bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Dual Nipkow Disk Confocal Microscope and High Speed Confocal Imaging: New Designs, New Cameras, and New Capabilities.
Authors: Peeters G A , Buchin MP 
Published: July 2020

Abstract
The introduction of a dual Nipkow disk scanning head should facilitate confocal microscopic study of live specimens. The use of micro-lenses in this design improves the Z-axis resolution, scan speed and overall optical efficiency of the original Nipkow disk design. Advantages of this device include the capability for high speed scanning (360 scans/sec), real time viewing, superimposed transmitted light and confocal imaging, together with reliable optical alignment, and a compact modular scan head that attaches to the video port of existing microscopes. When used with low light & light amplified cameras, lower power densities can be used to illuminate live specimens resulting in longer experiments being successfully completed.
The schematic diagram shows the optical path and specifications of the new scan head. A pre-aligned collimated light beam illuminates the upper micro-lens disk array. Each lens within the array focuses light through a small pinhole aperture on the lower Nipkow disk providing confocal multi-point scanning of the specimen. Fluorescent light emitted from the specimen passes back through the pinhole apertures to the dichroic mirror located between the two rotating disks. The dichroic mirror reflects the confocal image into the detector path. The confocal image can be observed directly through a 10x ocular or relayed to the camera port. The camera is chosen to achieve the desired combination of speed, dynamic range and spatial resolution. The acquisition, display, and recording aspects of the system are matched to the camera and are readily available. Limitations of the system include a fixed Z-axis resolution determined by the objective lens and pinhole size.
A Confocal Microscope with Spectrophotometry Detection
Authors: Calloway C B 
Published: July 2020

Abstract
The Leica TCS SP Confocal Microscope combines spectrophotometric detection with confocal microcopy. The result is a multi-channel confocal imaging spectrophotometer that significantly increases the flexibility and efficiency of the detection system.
In conventional point scanning confocal microscopes excitation light from laser(s) is delivered to the scan head via fiber optic, passed through a pinhole, reflected by a primary dichroic to the scanning system, and scanned onto the surface of the specimen. Light emitted from the specimen is descanned and passed to the detection system. The detection pinhole(s) are placed either between the primary dichroics and the detection system as in Leica confocal microscopes or closer to the end of the detection system. In the detection system emitted light is separated using a combination of dichroics, mirrors, and barrier filters before being passed to the photomultiplier detectors.
The Leica TCS SP Spectral Confocal microscope has the same beam path as the filtered system up to and including the detection pinhole. The detection system including the secondary dichroics and the barrier filters is replaced by the spectrophotometer detection system (SP). The light emitted from the focal plane that passes through the detection pinhole has both intensity and spectral information. In the SP system this light is passed through a prism. The prism splits the emitted light into a spectrum from 400 to 750 nm. The spectrum is directed at a PMT for detection. The physical dimension of this spectrum is such that the entire spectrum can be imaged onto the window of the PMT.
A New Generation, Fast 3D Fluorescence Microscope using Wavefront Coding Optics
Authors: Cogswell Carol J , Arnison Matthew R , Dowski Edward R , Tuckert Sara C , Thomas Catheyt W 
Published: July 2020

