bluuebunny/crossref_metadata_2025 huggingface dataset:
The Hayashi-nishi Method for Measurement of Mixing:Computer-Generated Morphologies and Impact-Modified Polymers
Authors: Tant M R , Calvert D B , Wehner P S
Published: July 2020

In this work we apply the Hayashi-Nishi method for quantitatively analyzing the state of mixing of a secondary component in a polymeric matrix. We (1) evaluate the method using computer-generated morphologies and (2) compare the state of mixing of a core-shell impact modifier in polymers having differing affinity for the impact modifier. In the Hayashi-Nishi method two numbers, related to dispersion and distribution, are required to quantify the state of mixing of a secondary phase in a polymeric matrix. The 5-parameter in the Hayashi-Nishi method is defined as

The Ai's are the areas of each individual secondary phase particle or aggregate and nis the number of such particles in the image. S is related to the size distribution of these aggregate areas and is a measure of dispersion of the secondary phase in that particular image.
Investigation of the Fracture of Resinated Single Wood Fibers in an Environmental Scanning Electron Microscope (Esem)
Authors: Egan A , Shaler S
Published: July 2020

Single fiber fracture is important in understanding the fundamental failure mechanisms in wood/polymer composite products such as medium density fiberboard (MDF). The mechanical properties and fracture behavior of individual wood fibers has only recently been observable using a combination of environmental scanning electron microscopy (ESEM), laser scanning confocal microscopy and digital image correlation (DIC). Previous work has shown that specific areas on the fiber such as microcompressions and pits acted as crack nucelators and induce a brash fracture across the surface of the fiber. Given the development of these procedures it is now possible to observe and measure the mechanical properties and fracture characteristics of the wood fiber/ polymer composite fibers.

Individual black spruce wood fibers were coated with diphenylmethane 4-4'diisocyanate resin containing Hostasol Red GG. The addition of the Hostasol Red flurochrome provided the option of quantifying resin coverage by fluorescence microscopy.
Mid-Infrared, Raman and NMR Imaging of Flax
Authors: Himmelsbach D S , Khalili S , Gamble G R , Akin D E
Published: July 2020

Multiple microspectroscopic imaging techniques have been employed to reveal the chemical and structural nature of flax (Linum usitatissimum L.) stem tissue, with the goal of finding the best technique by which to monitor retting processes for the production of linen fibers. Three of these techniques, which we have investigated for this purpose, are: mid-infrared (MIR), Raman and nuclear magnetic resonance (NMR) imaging. Initially, all three techniques were evaluated on the basis of their ability to reveal the nature of unretted flax stems. MIR imaging was found to be the most useful for this purpose. Thus, it was selected as the method of choice to follow retting processes to obtain flax fibers.

A Fourier transform (FT)-IR spectrometer equipped with microscope was used to acquire the MTR images. A FT-Raman spectrometer that was equipped with a Raman microprobe and utilizing a 1064 nm laser source was used to collect the Raman images.
A Novel Technique to Determine Platelet Deposition on an Experimental Nylon Membrane Using Lvsem and Tem
Authors: Murphy MA , DiOrio JP
Published: July 2020

Porous nylon fabric is used as a filter in blood devices. This study was undertaken to evaluate the interaction between an experimental nylon filter and the cellular elements which pass through it. Our investigation was mainly centered on determining the deposition of cellular material on the nylon filter using Low Voltage Scanning Electron Microscopy (LVSEM) and Transmission Electron Microscopy (TEM).

The filter was prepared for LVSEM by cutting a 2.5 cm2 of the nylon filter material with a razor and placing it in a nylon mesh bag which was stapled shut. The bag was rinsed three times for ten minutes each in fresh Sorensen's phosphate buffer. A graded alcohol series of 25%, 35%, 50%, 70%, 80%, and 90% was used to dehydrate the filter for ten minutes each. This was followed by dehydration twice for 20 minutes in fresh 100% ETOH. The filter was then Critical Point Dried (CPD) in the Tousimis AUTOSAMDRI-814.
The Effect of Focused Ion Beam (Fib) Specimen Geometry on X-Ray Fluorescence During Energy Dispersive X-Ray Spectroscopy (EDS) Analysis in the Transmission Electron Microscope (TEM)
Authors: Longo David M , Howe James M , Johnson William C
Published: July 2020

The focused ion beam (FIB) has become an indispensable tool for a variety of applications in materials science, including that of specimen preparation for the transmission electron microscope (TEM). Several FIB specimen preparation techniques have been developed, but some problems result when FIB specimens are analyzed in the TEM. One of these is X-ray fluorescence from bulk material surrounding the thin membrane in FIB-prepared samples. This paper reports on a new FIB specimen preparation method which was devised for the reduction of X-ray fluorescence during energy dispersive X-ray spectroscopy (EDS) in the TEM.

