bluuebunny/crossref_metadata_2025 huggingface dataset:
Transfer Cell Development And Immunolocalization Of A Senescence Associated Gene (Sag14) In Senescing Arabidopsis Thaliana Leaves.
Authors: Russin W A , Weaver L M , Amasino R M
Published: July 2020

Leaf senescence is an orderly, active process in which nutrients in a leaf are reclaimed and mobilized to other parts of the plant. It is considered to be the final stage in leaf development. During senescence leaves undergo highly coordinated changes in cell structure, metabolism, and gene expression. For example, chloroplasts undergo obvious structural modifications during leaf senescence. Regarding gene expression, genes with steady-state mRNA levels that increase during senescence are often referred to as senescence-associated genes, or SAGs.

In order to study ultrastructural changes that occur during senescence, portions of fully expanded, mid-senescent, and fully senescent Arabidopsis thaliana leaves were chemically fixed using vacuum-accelerated microwave processing. Thin sections were observed for ultrastructural changes such as development of transfer cells that are characterized by the presence of cell wall ingrowths. Transfer cell development is correlated with an increased capacity of tissues to take up materials, related partly to an increase in plasma membrane surface area.
Effects Of Varying Salinity On Leaf Ultrastructure Of Potamogeton Pectinatus L
Authors: Barnabas AD , Bunsi P , Naidoo Y , Przybylowicz WJ , Mesjasz-Przybylowicz J
Published: July 2020

Potamogeton pectinatus is a submerged halophyte which occurs in waters of low salinity (5% to 10%). Its upper salinity tolerance has been reported to be 19%. Reasons why P.pectinatus is unable to tolerate salinities in excess of 19%is important to our understanding of its biology. In the present study, leaf ultrastructure of plants growing at low salinity was compared with plants growing at high salinity in order to assess the effects of different salinities on the ultrastructure. Attention was focussed on ultrastructural changes occurring in the leaf epidermis, the main photosynthetic tissue.

Plants were grown in seawater at two salinities : 5%(low salinity) and 20% (high salinity). Pieces of mature leaf blades from both treatments were harvested and prepared for Transmission Electron Microscopy (TEM) following standard procedures. The overall distribution and concentration of chlorine (CI) in the leaves was ascertained since this element is the most abundant anion in seawater and is important in considerations of salt tolerance in submerged halophytes.
The Study Of Meiosis-I In Maize By Confocal Microscopy And Digital Deconvolution
Authors: Lee Thomas C , Walden David B , Cheng Ping-chin
Published: July 2020

Visualization is interdependent with gene mapping and DNA sequencing, because spatial relationships are important to understand function. The ability to visualize the chromosomes, especially in threedimensional (3D) space, has great benefits for the better understanding of a gene's function. For these reasons, a better technique is needed to study the chromosomes in 3D. Studying the stages of meiosis I provides an excellent opportunity to evaluate the capability of 3D imaging techniques. The loosening of the chromosomes in meiosis I exemplify the advantages of 3D imaging.

Tassel inflorescence from maize (Zea mays L., inbreed Ohio43) was collected from the field station at the University of Western Ontario in London, Ontario, Canada during the summer of 1998. Tassels at proper developmental stages were fixed in 3:1 (EtOH / acetic acid) solution. The anthers were stained with the Feulgen method and cleared in methyl salicylate.
Morphometric Analysis Of Polychlorinated Biphenyl 118-Induced Ultrastructural Alterations In The Rat Liver
Authors: Gallant TL , Singh A
Published: July 2020

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that bioaccumulate in the food chain and thus pose a health risk to human beings and other animals. The exceptional dielectric and lubricating properties of the PCBs resulted in their extensive use during the mid-1950s as lubricants in electrical generators and transformers. PCBs have a long half-life and persist in the environment causing concerns related to their toxicity. The present research on congener 118 (2,3',4,4',5-pentachlorobiphenyl) involves the quantification of hepatocyte alterations using stereology techniques. Application of a quantitative technique to estimate liver pathology confers more confidence in evaluating toxicity of a xenobiotic, and in the determination of its NOAEL (No Observable Adverse Effect Level).

