bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Resolution Microanalysis of Particles from the Human Lung
Authors: Bell David C , Rainey Lenore C , Vander Sande John B 
Published: July 2020

Abstract
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

Abstract
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

Abstract
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

Abstract
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 (&lt; 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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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]).