Figure 1 shows three membrane geometries that were investigated in this study on a single-crystal Si substrate with a RF sputter-deposited 50 nm Ni film. Membrane 1 is the most commonly reported geometry in the literature, with an approximately 20 urn wide trench and a membrane having a single wedge with a 1.5° incline.
Material Dependence of Sputtering Behavior During Focused Ion Beam Milling: A Correlation Between Monte Carlo Based Simulation and Empirical Observation
Authors: Prenitzer BI , Giannuzzi LA , Brown SR , Irwin RB , Shofner TL , Stevie F A
Published: July 2020

The focused ion beam (FIB) lift-out method is a high precision technique by which site-specific cross-section transmission electron microscopy (TEM) specimens may be rapidly prepared from virtually any material. The technique is particularly useful when the sample geometry or composition is complex (e.g., fibers, powders, composites and interfaces). In addition to the preparation of TEM specimens, FIB milling has also found widespread utility in micromachining and microfabrication applications as well as specimen preparation for scanning electron microscopy (SEM) and secondary ion mass spectrometry (SIMS).

As the applications of the FEB instrument continue to become more universally recognized, the need to understand the interrelationships between the target material, processing parameters, and process efficiency of the milling phenomena becomes more critical. Incident ion attack angle, target material stopping efficiency and sputtering yield, Y, are important variables governing the milling process. TRIM, a binary collision approximation Monte Carlo simulation code, is used to physically model variables that influence FIB sputtering behavior.
The Small Angle Cleavage Technique for XTEM Sample Preparation
Authors: Walck S D , McCaffrey J P
Published: July 2020

The Small Angle Cleavage Technique (SACT) is a relatively simple and inexpensive method of producing superior cross sectional TEM specimens. For speed of preparation, it is unsurpassed; for example, ten samples can easily be prepared in about an hour from a typical material. It is particularly well suited for rapidly examining coatings and thin films very soon after they have been deposited. A major limitation of the technique is that it does require the substrate material to cleave or fracture. For this reason, it has been applied almost exclusively to semiconductor materials, but the technique has been extended quite successfully to other substrates such as glass, silicon carbide, quartz, sapphire, and other hard materials. Several procedures have been added or modified to the original technique developed by McCaffrey that makes it much easier to get started in using the technique. A detailed pictorial outline of the technique has been described elsewhere by the authors.
The Basics of Microtomy for Materials Science Microscopy
Authors: Malis Tom
Published: July 2020

The common theme of most forms of thin specimen preparation for the physical sciences has been the controlled, gradual removal of material, whether by electrolytic dissolution, ion bombardment, careful mechanical dimpling or step-by-step tripod polishing. It is somewhat ironic that a “brute force” method, wherein the material is forced at a high rate against an atomically sharp diamond wedge so that an ultrathin slice, or section, is sheared, or fractured, from a ‘block’ of the material, should be viewed not only as acceptable, but with growing enthusiasm in the last several years. This process, known as ultramicrotomy, originated in life science TEM, but has been modified by materials scientists and applied to an impressive array of materials as illustrated in Table 1, culminating, perhaps, with the recent successful sectioning of diamond film.
Deconvolution in 3-D Microscopy: Applications and Limitations
Authors: Shaw PJ
Published: July 2020

Many imaging processes, including both conventional wide-field and confocal fluorescence microscopy, can be described to a good approximation as linear and spatially-invariant. Linearity means that the image of an extended specimen is simply the sum of the images of the parts of the specimen - in the limit the specimen can be regarded as a collection of points, and its image is the sum of the images of the points - i.e. weighted instances of the imaging system's point spread function (psf). In the case of spatially invariant imaging the psf is the same over the entire field of imaging. Mathematically, the image is the convolution of the specimen with the system psf. In principle, this convolution can be reversed to remove degradation introduced by the imaging process, a computational procedure often called deconvolution or, more generally, restoration.
Wide-Field Deconvolution for Time-Lapse 3D Microscopy Of Cell Locomotion: The Good, The Bad and the Artifactual
Authors: McNally JG
Published: July 2020

We are using wide-field deconvolution microscopy to study the locomotion of cells within a three-dimensional cell mass. How cells move within such an environment is poorly understood, even though such movement occurs widely, for example, during embryonic development or tumor cell invasion. In these and other cases, cells moving within a cell mass face challenges not encountered by isolated cells crawling on a flat substrate. Cells within a mass must clear a path for themselves, and also gain traction on other cells which themselves may be moving in the same or another direction. To understand the mechanisms of such 3D movement, we observe and analyze it in both normal and mutant cells. The mutant analysis sheds light on which molecules are important for 3D locomotion.

To visualize either normal or mutant cell locomotion within a three-dimensional cell mass, we require time-lapse 3D imaging at light levels low enough to maintain specimen viability.
Optimization of Microfluidics for Genetic Analysis
Authors: St. John Pamela M , Connell Kip , Woudenberg T M , Deshpande M , Gilbert J R
Published: July 2020

Novel microfluidic designs are needed for fluid delivery and transport in an integrated micro-genetic analysis system and these designs often lead to complex relationships between the shape and size of the microchannels and the behavior of multi-component fluids. We are studying both pressure-driven and electrokinetic flow in various microfluidic channel designs fabricated from both glass and silicone substrates to understand chemical containment and transport. In conjunction with the experiments, simulations are being performed to predict behavior in microdevices and models are being used to develop a CAD tool for designers.