Alterations in the volume fraction of cellular constituents in the hepatocytes induced by congener 118 were estimated relative to the sex of, and dose fed to a laboratory population of weanling Sprague-Dawley rats. The rats were given the PCB dose in concentrations of 2, 200, and 2,000 ppb to females and 10, 1,000 and 10,000 ppb to males; three animals were used in each dose group.
Primitive Patterns of Reproduction In The Foraminifera (Protists)
Authors: Goldstein Susan T
Published: July 2020

The Foraminifera are an enormously successful group of predominantly marine protists. They first appeared in the rock record >500 million years ago and currently are found in most planktonic and benthic marine and marginal marine environments. Though complete life cycles are known for only ˜30 of the >10,000 described extant species, these taxa illustrate reproductive patterns that are among the most diverse of all protists. The foraminiferal life cycle typically is characterized by an alternation of sexual and asexual generations during which meiosis occurs in the early phases of schizogony in the agamont. Sexual reproduction involves the production of biflagellated, triflagellated, or amoeboid gametes, and fertilization may be gametogamous, gamontogamous, or autogamous. Of these options, most Foraminifera for which sexual reproduction has been documented (44 species) produce many thousands of small biflagellated gametes that are shed directly into the surrounding seawater where gametogamous fertilization occurs.
Microscopic Examination Of Vascular Differentiation And Pattern Formation In The Inflorescence Stems Of Arabidopsis
Authors: Freshour G , Hahn M , Ye Z-H
Published: July 2020

Plant vascular system is the principal means to carry water and food throughout the plant body. It is composed of two types of conducting vascular tissues, xylem and phloem. Xylem carries water and minerals from the root to the shoot, whereas phloem transports photosynthates from leaves to other parts of the body. The evolution of vascular system which solved the problem of water and food transport was considered to be one of the key events for the successful emergence of vascular plants on the land from aquatic environments. Although vascular tissues in almost all vascular plants consist of xylem and phloem, diverse arrangements of vascular tissues within the bundles and of vascular bundles in the stele were evolved (1). The occurrence of diverse vascular patterns in vascular plants offers an excellent opportunity to study the evolutionary mechanisms controlling pattern formation. In this report, we present our studies on the vascular differentiation and pattern formation in the inflorescence stems of Arabidopsis thaliana.
Electron Probe Microanalysis Of Spironolactone Bodies
Authors: McNeil JP , Carter JE , Boudreaux CW , McDonald F , Tucker JA , King JAC
Published: July 2020

Spironolactone bodies (SB) were first described in 1963 by Janigan. These laminated, whorled structures are seen in cells of the adrenal zona glomerulosa in patients treated with the drug spironolactone. Spironolactone is an aldosterone antagonist. Hyperaldosteronism may result from excess production by the adrenal cortex. By both light microscopy and transmission electron microscopy (TEM), SB have a distinctive, laminated appearance. Kovacs, et al. observed that SB are composed of cellular constituents. To our knowledge, SB have not been analyzed using scanning electron microscopy (SEM) and electron probe microanalysis technology.

An adrenal gland with a 1 cm cortical mass was removed from a 39 year old female and received in 10% buffered formalin. Histologic examination of the mass showed a monotonous population of cells recapitulating zona glomerulosa cells. Intracytoplasmic structures compatible with SB were identified. Portions of the adrenal gland were processed for TEM and SEM analysis.
Left-Handed Z-Rna In Lens Epithelium: Preequatorial Zone
Authors: Gagna CE , Kuo HR , Lambert WC
Published: July 2020

Our goal was to determine the cellular localization of left-handed Z-RNA, within preeguatorial zone (PZ) epithelium of the normal adult dog ocular lens (1.5 yr) (Fig. 1), employing anti-Z-RNA IgG polyclonal antibodies. B-DNA has the ability to adopt the Z-DNA configuration in vitro(1). A-RNA can be transformed into Z-RNA under certain conditions (2). Z-RNA has been localized in cultured cells (3). Strong evidence supports the presence of Z-DNA in vivo (1). Elimination of DNA binding proteins by certain fixatives can initiate DNA supercoiling which stabilizes Z-DNA sequences (1). Z-DNA may play a role in regulating in vivo transcriptional enhancement (1).

Anti-Z-RNA antibody probes were produced in 3 rabbits immunized with injections of Z-RNA: Br-poly[ribosomal(G-C)]. Concerning light microscopy [immunohistochemistry (ABC method)], lens tissues were fixed in Carnoy's, embedded in paraffin and sectioned (3 μm) (Fig. 2). Image analysis was performed using a Leitz DM-RB microscope and Leica Quantiment 500 + image analyzer.
Localization Of Glutamyl/Prolyl-Trna Synthetase Within The Eukaryotic Multienzyme Complex By Immunoelectron Microscopy
Authors: Trempe Norcum Mona , David Dignam J
Published: July 2020

Aminoacyl-tRNA synthetases are the family of enzymes responsible for correctly linking tRNA molecules with amino acids so that the latter may be subsequently incorporated into growing polypeptide chains. A unique feature of aminoacyl-tRNA synthetases from higher eukaryotes is the existence of a high molecular mass multisynthetase complex. This approximately 1 × 106 Da particle measures approximately 27 nm in diameter and appears to be a cup or elongated “U“ shape as suggested by distinct orientations visible in negatively stained electron micrographs. A working model of the structure has been proposed which divides the particle into three domains. The composition of each domain as well as relative positions of individual polypeptides within each have been proposed, but to date there has been no precise localization of any specific component or site within the multisynthetase complex. This study describes the first localization of a particular polypeptide within the particle.
Huntingtin Localization In Rat Cortex And Striatum
Authors: William L'Amoreaux  , Nevins Sandra
Published: July 2020