Abstract
We are developing a “new-generation” fluorescence microscope that will allow very fast (milliseconds) acquisition of fully three-dimensional (3D) images for a wide spectrum of biological applications. This new system will overcome the slow image acquisition constraint of existing confocal and widefield deconvolution microscopes (the two most commonly used instruments for 3D fluorescence imaging) that has prevented them from being used for investigations of live-cell dynamics in three dimensions. Our new microscope incorporates the innovative techniques of optical wavefront coding, pioneered by W. T. Cathey and E. R. Dowski, University of Colorado. With this new system, as compared to the normal sequential plane-by-plane image acquisition requirement of confocal and widefield microscopes, we need acquire only a single CCD camera image to obtain an equivalent extended-depth-of-focus (EDF) rendering of a thick specimen, and a minimum of only two images for a 3D stereo view that has full depth.
Our microscope system uses a special-purpose optical element to uniformly “code” the information from all planes throughout the specimen volume onto a single CCD camera image. Specimen-independent digital processing is then used to “decode” this raw image. In effect, the coded raw image is blurred by a special type of aberration which produces an image that is nearly independent of focus. The system then uses a fast, non-iterative, digital filtering algorithm to remove this special blur so that a large volume of the specimen image appears sharply focused all at once.
A Multicolor Femtosecond Lightsource for (Multiphoton) Confocal Fluorescence Microscopy
Authors: Potma Eric , Kahya Nicoletta , de Boeij Wim P , Wiersma Douwe A 
Published: July 2020

Abstract
Recent advances in fluorescence microscopy add to the versatility of this optical technique and intensify its significance as an indispensable tool in biological research. Especially the use of multiphoton excitation offers the microscopist many advantages like inherent optical sectioning, reduced out-of-focus bleaching and higher penetration depths into the sample. In this regard, the commercial availability of pulsed solid-state lightsources like the Ti:Sapphire laser, that provide short pulses needed in the nonlinear multiphoton process, have paved the way for the routine implementation of multiphoton microscopy in the biologists laboratory. Although the spectral range of the commonly used Ti:Sapphire laser allows the application of two-photon fluorescence microscopy on chromophores that absorb in the range of 350-450 nm, a lasersource that enables the two-photon excitation of molecular probes at even shorter wavelengths (&lt;350nm) would be highly beneficial.
In this contribution we present a visible femtosecond optical parametric oscillator (OPO, Figure 1) that is ideally suited to excite molecular species at 285-335 nm by means of a two-photon process. Femtosecond pulses with durations as short as 30 fs can be generated within a tuning range from 570 to 670 nm. A cavity dumper incorporated in the laser cavity provides variable pulse repetition rates (single shot to 82 MHz).
Fluoronanogold as a Probe for High Resolution Correlation Between ImmunoFluorescence and Electron Microscopy
Authors: Takizawa T , Robinson J M 
Published: July 2020

Abstract
[Introduction] Immunocytochemical labeling of cryosections, especially immunofluorescence microscopy using semi-thin (0.5-μm) cryosections, has been a powerful technique for detection of cellular antigens in situ and has been widely employed in cell and molecular biology studies. In many cases, immunofluorescence provides sufficient resolution and sensitivity to answer the question being addressed. However, in certain cases the increased resolution of the electron microscope using ultrathin (90-nm) cryosections may be required to define more precisely the localization of specific molecules. Recently, a unique fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), has been developed for use as a secondary antibody in immunocytochemical applications. It consists of a Fab' fragment of an antibody to which a 1.4-nm gold particle and fluorochromes are conjugated. FNG permits correlative microscopic observation of a sample stained in a single labeling procedure by multiple optical imaging. Recently, we have shown FNG immunocytochemistry on ultrathin cryosections to be valuable for high-resolution correlation of immunofluorescence and immunoelectron microscopy. In the present study, we have examined the utility of FNG as a secondary antibody for immunolabeling of myeloperoxidase (a marker protein for the azurophillic granules) in ultrathin cryosectioned human neutrophils.
[Materials and Methods] Purified human neutrophils were fixed with paraformaldehyde, embedded in gelatin, infiltrated with sucrose, cut as ultrathin cryosections, and then collected on formvar film-coated nickel EM grids as described previously. Grids containing ultrathin cryosections were incubated with antimyeloperoxidase and then incubated with FNG.
Oxidative Injury by Oxygen and Nitrogen Radicals in Diabetic Retinopathy in the BBZ/WOR Rat: CytoChemical and ImmunoCytochemical Studies
Authors: Ann Ellis E , Grant Maria B , Guberski Dennis L 
Published: July 2020