Fluorescent flow profiles are generated using a charged caged dye which fluoresces upon irradiation with 355 nm light from a Nd:YAG laser. The starting position of the dye is determined by a tightly focused laser spot <20 μm in diameter.
Scanned Probe Microscopy (AFM, et al.): How to Choose and Use
Authors: Russell Phillip E , Batchelor A D
Published: July 2020

Virtually everyone associated with a science or engineering discipline has some baseline knowledge of optical microscopy; and most attendees at this conference have a reasonable exposure to at least some form of electron microscopy. The many developments in instrumentation and application require the modern microscopist to continuously follow the literature to stay aware of the ongoing improvements and advances in these microscopies. While electron and optical microscopes have been around for many decades, the family of microscopes known as Scanned Probe Microscopy (SPM) are just entering their second decade; and are actually in there first decade of widespread use.

The most commonly used forms of scanned probe microscopy are the force microscopes; commonly referred to as Atomic Force Microscopy (AFM). This includes a wide variety of microscopy modes that are generally made available as modifications of a basic AFM. Before describing the basic versions of AFM, the fundamentals of instrument design must be addressed.
Fixing and Embedding “Difficult” Biological Material
Authors: Mollenhauer Hilton H
Published: July 2020

Introduction: This is a personal account of the author's attempts to solve some of the problems associated with the fixation and embedding of difficult biological materials such as plant tissues, almost all insect tissues, and large pieces of tissues including whole organs.

Historically, the search began in the 1950's with the study of the maize root tip using the relatively new technique of electron microscopy. This system of cells, separated by thick and partially impermeable walls, did not respond well to the procedures most suitable for animal tissues. New approaches were needed and, in time, some were found that were at least useful if not ideal. A brief account of several of these is given in the following paragraphs.

Fixation of plant cells: Potassium permanganate was chosen very early in the study primarily because it worked better for plant cells than did any of the fixation procedures currently available.
The Pulmonary Circulation in Health and Disease
Authors: Aharinejad S , Schraufnagel DE
Published: July 2020

SEM analysis of pulmonary vascular casts shows basket-like alveolar capillaries that are about 6 μm in diameter. Generally pulmonary arteries are adjacent to airways and veins are not. The pleural surface and the airways capillaries have a planar meshwork with few large vessels. Uncommonly are lymphatics cast in normal lungs but they are regularly found in casts of edematous lungs. Casts of pulmonary veins in several species have smooth surfaces with a slight notch occurring at regular intervals; these notches will deepen after physiological interventions and are sharply constricted in spontaneously hypertensive rats (SHR) with pulmonary hypertension and cattle. The circular bands are caused by tufts of smooth muscle cells and have been called pulmonary venous sphincters (Fig. 1). The depth of the constrictions can be measured to show the effect of an experimental intervention on individual small veins, which is important in understanding the pulmonary microcirculation and its control.
Using Correlative Microscopy in the Study of Biological-Biomaterial Interactions
Authors: Albrecht RM
Published: July 2020

The application of two or more imaging-based technologies to portions of a sample prepared in parallel or preferably to the exact same sample in series is the basis for correlative microscopy. Simultaneous visualization of structure in conjunction with other cellular parameters such as cell motility, intracellular ion concentration, receptor expression, and protein distribution/movement can be followed in living cells by various forms of photon, acoustic, and force based microscopy. The same cells can subsequently be prepared by various physical or chemical based procedures and reexamined by other microscopic methodologies to gain additional qualitative and quantitative information often at significantly increased levels of resolution. Recent advances key to correlative microscopy have been the development of high sensitivity, high resolution video cameras, and computer image processing, image enhancement, image analysis, and reconstruction capabilities. New instrumentation such as video enhanced, rectified interference-based light microscopy,LM; confocal LM, and two and three photon LM also have been critical.
A Novel Approach for Characterisation of Adhesive Toothbiomaterial Interfaces by ATM.
Authors: Van Meerbeek B , Yoshida Y , Snauwaert J , Hellemans L , Lambrechts P , Vanherle G , Wakasa K , Pashley DH
Published: July 2020

The ability to adhere biomaterials to tooth tissue has drastically changed today's dental restorative practice. Modern adhesive techniques enable dentists to restrict operative procedures to the sole removal of diseased tooth tissue without further destruction of the remaining sound tissue. However, adhesive restorations have only a short lifetime of ca. 5 years. Extending the restoration lifetime is therefore one of the primary goals of present research in dental materials science. In practical relevance and direct benefit to that objective are studies that contribute to a better understanding of the interfacial phenomena that take place between restorative material and tooth tissue. Therefore, the resolution of instruments available to elucidate this interfacial interaction is essential. Besides the transmission electron microscope (TEM), a more recent powerful tool to study tooth-biomaterial interaction with high resolution is the atomic force microscope (AFM).
Imaging of Explanted Mechanical Heart Valves
Authors: Goodman SL , Harasaki H , Wika KE , Brendzel AM
Published: July 2020

Prosthetic mechanical heart valves (MHVs) made from pyrolytic carbon (PYC) are a highly successful biomaterial application due to their excellent durability and clinically adequate blood compatibility. Their thromboresistance has been attributed to a combination of hemodynamics, material inertness, and surface properties which minimize platelet adhesion and activation. These surface properties include that polished PYC is smooth and that it strongly adsorbs plasma albumin, which is well known to reduce platelet adhesion. However, recent evidence suggests that this explanation is incomplete. Low voltage SEM shows that PYC valve leaflets are actually quite rough at submicron scales and that PYC induces extensive platelet spreading in vitro in the presence of albumin. Surprisingly few studies have examined MHVs explanted from humans or research animals for platelet adhesion and thrombosis using SEM. Moreover, in most studies it appears that even routine sample preparation methods for the preservation of biological samples for SEM,
CTF Corrected Structure of the Closed-State Calcium Release Channel
Authors: Ludtke S , Serysheva I , Hamilton S L , Chiu W
Published: July 2020