Huntington’s Disease (HD) is a progressive neurodegenerative disease that is characterized by loss 19 of motor function and a decline in cognitive functions. HD is characterized morphologically by loss of neurons in the basal ganglia. During the progression of HD, striatal projection neurons die but cholinergic and somatostatin-containing intemeurons survive. The mutated gene for HD has been described and the protein transcribed by the disease (huntingtin or HT) described. Within the striatum, there is a lack of correlation between presence of HT and neuronal vulnerability. HT has been shown to be associated with intracellular vesicles in human tissues.

Female rats were transcardially perfused with 4% paraformaldehyde, 0.1 M lysine, 10 mM sodium periodate (PLP) in 0.1 M phosphate buffer. Following fixation, the brain was removed and the cortex and striatum removed from the brain as 5 mm3 pieces and transferred to fresh PLP containing 0.25% glutaraldehyde.
High Pressure Freezing And Freeze Substitution As Tools For Post-Embedding Immunocytochemistry In Monkey Brain Sections
Authors: Paré J-F , Charara A , Smith Y
Published: July 2020

For the past ten years, research in our laboratory has focused on the synaptic circuitry of GABAergic and glutamatergic networks in the monkey basal ganglia, a group of subcortical brain structures involved in the control of motor and psychoaffective behaviors. Elucidating the distribution and synaptic localization of different subtypes of glutamate and GABA receptors and transporters is essential to understand the basic functioning of these brain structures and develop better therapies for neurodegenerative diseases such as Parkinson's and Huntington's diseases. Although immunoperoxidase labeling allows to localize receptors at the cellular level, this method is generally not suitable for quantification and exact synaptic location of receptors due to the diffuse nature of the peroxidase reaction product. Here, we describe a highly sensitive post-embedding immunogold technique on Lowicryl-embedded ultrathin sections to analyze the subsynaptic distribution of these receptors at the electron microscope level.
A Microscopic Investigation Of The Interaction Between Borrelia Burgdorferi And Human Astrocytes
Authors: Karpiak Veronica C , Garon Claude F
Published: July 2020

Borrelia burgdorferi is the causative agent of Lyme disease. In addition to the characteristic effects of acute infection, Lyme disease can cause chronic effects including cardiac and neurological complications [1]. Several laboratories have shown the ability of B. burgdorferi to attach and invade several cell types such as lymphocytes [2] and fibroblasts [3]. Cultured normal human astrocytes were used to investigate the possible interaction between a cell type of the central nervous system and the Lyme disease spirochete.

Initially scanning electron microscopy was used to monitor and characterize the surface interaction between astrocytes and spirochetes. Astrocyte cell cultures were co-incubated with low passage B31 cultures over a time course from 1 hr to 18 hr. The co-cultures were washed extensively to discourage non-specific binding, glutaraldehyde fixed, critical point dried and mounted for field emission scanning electron microscopy. A time dependent association between the cultured astrocytes and B. burgdorferi were found to range from approximately 20% after 1 hr to almost 100% after 18 hr.
Apoptosis Signaling In Retinas Of Mice With Inherited Photoreceptor Degeneration.
Authors: Rich K A
Published: July 2020

The signaling pathways which lead to the wave of photoreceptor cell death in the rd mouse during the second week after birth have yet to be defined. The photoreceptors of these mice develop normally until they begin to elaborate phototransducing outer segments at around postnatal day (P)8. At this point, their differentiation slows as intracellular levels of cGMP rise due to the inherited mutation in the phosphodiesterase-β subunit.

We have used an immunocytochemical approach to investigate potential signaling pathways leading to photoreceptor cell apoptosis in this mutant. The earliest change detected in the photoreceptor layer was an induction of CREB immunoreactivity in photoreceptor nuclei, commencing at P8. Although CREB is generally thought to be constitutively expressed and its function controlled primarily by its phosphorylation, we find that immature photoreceptors of normal mice are immunonegative for CREB until the time of eye opening and the initiation of phototransduction at around P14.
Specimens For Observing Single Events Of Dna Metabolism By Fluorescence Microscopy
Authors: Sun M , Serwer P
Published: July 2020