Abstract
Previous studies have documented NADH oxidase as a source of the oxygen radicals, superoxide (O2) and hydrogen peroxide (H2O2), at sites of disruption of the blood retinal barrier (BRB) in a rat model of noninsulin dependent diabetes (NIDDM). Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the complications of diabetes. The effects of NO depend on its microenvironment and one mechanism for its role in complications of diabetes is interaction with oxygen radicals to form the toxic species, peroxynitrite (ONOO−). Since NO is a highly labile molecule, its potential localization is best achieved by immunocytochemical studies of NOS. The activity of iNOS can be detected by localization of nitrotyrosine, a marker indicative of the formation and activity of ONOO−.
Colloidal gold labeled immunocytochemical studies of iNOS and nitrotyrosine were done on sections of neural retina in eyes of obese, NIDDM BBZ/Wor rats in which NADH oxidase had been localized with cerium perhydroxide. Disruption of the BRB was documented by immunocytochemical localization of extravasated endogenous serum albumin. Age matched, nondiabetic control retinas were from eyes from BBDR/Wor rats.
Gold-Based Autometallography
Authors: Hainfeld James F , Powell Richard D , Stein Joshua K , Hacker Gerhard W , Hauser-Kronberger Cornelia , Cheung Annie L M , Schöfer Christian 
Published: July 2020

Abstract
Gold labels such as Nanogold® and colloidal gold are enlarged and visualized in the electron microscope or optically by the selective deposition of silver onto their surfaces. This process, known as autometallography (AMG), silver amplification or silver enhancement, is initiated by exposing the particles to a solution containing silver (I) ions and a reducing agent such as hydroquinone or npropyl gallate. Particles may be enlarged to between 30 and 100 nm in diameter, giving a distinctive black, punctate staining in the light microscope. Nanogold® labeling with silver amplification is one of the most sensitive methods available for histopathology applications such as in situ hybridization. With Catalyzed Reporter Deposition (CARD; also called Tyramide Signal Amplification, or TSA® ; NEN Life Sciences, Boston MA), it has been used to detect as few as 1-2 copies of viral DNA or RNA per cell. However, its uses are restricted by reactions of silver (I) with halides and other elements in tissues. Also, after signal development, self-nucleation and non-specific background deposition begin quickly, which can make end-point selection difficult or prevent incorporation into automated procedures.
Multiple Labeling for EM using Particles of Different Shape and Metal Composition
Authors: Meyer DA , Albrecht RM 
Published: July 2020

Abstract
Multiple labeling for electron microscopy (EM) is typically accomplished by using colloidal gold (cAu) particles of different sizes. The size distribution of each cAu preparation, which may vary by up to 15%, is the principal factor that limits the number of labels which can be used simultaneously. Furthermore, there is no effective way to use multiple labeling for quantitative, high resolution EM studies. Such analysis requires the use of a single Fab antibody fragment conjugated to a single cAu particle in order to ensure that each particle corresponds to only one antigenic site. Whole antibody molecules cannot be used for quantitative analysis because they are at least divalent, and some, such as dimeric IgA or pentameric IgM, have even more antigen-binding sites. Consequently, it is impossible to deduce whether the presence of one whole antibody molecule corresponds to the presence of one, two, or more targets. Particle diameters ranging from 3 to 5nm are optimal for quantitation because one Fab fragment adsorbs to one particle, more than one Fab fragment may adsorb to a single particle larger than 5nm, and, when smaller than 3nm, several particles may bind to a single Fab fragment.
We are evaluating parameters apart from particle size variation to accomplish multiple labeling for both qualitative and quantitative analyses. One method relies on electron energy loss spectroscopy (EELS) to distinguish particles of several metallic compositions, including Au, Ag, Pt, Pd, Rh, and Ru. EELS is performed using a LEO 912 energy filtering transmission electron microscope (EFTEM) with an in-column Omega spectrometer.
Localization of Actin Filaments in the Central Nervous System Using Phalloidin and Correlative Light and Electron Microscopy
Authors: Capani Francisco , Martone Maryann E , Deerinck Thomas J , Ellisman Mark H 
Published: July 2020