Several structures of the Ryanodine receptor in different functional states with various bound ligands have been determined using electron cryo-microscopy in recent years. These structures have varied in resolution from 25-40Å, however, to date, none have been corrected for the CTF (contrast transfer function) of the microscope. The CTF can have a significant effect on the detailed structure of the molecule, causing features in the 40-70Å range to be unnaturally emphasized. This causes small holes to become larger and small protrusions to become elongated. Unfortunately due to zero points in the CTF, correction cannot be properly accomplished with individual images. Several approaches to correcting the CTF have been proposed and/or used by combining information from images taken at different defocus settings. We have taken the approach of performing CTF correction as a preprocessing step. There are both advantages and disadvantages to this method. There are 2 primary advantages: First, by combining data from identical regions of focal pairs,
FEISEM, Form and Function in the Nuclear Pore Complex
Authors: Allen TD , Bcnnion G R , Rutherford S A , Kiscleva E , Goldberg M W
Published: July 2020

Recent initiatives have resulted in a considerable increase in our understanding of the structure of the nuclear pore complex (NPC). The biochemical factors involved in both import and export have been rapidly characterised, with steady progress in the molecular dissection of the structural elements of the NPC, which is a unit of considerable molecular architecture (MW 125 kD), comprising an estimated 50- 100 different proteins. Despite this progress, the crucial molecular interactions involved in the mechanics of transport through the central transporter of the NPC remain unclear. NPC structure in Diptera, fish, (Fig 1) amphibians, birds and mammals shows a high degree of evolutionary conservation. 3D reconstructions of isolated yeast NPCs, show that the core structure is very similar to ‘higher’ organisms, but peripheral structures may be considerably reduced in structural complexity (1).

Individual NPC components have been accessed in FEISEM by a variety of methods, including proteolysis,
Structural Studies of Translocation Channels: The Nuclear Pore Complex and the Translocon
Authors: Yang Q , Ménétret J-F , Akey IV , Plath K , Rapoport T A , Akey C W
Published: July 2020

Protein translocation plays a critical role in the targeting of both soluble and membrane proteins to their correct intra- and inter-cellular compartments. We are studying the 3D architecture of two rather different translocation machines, the Nuclear Pore Complex (NPC) and the ribosome-Sec61p complex (translocon), with the aim of understanding their physical mechanisms of gating and transport. Towards this end, we are using single particle electron cryomicroscopy and 3D reconstruction of frozen hydrated specimens to obtain interpretable maps that are biologically relevant.

Previous work suggested that a central channel complex (termed the transporter) is present in vertebrate NPCs. Based on classification studies of the transporter and STEM images of Chironomus NPCs caught translocating large mRNPs, we have hypothesized that the transport mechanism utilizes a double iris-like gating mechanism, in which oppositely facing gates located at either end of the transporter open asynchronously. Recently, we have extended our studies to yeast NPCs and shown that this organelle is markedly smaller than its vertebrate cousin.
Cytomegalovirus Capsid Structure and Tegument Binding
Authors: Steven A C , Gibson W , Cheng N , Trus B L
Published: July 2020

Herpesviruses form an extensive family of DNA viruses, in which three subfamilies called alpha-, beta- and gammaherpesviruses, respectively, are distinguished on the basis of biological properties. In terms of overall structure, all herpesviruses conform to a common plan: the virion consists of a thick-walled capsid, ∼ 1250 Å in diameter, containing the genome, surrounded by a complex layer of proteins called the tegument (a feature unique to herpesviruses), and a lipoprotein envelope. At a more detailed level, it has not been clear whether the three subfamilies are also differentiated structurally. The molecular architecture of alphaherpesvirus capsids has been studied by cryoelectron microscopy of herpes simplex virus 1, revealing its icosahedral (T=16) surface lattice and the locations of the four abundant shell proteins. To ascertain how closely these features are emulated in a betaherpesvirus, we have studied capsids of simian cytomegalovirus (SCMV) by similar methods. By comparing the structures of nuclear capsids and cytoplasmic capsids (which have bound some tegument proteins),
Cryo-Electron Microscopy and Image Processing Methods for Studying the Ribonucleoprotein Vault
Authors: Kong L B , Rome L H , Stewart P L
Published: July 2020

Vaults are highly conserved ribonucleoprotein complexes found in thousands of copies per cell in various tissues. The major vault protein, pi 04, accounts for over 70% of the particle mass and has been recently found to be involved in cancer cell multidrug resistance. We have utilized cryoelectron microscopy (cryo-EM), and three-dimensional image reconstruction to generate a 3-D structure of the vault particle. Individual particle images were isolated using the QVIEW software package, which simultaneously performs density exclusion, planar background subtraction, and application of an elliptical mask. The IMAGIC-5 software package was used for the subsequent particle orientation and reconstruction steps (Fig. I).