Single stained DNA molecules can be visualized by fluorescence microscopy (1). Therefore, perhaps single events of DNA metabolism can also be visualized by fluorescence microscopy. Problems to solve include (a) photobleaching of stained DNA, (b) photolytic damage to macromolecules necessary for metabolism, including DNA, (c) inhibition of metabolism by the process of preparation of the specimen, and (d) interference with image formation by macromolecules that either must be or by chance are present during metabolism. These problems have been solved, at least in part, during a study of the in vitro packaging of bacteriophage T7 (2). Packaging occurs in T7 capsids that had previously been assembled without assistance of DNA. The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the terminally repetitious, nonpermuted T7 DNA. The metabolic event observed in ref. 2 was capsid-dependent specific cleavage of concatemers during packaging. The concatemers had formed a DNA network before cleavage.
A Covalently Linked 10 Nm Gold Immunoprobe
Authors: Gutierrez Edmund , Powell Richard D , Hainfeld James F , Takvorian Peter M
Published: July 2020

Chemically functionalized metal cluster compounds have demonstrated important advantages over colloidal gold as biological microscopy labels. They are covalently cross-linked to the targeting biomolecule, and therefore may be conjugated to a wide range of molecules which cannot be labeled with colloidal gold. The 1.4 nm Nanogold® cluster has been conjugated to peptides, lipids and oligonucleotides, some of which have been proposed as elements of novel molecular wires and novel materials. Dissociation of colloidal gold particles from the conjugate probe, a source of error in quantitative immunogold studies, is greatly reduced by covalent cross-linking. Nanogold® is an uncharged molecule, and because its surface is completely coordinated by organic ligands, nonspecific binding is greatly reduced. Nanogold® conjugates also show greatly enhanced penetration into cells and tissue sections. However, gold probes larger than Nanogold® are desirable for improved visualization in specimens with electron-dense regions or staining, or for applications such as double labeling studies with different sized gold labels, or visualizing wider antigen distributions.
Cellular Redistribution Of Taurine In Osmotically Swollen Rat Hippocampus
Authors: Taylor CA , Olson JE , Kreisman NR , J.Leasure
Published: July 2020

Taurine is transported from Purkinje cells into perineuronal glial cells during hypoosmotic swelling and is lost first from pyramidal cells during hypoxia/ ischemia, suggesting in situ cellular redistribution. Cultured isolated astrocytes or neurons and slices from rat hippocampus were treated first in 290 mOsm saline (ISO), then in 200 mOsm (HYPO) with or without taurine for 15 and 60 minutes.

Cells and slices were then fixed in 80 mM phosphate buffered (pH 7.4) fixative containing 1.25% paraformaldehyde and 2.5% glutaraldehyde followed by immuno-cytochemistry.Immunostaining was done either directly on the cell layers or on 1 micron araldite/eponate sections (Pelco). After incubation with antibody to taurine, GFAP, SNAP-25, or normal serum, immunoproduct was visualized using step-avidin-DAB (DAKO:LSAB+). Measurements of pyramidal cell diameters were made on toluidine blue-stained sections containing cells with prominent nucleoli.

The data suggest that taurine is initially lost from pyramidal cells within 15 minutes in HYPO to maintain normal cell volume, while taurine efflux from glia occurs over a 60 minute time peroid.
Microscopic Evidence Of Biofilm Formation In The Gut Of Two Pest Tephritidae
Authors: Potter SE , Lauzon CR , Smith NR
Published: July 2020

Tephritidae are the true fruit flies, which include the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), the walnut husk fly, Rhagoletis completa Cresson, the Mexican fruit fly, Anastrepha  ludens (Loew), and the apple maggot fly, Rhagoletis pomonella (Walsh). Fruit fly infestation causes major damage to many economically-important food crops and is of major concern in agriculture worldwide.

Tephritidae are controlled primarily by repeated applications of organophosphate pesticides. Health concerns, environmental pollution, and costs associated with pesticide use press researchers to find new methods for control. One approach to finding new control methods involves investigating the associations that exist between tephritids and microorganisms. Currently, bacterial odors are being used to lure these pest tephritids into traps. These flies are specifically attracted to odors from certain bacteria found to inhabit their gut. Data suggest that these bacteria are likely involved in important aspects of fruit fly nutrition and reproduction.
Effectiveness Of Immunolabeling Gfap For Estimating Astrocytic Shape And Volume
Authors: Bushong E , Martone M E , Foster C , Ellisman M H
Published: July 2020

