemed clinically dead. However, several workers, for example, Montel et al., Suzuki and Adachi, have recently demonstrated new life for the chemical method. Using, the same theoretical basis as the chemical method, geologically reasonable dates often can be obtained by electron microprobe analysis of U-Th rich monazite (Ca-REE-U-Th phosphate). Other U- or Thrich minerals sometimes can be used to obtain microprobe U-Th-Pb dates, e.g., zircon, xenotime, uraninite.

Microprobe U-Th-Pb dates of uraninites from the McAllister Ta-Sn pegmatite, Coosa County, Alabama, are reported here. The McAllister pegmatite is a NW-SE dike-like body in the western part of the Rockford granite pluton. Polished grain mounts were made from screened table runs of heavy mineral separates. Wodginite (Mn, Sn, Nb, Ta-oxide), tantalite-columbite, zircon, cassiterite comprise most of the grains in the samples. Uraninite occurs as inclusions, generally <0.01 mm, in these grains (Figure 1).

U, Th, Pb concentrations were measured using a JEOL 8600 Superprobe. Operating conditions were 100 nA, 20 kV, and count times up to 200 seconds for peak plus background. The U Mβ, Th Mα, and Pb Mα peaks were measured. Standards were UO2, ThO2, and PbS.
The Golden Age of Microanalysis.
Authors: Heinrich Kurt F J
Published: July 2020

The main initial source of information in the USA concerning the electron probe was a meeting at the Naval Research Laboratories in February 1958, followed in 1960 by a summer school at MIT organized by Prof. Norton and R. Ogilvie. Further visits to the USA and publications in English by the inventor of the instrument, R. Castaing, also contributed to the attention given here to the new technique. Since numerous research laboratories in the USA were keenly interested in the new device, the development of instruments and the contributions to its use were considerable. The relatively small number of active researchers in the field rendered personal connections and cooperation possible and attractive.

Before the availability of American-made commercial microprobes, many investigators built their own instruments. Most of them were not trained as instrument builders, and the usefulness of the resulting devices was somewhat limited. These improvisations in instrument construction ceased once commercial microprobe manufacturers entered the field. Yet, in the process, much was learned and the American investigators contributed significantly to the theory of microanalysis. We realized that, with the limited calculation facilities then available, the complex interactions leading to the x-ray signal had to be treated in simplified ways, and we described ‘approximations’ rather than ‘laws’ in our efforts to understand and quantitatively describe the physical facts. We also came to realize that even where the physical processes were well known, we often lacked the knowledge of the physical parameters required for quantitation and the effect of their uncertainties on the accuracy of the analytical results. Another area in which advances were needed, and soon achieved, was the application of the technique to a wide range of technical and scientific problems. The interaction between manufacturers and users greatly stimulated improvements in the instrument design.
Crystal Spectrometers and Monochromators in Microanalysis
Authors: Wittry D B
Published: July 2020

An x-ray spectrometer is actually a scanning monochromator because two conditions must be satisfied, namely: (1) nλ, = 2d sinθ and (2) θI{= θR to within the rocking curve of the crystal 2Δθ. Before Castaing (B.C.) there were four known types of focussing monochromators, namely: Johann, Johansson, Cauchois, and von Hamos. Castaing was fortunate in having Andre Guinier (well known for his work in x-ray crystallography) as his thesis advisor. Thus, he wisely chose to use a spectrometer of the Johansson type. Had he used a flat crystal spectrometer, the result would have been unsatisfactory and the history of microanalysis would have been significantly different. Many of the spectrometers that were used in the early instruments were either of the Johansson type, in which the crystal planes are curved to a radius of 2R and the surface has a radius of R, the focal circle radius. This is sometimes called exact focussing because rays in the plane of the focal circle converge exactly to a point focus. For simpler construction, the Johann crystal which has the surface parallel to the atomic planes (also curved to a radius of 2R) was sometimes used. While both types provide one-dimensional focussing, the Johansson type is preferred because the effective width of the crystal can be greater.

In addition to the type of geometry used, early instruments developed after Castaing (A.C) also had variations in the mechanism of the crystal movement. Many used a relatively simple mechanism with the crystal and detector moving about a fixed center for the focal circle as shown in Fig. 1A. Some later instruments used curved crystals that were fixed in position, and scanning was accomplished either by rotating the crystal as in Fig. 1B or by a mechanism that flexed the crystal and changed the radius of the focal circle as in Fig. 1C. The most satisfactory arrangement and the one subsequently adopted by most manufacturers of commercial microprobes utilized a linear motion of the crystal carriage with a provision for tilting the crystal so that the focal circle always passed though the x-ray source as shown in Fig. 1D. If the detector moves so that it remains on this focal circle, its motion is along a leaf-shaped curve. With this design it is easy to have a readout proportional to wave length.