A symmetry self-search test was used to measure the signal strength for a variety of point groups and the highest correlation was found for the cyclic 8-fold point group. We have utilized the sinogram correlation algorithms in IMAGIC-5 to find the Euler angles of both 1 μm and 2 μm defocus particle images.
Protein Unfolding and Degradation by the CLP Family of Proteases
Authors: Kessel Martin , Beuron Fabienne , Booy Frank , Kocsis Eva , Maurizi Michael , Steven Alasdair
Published: July 2020

ATP-dependent proteases play a major role in regulatory protein degradation in both prokaryotic and eukaryotic cells. ATP-dependent proteases in E. coli fall into two classes. The first class requires the interaction of structurally separate proteases with an ATPase, whereas in the second class both the protease and ATPase are formed from regions of the same polypeptide chain. We have studied the structure of several of these protein degrading complexes in E. coli and have found a remarkable similarity in the architecture of these macromolecular assemblies.

The prototypical protease of the first class has as its proteolytic component ClpP, a 14 subunit (MW 21,500) complex arranged as two lOnm-diameter stacked rings of seven subunits each. ClpP can interact with either one of two ATPases, ClpA or ClpX, each with unique substrate specificity. ClpA has two ATP-binding sites per subunit (MW 84,000), and its subunits are arranged as a 13nm (diameter) hexameric ring (MW -500,000).
On the Structure-Function Relationships of the Unique Proteinase Inhibitor Human α2 Macroglobulin
Authors: Stoops James K , Kolodziej Steven J , Qazi Usman , Nolasco Norman J , Gettins Peter GW , Strickland Dudley K
Published: July 2020

Human α2 macroglobulin (α2M) has structural and functional properties that contribute to its uniqueness as proteinase inhibitor. It is the largest known (Mr=720,000) and the only natural proteinase inhibitor which has a broad range of reactivity and for which the reaction is irreversible. It has a vital role in the clearance of proteinases from the circulation and in regulating their activity in fibrinolysis, coagulation and complement activation.

An α2M molecule can entrap two proteinase molecules such as chymotrypsin and trypsin and can therefore be considered to contain two functional domains. Each subunit in the homotetramer has a bait region with cleavage sites for nearly all known endoproteinases and an internal thiol ester bond. A proteinase cleaves the two bait regions within both functional units leading to an activation and cleavage of the thiol ester bonds. Consequently, α2M undergoes a major structural change resulting in the irreversible entrapment of the proteinase.
Fluoronanogold: An Efficient Labeling Reagent for Immunocytochemistry
Authors: Robinson JM
Published: July 2020

The field of immunocytochemistry comprises a large body of methodologies whose purpose is to obtain spatial and temporal information about biological samples with a high degree of chemical specificity. The major differences in immunocytochemical methods are: (1) type of sample preparation (i.e., pre-embedding or post-embedding) and (2) type of detection system (i.e., chromogenic, fluorochromes, or particulate). The history of advances in immunocytochemistry can be understood, to a large extent, through the development of the various detection systems. All of these labeling systems have their appropriate applications and are in use today.

The introduction of colloidal gold as a particulate immunoprobe revolutionized immunocytochemistry, particularly at the ultrastructural level. The fact that these probes are discrete particles that are electron dense contributes to their wide acceptance. Due to the particulate nature of these probes, labeling density over a given structure or region of a cell can be determined by simple counting procedures.
Fluorescent Antenna Proteins from the Bioluminescent Bacteria
Authors: Lee John
Published: July 2020

The emission spectrum from bioluminescent bacteria has been observed to depend on the type of bacteria. Photobacterium phosphoreum species usually show bioluminescence maxima around 472 nm and Photobacterium leiognathi species to slightly longer wavelength. A certain strain (Yl) of Vibrio fischeri, has a yellow bioluminescence with maximum at 542 nm. These differences have been explained as due to the bioluminescence originating from the fluorescence transition of an “antenna” protein, participating in the bioluminescence reaction along with the enzyme bacterial luciferase. The bioluminescence from a number of coelenterates involves a similar participation of an antenna protein, the famous “Green-Fluorescent Protein” being the origin of the bioluminescence emission from these organisms.
Quantitative Imaging Of the Green Fluorescent Proteins
Authors: Piston David W , Patterson George H , Knobel Susan M
Published: July 2020

The cloning and expression of GFP in heterologous systems introduced a fantastic tool for studying specific gene expression and protein localization inside living cells. However, one aspect of GFP that has not been exploited to its full potential is its use as a quantitative imaging tool. To determine its quantitative usefulness, we have addressed five points that are important in GFP imaging: detectable signal over background, photostability, pH stability of the molecule, temperature dependence of chromophore formation, and estimation and normalization of GFP levels.

To determine the quantitative limits of GFP in cells, several GFP versions (wtGFP, αGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F)) were compared by imaging of GFP expressing cells or by spectroscopic measurements of purified proteins. When imaged, the GFP signals are contaminated by the naturally occurring background autofluorescence, but improved detection can be achieved for each green GFP by combination of confocal microscopy using 488 nm excitation, a rapid cut-on dichroic mirror, and a narrow bandpass emission filter (Figure l).
GFP-Tagged Protein Domains as Tools to Study Localized Second Messenger Function in Living Vells
Authors: Meyer T , Oancea E , Stauffer T , Teruel M N
Published: July 2020