Each astrocyte forms an extensive network of fine processes within the surrounding neural tissue, interacting extensively with neighboring neurons and blood vessels. Fine glial processes surround synapses and probably modulate synaptic transmission. Glial endfeet on capillaries are responsible for transport of ions and metabolites and possibly control blood flow. Alterations in these fine structures may be of significance in brain function and disease. Glial fibrillary acidic protein (GFAP) is an intermediate filament found in astrocytes of the central nervous system. GFAP is commonly found in the perikarya and processes of protoplasmic and fibrous type astrocytes. Immunohistochemical labeling of GFAP is extensively used as a means of determining the location and shape of astrocytes. However, its labeling pattern varies with brain region (e.g. cortex vs. hippocampus), with cell state (natural vs. reactive astrocytes), and with the specific α- GFAP antibody used. Furthermore, Golgi-stained or dye-filled astrocytes show numerous small appendages or vellate structures that conform to the surrounding tissue and do not stain for GFAP.
Wax Particle Morphology And Ultrastructure Of Two Species Of Adult Insects: Prey And Predator
Authors: Freeman T , Nelson D , Buckner J , Jackson G
Published: July 2020

Some insects cover themselves with waxy particles including all parts of their body except the eyes. Two examples of insects that produce waxy particles from specialized glands on their abdominal surfaces are the adult stages of the silverleaf whitefly (Bemisia argentifolii) and one of its predators, the dusty wing (Semidalis flinti). In both species, the production of the waxy particles begins at adult eclosion. The silverleaf whitefly, a major plant pest, produces ribbons of waxy material that extrude from the numerous microtrichia that cover the surface of abdominal wax plates. Silverleaf whitefly males have 4 pair of wax plates and females have 2 pair. The adults use their tibia to break off the extruding ribbons, forming semicircular waxy particles about 1 micrometer in diameter. The semicircular waxy particles are composed of a mixture of long-chain aldehydes and alcohols, 34 carbons in length.

For the dustywing predator, the particles are formed by wax producing pores that cover most abdominal surfaces of the adults.
Uses Of Microscopy In The Crime Lab
Authors: Mc Adam T
Published: July 2020

Recent celebrated criminal cases have raised the public's awareness of forensic science. The advent of DNA technology, in particular, has led to a perception of instrumental techniques that lead to a mathematical probability of guilt that approaches certainty. Mostly passed over in press accounts of forensic operations is the use of the microscope in the examination of physical evidence. By the use of an example from an adjudicated criminal case it is hoped that a greater appreciation is gained of the microscope, that in its various forms, is by far the most widely used instrument in a crime laboratory today.

A middle-aged woman was reported missing by her son after the Memorial Day weekend in 1997. An examination of her apartment by members of the Washington State Patrol Crime Scene Response Team revealed a small amount of blood on her bedding. This bedding, together with other items, was taken into evidence.
Detecting And Optimizing Fluorescence Signals
Authors: Metcalfe Walter K
Published: July 2020

Classical fluorphores have been widely utilized in biological research, and have been extensively modified to promote particular qualities. Here we review practical methods of optimizing fluorescence signals for microscopy, discuss experimental solutions to common laboratory applications of fluorescent probes, and describe advantages of the sulfonated rhodamines.

Fluorescence output of dyes The usefulness of a fluorescent probe depends critically on how bright it is and on its ability to resist photobleaching.

The brightness of a dye depends on two factors: first, its efficiency in capturing excitation photons, called the molar extinction coefficient, and second, on the number of photons emitted for each photon absorbed, called the quantum yield. The product of these two factors describes the brightness of a dye. Both the molar extinction coefficient and the quantum yield are constants under specific environmental conditions. Consequently, this is a measure that is useful to compare the brightness of dyes.
Factors Which Influence The Development And Success Of An Academic Electron Microscope Facility: A Case Study
Authors: Wheatley J C
Published: July 2020

There are many electron microscope centers around the world and it is well recognized that there is no single “right” way to develop successful research and teaching electron microscope organizations.

The model for this discussion is that of the Center for High Resolution Electron Microscopy (CHREM) at Arizona State University. Several factors, including but not limited to those discussed below, have contributed to the continuing growth and development of this Center.

The Center serves students and faculty from eight separate academic units. It is also accessible to a significant number of microscope users from outside the university. These include researchers from industry as well as other academic institutions. The number of active instrument users typically averages eighty per year. There are ten instruments available to those who successfully complete the requirements for microscope use. It is not necessary to discuss the physical plant which houses the Center's instruments. Although very important, this topic has been adequately discussed elsewhere by experienced researchers.
Atomic Resolution Electronic Structure in Device Development
Authors: Batson PE , Domenicucci AG , Lemoine E
Published: July 2020

Recently, we have seen that the STEM can produce 0.12-0.26nm atomic resolution image detail in Annular Dark Field (ADF) mode even at modest acceleration voltages and in partnership with EELS for bonding studies. In the semi-conductor field, we are in a good position to take advantage of this imaging capability, because several atomic plane spacings range from 0.14 - 0.26 nm in the [100], [011] and [111] projections of Si and Ge. Figure 1. shows a result for the Si [111] projection obtained in the IBM VG Microscopes HB501UX, modified to produce a 0.19nm probe at 120KeV. The image has been filtered to remove spatial distances shorter than 0.15 nm, yielding the inset power spectrum. Each bright spot in the image corresponds to a single atomic column, separated from its neighbors by 0.19 nm. At 20Mx, the atom columns show enough contrast on the screen to allow astigmatism correction. These are similar to those discussed previously.
Digitally Enhanced, Polarization-Based Microscopy: Reality and Dreams
Authors: Inoué Shinya
Published: July 2020