Abstract
Determination of the fine organization of actin networks is important for understanding different functional aspects of the central nervous system (CNS) such as plastic events. Early studies investigating the localization of actin filaments at the ultrastructural level relied on structural methods or antibodies raised against actin isoforms. More recently, many investigators have employed the actin-binding peptide phalloidin conjugated to various fluorescent molecules. By conjugating phalloidin to the fluorophore eosin, we have been able to localize f-actin at the electron microscopic level using photooxidation of diaminobenzidine (DAB) by eosin. The goal of this study is to provide a better description of the cellular and subcellular localization of actin filaments at the light and electron microscopy level based on the high binding affinity of phalloidin for actin and utilizing this novel method.
Tissues were obtained from male Sprague Dawley rats that were perfused transcardially under deep anesthesia with normal rat Ringer's solution at 35°C followed by fixative. The fixative contained 4 % paraformaldehyde and different concentrations of glutaraldehyde, ranging from 0.1- 2.5%. Sections of cerebellum, striatum and hippocampus which were cut at a thickness of 50-80 μm with a Vibratome were incubated with phalloidin-eosin in a solution of 0.05% in 0.5% cold water fish gelatin/50mM glycine-PBS (“working buffer”) for 2 hrs. As a control, phalloidin-eosin was omitted for one set of tissues.
Novel Methods in Fluorescence Sensing
Authors: Lakowicz Joseph R , Gryczynski Ignacy , Gryczynski Zygmunt , Tolosa Leah , Rao Govind , Dattelbaum Jonathan , Eichorn Lisa 
Published: July 2020

Abstract
Chemical sensing based on fluorescence is presently undergoing rapid development. As one example, fluorescence lifetime measurements are now being used for measuring cation and anion concentrations in clinical chemistry, cellular imaging and flow cytometry. Additionally, there is an increasing use of proteins as sensors because of their high affinity and specificity for ligands.
In this report we describe some recently developed approaches to fluorescence sensing. These new methods are based on the use of a fluorescence reference either within the sample or external to the sample, but within the optical path. Two types of references are used; microsecond lifetime metalligand complexes (MLCs) or oriented films of ns decay time fluorophores. A protein sensor for glucose was developed using the glucose-galactose binding protein (GGBP) from E.coli (Figure 1).
Analisis of PKA Binding to A-Kinase Anchoring Proteins Using Fluorescence Resonance Energy Transfer
Authors: Ruehr M L , Bond M 
Published: July 2020

Abstract
The cAMP dependent-protein kinase (PKA) signaling pathway plays a key role in the sympathetic regulation of muscle contraction in adult cardiac myocytes. This pathway may be regulated via compartmentalization of its components, whereby A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular areas to give each receptor binding event specificity. The purpose of this study is to investigate the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid, and the regulatory subunit of PKA (R).
Fluorescence resonance energy transfer (FRET) was used to monitor binding events between the type II regulatory subunit of PKA (RE) and Ht31 or Ht31P, a mutated form of Ht31 which does not bind RII. Each protein was fused to a derivative of the green fluorescent protein (GFP) so that the excitation-emission spectra of the two fluorescent proteins overlap.
A Tutorial of the Fib Lift-Out Technique for TEM Specimen Preparation
Authors: Giannuzzi Lucille A 
Published: July 2020