We have investigated the translocation of different GFP-tagged protein domains in response to various receptor stimuli. Confocal fluorescent imaging was used to study the localization of the GFP-tagged protein domain before, during and after the receptor stimuli was applied. A minimal cysteine-rich domain from protein kinase C was shown to be a useful tool to study localized diacylglycerol changes. Furthermore, the calcium-induced plasma membrane translocation of the GFP-tagged C2-domain from PKC could be used to study receptor-mediated changes in near plasma membrane calcium concentration. In addition, different pleckstrin homology domains (PH-domains) could be employed to measure changes in the concentration of phosphatidylinositol 4,5-bisphosphate (PI4.5P2) as well as of P13,4P2and PO,4,5P3. In combination, these fluorescent translocation probes provide a new means to investigate the local and temporal control of cell signaling processes in individual living cells.
Dynamin Mediates Membrane Vesiculation
Authors: Sweitzer Sharon M , Hinshaw Jenny E
Published: July 2020

Dynamin, a 100 kDa GTPase, is essential for receptor mediated endocytosis and synaptic vesicle recycling; however its mechanism of action is unknown. The requirement for dynamin was first elucidated by the discovery that the shibire gene product in Drosophila melanogaster was homologous to mammalian dynamin-1 (1,2). The shibire flies exhibit a depletion of synaptic vesicles and an accumulation of collared clathrin-coated pits at the plasma membrane of their nerve termini (3). It was later demonstrated that endocytosis was inhibited by the overexpression of dominant negative mutants of dynamin (4,5), and that purified dynamin can self-associate to form spirals which resemble the collars of shibire and structures seen in synaptosomes treated with GTPγS (6,7). These observations led to the speculation that dynamin pinches the clathrin-coated bud from the plasma membrane. In support of this hypothesis, we show that purified recombinant dynamin can bind to a lipid bilayer in a regular and repeating pattern to form helical tubes which vesiculate upon the addition of GTP.
Transport Through the Secretory Pathway: Observations of Cargo and Peripheral Coat Proteins
Authors: Presley John F , Cole Nelson B , Lippincott-Schwartz Jennifer
Published: July 2020

We have used green fluorescent protein (GFP) chimeras to examine the dynamics of the early secretory pathway and the role of the peripheral coat protein COP I. To describe the overall properties of ER to Golgi transport we used the temperature sensitive viral glycoprotein, ts045 VSVG, tagged with GFP at its cytoplasmic tail. VSVG-GFP retained the temperature sensitive phenotype of its parent: it reversibly misfolded and was retained in the ER at 40°C. Upon shift to 32°C it was rapidly exported from the ER, moving as a synchronous pool into the Golgi complex and then to the cell surface. Using time-lapse imaging of living cells expressing VSVG-GFP we found that the carriers for ER to Golgi traffic are tubulovesicular pre-Golgi intermediates that move centrosomally to the Golgi at speeds of>1 μM2/sec and then fuse with the cis face of the Golgi complex. These movements are dependant on microtubules and the dynein/dynactin complex.
Neutrophil Microtubules Display Unusual Properties and are Highly Sensitive to Taxol
Authors: Robinson JM , Vandré DD
Published: July 2020

The microtubule (MT) cytoskeleton plays a crucial role in a number of cellular functions including cytoplasmic organization, intracellular transport, cellular polarity, and cell division. Microtubules are linear polymeric structures composed of heterodimers of α- and β-tubulin with a class of specialized proteins, the microtubule-associated proteins, typically being present. Microtubules are dynamic structures that undergo repeated rounds of assembly/disassembly. The half-life of MTs, an index of their dynamics (i.e., assembly-disassembly), has been estimated from studies of single cells in culture and typically ranges from ≈ 5 - 20 minutes depending on the cell type. These dynamic properties of MTs have been explained in terms of the “dynamic instability” model. Five parameters have been used to describe dynamic instability of MTs: (1) elongation velocity; (2) shortening velocity; (3) nucleation frequency; (4) catastrophe frequency; and (5) rescue frequency. Catastrophe is the transition from elongation to shortening while rescue is the transition from shortening to elongation.
Molecular Regulation of Taxane-Induced Apoptosis
Authors: Bhalla K
Published: July 2020

Following intracellular uptake, taxanes bind to β tubulin and suppress microtubular dynamics, resulting in stable microtubule bundles and mitotic arrest of cancer cells. This proximal taxane- β tubulin interaction depends on the amount of intracellular accumulation of taxanes. Since taxanes are substrates for the membrane transporter P-glycoprotein (PGP) but not the MRP, overexpression of PGP but not MRP reduces the intracellular accumulation and cytotoxicity of taxanes. Recent reports have also suggested that alterations in the levels of specific β tubulin isotypes can reduce the effects of taxanes on microtubule dynamics, thereby inhibiting the mitotic effects and resulting cytotoxicity of taxanes.6 For example, increased βIV and (βIII tubulin levels may confer resistance against microtubule and cytostatic effects of taxanes. Taxane-induced G2/M arrest is preceded by the activation of cell-cycle mitotic kinase CDK1, which promotes the G2/M transition. Recent studies have indicated that the loss of cell-cycle Gl checkpoint due to p53 mutations may enhance taxane-induced mitotic arrest and cytotoxicity.
Taxol-Induced Apoptosis May Occur Via a Signaling Pathway Independent of Microtubule Bundling and Cell Cycle Arrest
Authors: Fan Weimin , Miller III Merrill C , Cheng Lirong , Willingham Mark C
Published: July 2020

Taxol, a plant-derived antimicrotubule agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. This naturally occurring antineoplastic drug has been demonstrated to possess broad activity against a variety of human solid tumors, particularly in drug-refractory ovarian cancer and metastatic breast cancer (1). However, the exact mechanism of taxol's cytotoxicity against tumor cells is not entirely clear. Recent studies have demonstrated that taxol, besides causing microtubule bundling and mitotic arrest, is able to induce internucleosomal DNA fragmentation and typical morphological features of apoptosis in a number of solid tumor cells. These results clearly indicate that taxol, in addition to its classical activity against microtubules and cell cycle arrest, also possesses significant cell-killing activity by induction of apoptosis.