Polarized light microscopy is used to identify and image optically anisotropic regions of the specimen; to determine their optical character; and to explore the arrangement of the molecules, fine structure, or atomic lattices that are responsible for the anisotropy. These studies can be carried out non-destructively in real time, and reveal events or structures that lie far below the resolution limit of the light microscope, or indeed at times even the electron microscope.

In biology, to study the dynamically changing, minute and weakly anisotropic domains within living cells, the polarizing microscope must be able to detect and measure birefringence retardances to a fraction of a nm, record the image with high microscopic resolution at nearvideo rate, and do so while the cell remains active.

Over the years, the extinction property and imaging capability of the basic polarizing microscope have been substantially improved by advances in optical design. More recently, video and CCD imaging and digital electronic processing have further enhanced the quality of the polarizing microscope image and our ability to rapidly detect and measure weak anisotropy.
Spectral Imaging for Separation of Fluorescent Signals from Autofluorescence
Authors: Levenson Richard M
Published: July 2020

Autofluorescence, also known as adventitious fluorescence or background fluorescence, often poses a significant problem in many applications of fluorescence microscopy. It contributes to unwanted noise and can overwhelm the desired signal. Particularly difficult samples to image include many pathology specimens that have been processed using crosslinking fixatives (typically formaldehyde). This procedure dramatically increase the autofluorescence level, leading to bright, broad spectrum emissions, particularly from connective tissue components. Unprocessed plant tissue and neuronal tissue also have extremely high levels of endogenous autofluorescence that can make many convenient labeling strategies, including most green fluorescent protein (GFP) labels, extremely problematic. Various solutions have been proposed for the reduction or elimination of autofluorescence. These include using narrow bandpass emission filters to try to isolate the desired fluorescence signal, the use of labels that can be excited at wavelengths that are much less likely to induce autofluorescence (moving the excitation towards the NIR is effective), and post-processing aldehyde-fixed samples with such reagents as sodium borohydride or toluidine blue to chemically suppress the autofluorescence signal. However, in many cases, these approaches are either infeasible or ineffective. Spectral imaging, that is, the acquisition of a high-resolution optical spectrum at every pixel of an image, offers another approach to elimination of the contribution of autofluorescence. This has recently become possible due to the development of a number of technologies that allow the collection of spectral datasets from fluorescently labeled samples using fluorescence microscopy. Such technologies include liquid crystal tunable filters (LCTFs) and Fourier transform imaging spectrometers, both of which are now commercially available.
High Performance Hyperspectral Imager for Microimaging
Authors: Malak Henryk , Herman Petr , Moore Wayne , Vecer Jaroslav
Published: July 2020

A high performance hyperspectral imager with high light throughput suitable for microscopy and analytical imaging was built and tested. The imager utilizes phenomenon of optical activity. The new technique provides a continuous spectral range of several hundreds of nanometers starting in deep UV. Similar spectral range starting in the near IR is also achievable.

A performance of a low cost implementation of the new technology is presented. The imager has a form of a microscope adaptor, which is inserted between the microscope and a low-cost 8-bit CCD camera. The resulting instrument is simple, robust, and highly compact. The imager module is placed in-line to the microscope imaging system and does not introduce observable image aberrations. Advantageously, the imager is transparent to conventional imaging operations, thus with the imager in-place there is no need for reconfiguration of the microscope to switch between conventional and hyperspectral video/digital imaging modes.

The presented spectral imager answers the need for a practical, sensitive, compact, and affordable imaging spectrometer. The instrument is well suited for a broad range of applications requiring rapid parallel acquisition of highly resolved concurrent spatial and spectral information such as high throughput screening, biochip analysis, remote sensing or nondestructive semiconductor testing.
Mammalian Apoptosis in Whole Neonatal Ovaries using Confocal Laser Scanning Microscopy
Authors: Zucker Robert M , Jeffery Susan C , Perreault Sally D
Published: July 2020