Abstract
The focused ion beam (FIB) instrument has been developed and exploited by the microelectronics arena for specimen preparation for both scanning and transmission electron microscopy (TEM). The inception [1] and subsequent development [2] of the FIB TEM lift-out (LO) technique has enabled electron transparent membranes of generally uniform thickness to be produced for TEM analysis. The primary advantage of the FIB technique is that site specific cross sections (or plan view sections [3]) may be fabricated quickly and reproducibly. The FIB LO technique has been used extensively in our laboratory for a wide range of materials [4] and biological applications [5] which are summarized in figure 1.
The FIB LO method consists of milling a series of trenches around an area of interest. Then the bulk sample is tilted up to ∼60 degrees to allow the beam to impinge on the lower portion of the specimen surface so that cuts can be made along the bottom edge and the lower 2/3 of the distance up one side of the specimen.
Bridging the Resolution Gap: Correlated 3D Light and Electron Microscopic Analysis of Large Biological Structures
Authors: Martone Maryann E 
Published: July 2020

Abstract
One class of biological structures that has always presented special difficulties to scientists interested in quantitative analysis is comprised of extended structures that possess fine structural features. Examples of these structures include neuronal spiny dendrites and organelles such as the Golgi apparatus and endoplasmic reticulum. Such structures may extend 10's or even 100's of microns, a size range best visualized with the light microscope, yet possess fine structural detail on the order of nanometers that require the electron microscope to resolve. Quantitative information, such as surface area, volume and the micro-distribution of cellular constituents, is often required for the development of accurate structural models of cells and organelle systems and for assessing and characterizing changes due to experimental manipulation. Performing estimates of such quantities from light microscopic data can result in gross inaccuracies because the contribution to total morphometries of delicate features such as membrane undulations and excrescences can be quite significant. For example, in a recent study by Shoop et al, electron microscopic analysis of cultured chick ciliary ganglion neurons showed that spiny projections from the plasmalemma that were not well resolved in the light microscope effectively doubled the surface area of these neurons.
While the resolution provided by the electron microscope has yet to be matched or replaced by light microscopic methods, one drawback of electron microscopic analysis has always been the relatively small sample size and limited 3D information that can be obtained from samples prepared for conventional transmission electron microscopy. Reconstruction from serial electron micrographs has provided one way to circumvent this latter problem, but remains one of the most technically demanding skills in electron microscopy. Another approach to 3D electron microscopic imaging is high voltage electron microscopy (HVEM). The greater accelerating voltages of HVEM's allows for the use of much thicker specimens than conventional transmission electron microscopes.
Application of Electron Microprobe Scanning Techniques to Resolve Trace Element Variations in Biogenic Aragonite.
Authors: Haywick Douglas W , Bersch Michael G 
Published: July 2020

Abstract
Aragonite (CaCO3) is one of the principal carbonate minerals utilized by metazoans to form shells, tests and other skeletal elements. It may also be inorganically precipitated under certain geological conditions. Aragonite is unstable in meteoric diagenetic environments (relative to its polymorph calcite), and is prone to dissolution and/or replacement by other minerals (usually calcite). The mobilization of Ca2+ and CO3 2− during aragonite alteration is largely responsible for early diagenetic cementation and the formation of limestone from unconsolidated carbonate sediment. Our study has been investigating the very earliest stages of aragonite alteration. To date, we have limited our work to bivalves of the genera Donax, Chione and Austrovenus collected from, respectively, southern Alabama, southwestern Florida and eastern North Island, New Zealand. Chione and Austrovenus are closely related genera which should permit direct comparison of results. The Florida and New Zealand study locations were chosen because they were near fossil localities containing identical Tertiary-aged specimens that had been subjected to more prolonged alteration. Our results confirm that initial aragonite alteration in meteoric environments is driven by dissolution. Outer surfaces begin to dissolve very rapidly (laboratory experiments suggest that this may occur after only a few months of exposure to meteoric water); leading to a chalky consistency along the outer extremities of the shell. Simultaneous selective leeching of some trace elements (notably Sr2+) also occurs. More prolonged dissolution results in progressive loss of mass and Sr2+ as well as penetration of the chalky “zone” into the interior of shells. Highly altered shells consist almost entirely of chalk.