Although it is well recognized that taxol can cause both mitotic arrest and apoptotic cell death, it remains unclear whether taxol-induced cell death is a secondary event resulting from mitotic arrest or represents a novel mechanism of taxol against tumor cells.
Towards Understanding the Structural Requirements for Endogenous Transcription in Rotavirus
Authors: Lawton J A , Estes M K , Venkataram Prasad B V
Published: July 2020

Rotaviruses are complex, relatively large (1000 Å) nonenveloped icosahedral viruses. They are the major causative agents of severe diarrhea in children and animals. Annually an estimated 1 million children die from rotavirus-induced gastroenteritis worldwide. Medical relevance, intriguing structural complexity, and several unique strategies in the morphogenesis and replication have provoked extensive molecular biological and structural studies on rotaviruses in recent years.

Rotaviruses are double-stranded RNA (dsRNA) viruses belonging to the family Reoviridae. Each of the 11 segments codes for one protein. Of the eleven proteins encoded by the genomic RNA, six are structural and five are non-structural. Structural studies have shown that mature rotaviruses are triple-layered particles (TLPs). During the process of cell entry the outer-most layer consisting of VP7 and the spike protein VP4 is lost and the resulting double layered particles (DLPs) become transcriptionally active (Fig. 1 A). During the process of transcription, the DLPs maintain their structural Integrity.
The Application of Microscopy and Other Imaging Techniques to the Study of Disease in the Fetus.
Authors: Blackburn WR , Hudson J , Cooley,Jr NR
Published: July 2020

The application of both classical and advanced microscopy to the exploration of abnormal, human, fetal development (birth defects) and to other forms of human, fetal disease has generated a wealth of information, often documented by dramatic images. We have studied, at varying levels of inquiry (simple inspection to molecular analysis), some 10,000 normal and abnormal specimens. For presentation purposes, we first demonstrate the use of imaging in explorations of the fetal habitat (the gestational sac). Our studies reveal a variety of fetal surface encounter lesions (skin-to-amnion). Many of these lesions in the past have been erroneously interpreted. These include fetal amputations, strangulations, and also the reimplantation of amputated parts to ectopic sites. We have also explored, by various imaging techniques, developmental abnormalities within a variety of fetal organs.
A Morphometric Analysis of Early Effects of Ethanol on Hepatocyte Organelles from Peromyscusmaniculatus
Authors: Ellzey J T , Drake J P , Dader L , Boentges P
Published: July 2020

Pathological changes of hepatocytes from rats fed a 30% ethanol-derived calories diet for three weeks include noticeable ultrastructural changes including steatosis and hypertrophy of the smooth endoplasmic reticulum. We sought to examine hepatocytes of deer mice administered ethanol in an inhalation chamber for two weeks to determine if subtle changes occur in hepatocyte organelles prior to steatosis.

Two strains of Peromyscus maniculatus, ADH-positive possessing hepatic cytosolic alcohol dehydrogenase and ADH-negative deer mice lacking this enzyme were purchased from the Peromyscus Genetic Stock Center (Univ. of South Carolina). They tested negatively for Hanta viruses. A morphometric analysis of the ultrastructure of ADH+(n=14) and ADH- (n=14) controls as well as experimentals exposed to chronic, intoxicating levels of ethanol was conducted. Blood ethanol levels were maintained between 1.25-1.75 mg/ml for two weeks in the experimentals.
Sertoli Cell Adhesion Molecules: Comparative Expression of Lselectin and Other Cams in Primary Cells And Immortalized Sertoli Cell Lines
Authors: Broome Ann-Marie , Millette Clarke F
Published: July 2020

Cell adhesion and cell adhesion molecules (CAMs) play a crucial role in testicular development and function. The seminiferous epithelium, the functional unit of the testis, represents a three dimensional architecture of supporting Sertoli cells (SC), and developing germ cells (GC). The seminiferous epithelium, therefore, must be receptive not only to individual cell growth and differentiation, but also to cell-cell interactions. Morphologically distinct cell-cell interactions occur between SC and GC and also between SC.[1] In general, these junctions can be categorized into three types: adhesive, occluding, and gap junctions. The orientation and function of these junctions are interaction dependent. For example, desmosome-like junctions (spot desmosomes) are found between SC and GC. These junctions are present in the basal and intermediate compartments of the testis and serve to translocate developing GC. SC-SC interactions, like the zonula occludens (tight junction), function as vectorial mediators, maintaining the blood-testis barrier and SC polarity.
Nitric Oxide Synthase in Diabetic Retinopathy in the BBZ/WOR Rat: An Immunocytochemical Study
Authors: Ellis E Ann , Grant Maria B , Guberski Dennis L
Published: July 2020

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and the highly reactive free radical species, NO. Two major constitutive isoforms, neuronal and endothelial NOS, have been ented in the retina. These constitutive isoforms produce small amounts of NO while the inducible isoform, iNOS, produces high levels of NO. Excess production of NO by iNOS has beem implicated in the complications of diabetes. Since NO is a highly labile molecule, its potential localization is best achieved by immunocytochemical studies of NOS.