Confocal Laser Scanning Microscopy (CLSM) is being used to visualize folliculogenesis and presumed apoptotic events in whole neonatal mouse/rat ovaries. Our laboratory has previously demonstrated that apoptosis can be visualized in 8-9 day whole mouse embryos, 12 day fetal mouse eyes or 12-15 fetal rat limbs stained with the fluorescent lysosomal stain, LysoTracker Red (LT). The observed LT staining has been correlated with Nile blue staining (lysosomes and phagocytosis), TUNEL staining (DNA breaks) and apoptotic tissue morphology in rat and mouse developmental systems. This increased LT staining intensity appeared to be associated with acidic tissue, lysosomes, phagocytosis and dying cells. Here we describe recent progress using LT to evaluate the apoptotic process in whole ovaries. We are currently optimizing sample preparation and confocal machine operation to improve the image quality. to increase depth of visualization, ovaries derived from 10-45 day old mice/rats are fixed with an aldehyde and then made transparent by MEOH dehydration followed by benzyl alcohol/benzyl aldehyde (BABB) clearing. Such whole ovaries or tissue sections are viewed by CLSM using 568-wavelength laser light to obtain images of oocytes and follicles. Follicles showing gross morphologic changes typical of atresia were also characterized by intense LT staining of granulosa cells. This increased LT staining intensity appeared to be associated with acidic tissue and dying cells contained within the follicle.
Spectral Imaging: Fluorescence and Brightfield
Authors: Levenson Richard
Published: July 2020

Spectral imaging is a relatively new technique that provides images of a scene at multiple wavelengths and can generate precise optical spectra at every pixel. Mathematical approaches may then be used to extract the maximum information from the spectral image. Spectral imaging is routinely used in “remote sensing”, that is, the analysis of distant landscapes and structures from airplanes or satellites. Minor differences in spectra can be used to detect different crops, or mineral deposits, for example. Closer in, spectral imaging has uses in industrial process control, detection of otherwise invisible bruising in fruit, vascular integrity in transplanted organs, and so on. Finally, spectral imaging has newly been adapted for easy use in microscopy, with applications in surgical pathology, multicolor fluorescence and immunohistochemistry, and in DNA expression arrays, among others. A variety of technologies can be used to perform imaging spectroscopy, including Fourier transform interferometry, tunable filters, line-scanning prism or gratings-based devices, and others based on polarization effects. Recently, CRI has developed a novel approach for brightfield microscopy which uses a spectrally agile light source. The latter has a number of advantages over previous techniques.
A Close-To-Native System to Study Membrane Protein Dynamics by Single-Molecule Optical Microscopy: an Application to Bacteriorhodopsin
Authors: Kahya Nicoletta , Pécheur Eve I , Wiersma Douwe A , Hoekstra Dick
Published: July 2020

Biological membranes are not just a passive component of the cells, they actively support their functioning as the imbedded protein machineries carry out a wide range of crucial biochemical processes. Although studies in vivo are becoming more and more accessible to single-molecule optical microscopy, in vitro studies are still very much informative for the understanding of individual biological machineries. One of the major goals is to define the minimum number of components of a machinery that is necessary for a particular step, thereby allowing detailed studies of the mechanism of action. Reconstitution of a transmembrane protein system in artificial membranes (liposomes) is the main method for such a strategy, which thus may provide the option to investigate the functioning of transport proteins, ion channels, fusion machineries, and signal transducers in relation to their environment.

We present a novel procedure in order to reconstitute transmembrane proteins in chemically well-defined and close-to-native lipid bilayers, providing an in vitro system for single-molecule optical microscopy. Furthermore, an application of this technique is shown in the case of a single-molecule study of protein-protein and protein-lipid interactions for the light-induced proton pump bacteriorhodopsin.

In this study, Giant Unilamellar Vesicles (GUV), 10-100 μm sized, are used as lipid bilayer models for several reasons.
Application of Novel Laser Capture Microdissection and RT-PCR to a Functional Analysis of Osteopontin in Mouse Growth Plate Cartilage
Authors: Jacquet R , Hillyer J , Zhang J , Landis W J
Published: July 2020

The long bones of vertebrates, such as the tibiae and femurs of humans, extend in length by means of genotypic and phenotypic changes orchestrated by the chondrocytes comprising growth plate cartilage. Among the constituents synthesized by these cartilage cells, osteopontin (OPN), a phosphorylated glycoprotein, is thought to play a critical role in events leading to normal growth plate function and ultimate mineralization of the deeper zones of this cartilage region. The precise role of OPN, however, is uncertain with regard to mineralization, and present evidence supports the alternative possibilities that the protein may be either facilitative or inhibitory to mineral deposition. in order to investigate OPN function in a model growth plate, cartilage from normal 1-11 day old postnatal mice was examined by the novel techique of laser capture microdissection (LCM) followed by RT-PCR to obtain a measure of OPN gene expression by chondrocytes of known age and specific location in the plate. LCM permits identification of individual or clusters of cells within a tissue section and subsequent unique isolation (“capture”) of such cells for a variety of molecular analyses.