Semiquantitative colloidal gold labeled immunocytochemical studies of endothelial NOS (eNOS) and iNOS were done on sections of neural retina in eyes of obese, noninsulin dependent diabetic BBZ/Wor rats with diabetes of five months to twelve months duration. Age matched, nondiabetic control retinas were from eyes from BBDR/Wor rats.

Levels of iNOS were elevated in diabetic (33.9 ± 10.0 gold particles) as compared to age matched nondiabetic control (3.5 ± 2.8 gold particles) retinas.
The Effects of Cigarette Smoke on Porcine Airway Epithelium
Authors: Morgan Darrell D , Moss Anthony G
Published: July 2020

Cephalad pulmonary mucociliary clearance driven by cilia of the ciliated airway epithelium provides probably the most important line of defense against inhaled toxins and particulate material for organs of the thoracic cavity. We demonstrate the reorganization of the cytoskeleton and the endoplasmic reticulum of airway epithelial cells upon cigarette smoke inhalation by employing DIC, LSCM, TEM and widefield fluorescence microscopy and have correlated this reorganization to changes in ciliary beat frequency (CBF) via FFT analysis. Neonatal pigs were used to provided healthy tracheal epithelial tissue. Exposure to cigarette smoke causes rapid ultrastructural changes including: ciliary distortion and detachment, ciliary abscission and severe alteration in endoplasmic reticulum structure suggesting profound disruption of essential membrane-cytoskeletal linkages.

To examine changes in CBF we describe a simple approach, using a laser scanning confocal microscope (LSCM) and an analog-to-digital computer converter/analyzer for the acquisition of data from biological systems that undergo rapid periodic movement, such as ciliary motion.
ION-Channel Activator-Induced Ultrastructural Changes in the Dog Retina
Authors: Megill JR , Monticello TM , Wasserman AJ , Kelly WA , Durham SK
Published: July 2020

The sensory portion of the retina is composed of multiple cell layers with complex functions and interactions vital to ocular homeostasis. Xenobiotics can induce physiological and morphological perturbations of these cell layers, and subsequently compromise vision. We describe an ion channel activator (ICA)-induced retinopathy observed in a preclinical drug safety study in dogs. Purebred beagle dogs received either ICA in vehicle (0.75% Methocel® and 0.05% Tween 80) or vehicle alone (control) for nine months. During the study, visual deficits and fundic changes were observed clinically, leading to premature termination of the study at eight months. The posterior globe of the left eye was fixed by the method of Acland and Aguirre, dehydrated in a series of graded methanol baths, and embedded in epoxy resin. Semi-thin (l-μm thick) sections of the retina were stained with toluidine blue, and examined by light microscopy. Ultrathin (60-90-nm thick) sections of the retina were mounted on copper grids,
Electron Microscopic Immunolocalization of Basic Fibroblast Growth Factor-Like Molecules in Capillary Endothelial Cells
Authors: Aktas Ranan Gullhan , Kayton Robert J
Published: July 2020

Basic fibroblast growth factor (bFGF) is a potent angiogenic polypeptide. It promotes angiogenesis in vivo and in vitro by stimulating migration, proliferation and proteolytic activity of endothelial cells. Whereas several effects of exogenous bFGF on endothelial cells have been described, it has remained unclear how endogenous bFGF produced by vascular endothelial cells regulate angiogenesis.

To further investigate functional implications of the distribution of bFGF, we undertook the present study. Our aims were (i) to identify the specific location of bFGF in endothelial cells using electron microscopy immunogold labeling technique (ii) to determine the distribution of bFGF in capillaries of different types of tissues.

Tissue samples from sciatic nerve, hippocampus, adrenal gland and kidney of normal adult rats were fixed in 4% paraformaldehyde/1 to 5% glutaraldehyde and embedded in Spurr's resin. Ultrathin sections were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-ZymoGenetics) specific for bFGF using a two-step immunogold labeling method.
Acid Phosphatase Activity in Spiral Ganglion Neurons of C57BL/6 Mice
Authors: Cohen Glenn M
Published: July 2020

C57BL/6 mice, along with several other mouse genotypes, have served as models for human presbycusis (age-related hearing losses). C57BL/6 mice and their genetic substrain C57/M6 show progressively severe hearing losses, starting as early as 30 days postnatally. The hearing losses result from sweeping degeneration of sensory (hair) cells and neurons that begins in the basal end of the cochlea and advances apically. Although the underlying mechanisms orchestrating sensory and neural degeneration are not known, it is possible to correlate degenerative events with the cytoplasmic levels and distribution patterns of a marker molecule, such as acid phosphatase (AP). AP, a representative lysosomal enzyme, plays a role in both normal cellular metabolism and degenerative changes (trauma and senescence). AP activity is visualized histochemically at the light and electron microscopic levels by the presence of dense deposits within lysosomes.