In this study, mouse tibiae were dissected, placed in RNAlater (Ambion, Austin, TX) to preserve message, and stored at −20°C. Sections (5 μm thick) of fresh frozen developing epiphyseal growth plates were obtained in a cryostat maintained at −20°C, fixed briefly in 70% ethanol, and stained with eosin. Sections were examined in a Pixcell LCM system (Arcturus Engineering, Mountain View, CA) where chondrocytes were attached to the surface of polymer film substrates and lifted free of sections. in separate experiments, ∼200-1200 cells were captured and analyzed. Substrates were transferred to Eppendorf tubes containing RNA extraction buffer. RNA was extracted from cells by microisolation (Stratagene, La Jolla, CA), DNAse-treated, reverse-transcribed, and then subjected to PCR (40 cycles) with AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA). Ethidium bromide agarose gels revealed OPN mRNA from groups of chondrocytes isolated from whole cartilage and from resting, proliferating, and hypertrophic growth plate zones from the mouse tibiae. Brain cells captured by LCM from the same mouse sections served as positive controls and reactions containing no reverse transcriptase were negative controls. 18S rRNA was used as a marker for semiquantitation and standardization of expressed message from captured cells.
[URE3] Prion forms Filamentous Networks in Yeast Cytoplasm
Authors: Speransky Vladislav V , Taylor Kimberly L , Edskes Herman K , Wickner Reed B , Steven Alasdair C
Published: July 2020

The concept of prion (infectious protein) originated in studies of transmissible spongioform encephalopathies (TSE) in mammals, but more recently, two nonchromosomal genes of yeast- [PSI] and [URE3] - were identified as prions. While the agents of TSEs kill infected cells and [URE3] or [PSI+] only slow growth at most, these infections are believed to have similar mechanisms, i.e. self-propagating amyloids. TSEs are often associated with amyloid deposition in infected tissues, and both Sup35p and Ure2p have been shown to form amyloid in vitro.

[URE3] is a prion of the Ure2 protein, that normally regulates nitrogen catabolism. Its ‘prion’ domain (residues 1-65) is necessary and sufficient for propagation of the prion, whereas the C-terminal portion (residues 81-354) is sufficient to carry out the nitrogen regulation function. The prion domain peptide spontaneously forms amyloid filaments in vitro. Full-length native Ure2p is a stable soluble dimer, but forms co-filaments when the prion domain peptide is added. This in vitro amyloid formation is highly specific and self-propagating, thus providing a possible explanation for the [URE3] prion. We have sought to clarify this hypothesis by examining the state of Ure2p in [URE3] cells by thin-section electron microscopy.

Yeast cells with the [URE3] prion and control [ure-o] cells were thin-sectioned after fixation and embedding in an epoxy resin. We found distinctive filamentous aggregates in the cytoplasm of [URE3] cells of a strain that overexpresses Ure2p (Fig. 1). These aggregates were seen in some cell profiles represented in 50-70 nm sections, and were never seen in control sections of [ure-o] cells. in cell sections showing these structures, there was typically one such aggregate, which could be quite large - up to several μm across - and approximately globular in outline. They contain irregularly associated filaments about 25 nm in diameter.
Tensegrity Architecture of an Agglutinated Foraminiferan Shell
Authors: Kinoshita Roy K , Rivas-Rivera Karla M , Bowser Samuel S
Published: July 2020

Shells of agglutinated foraminiferan protists are composed of mineral grains bound by secreted adhesives. As such, they are useful models for examining the evolution of “primitive” exoskeletons. Previous studies revealed the ultrastructure of shells in the giant Antarctic foraminiferan Astrammina rara and demonstrated that shucked specimens would reconstruct shells using glass beads. Here we further investigate shell architecture in this model species.

For micromechanical testing, an intact A. rara shell was placed between a fixed plate and a facing plate in series with a calibrated load cell. Displacement was effected by a high-precision drive, and 2-3 loading cycles were used to determine shell material properties. to assay tensile properties of the adhesive matrix, a network of pseudopodia and extracellular matrix fibers (i.e., the shell adhesive component) was obtained by incubating shucked cell bodies on 200-mesh gold grids. Pseudopodia were subsequently removed by detergent washes. Fibers in the resultant isolated matrix were severed with a Nd: YAG laser using an inverted DIC light microscope equipped with a 60× objective lens. Preliminary loading experiments using glass needles showed that Sepharose 2B beads were suitable strain gauges to assess compression within reconstructed shells (Fig. 1). in this assay, shucked cell bodies were incubated with a mixture of glass and Sepharose beads, and the reconstructed shells were examined by SEM.

Repeated loading and unloading demonstrated the elastic behavior of intact shells (Fig. 2). Adhesive matrix fibers snapped towards their attachment sites within 2 sec after cutting with a laser (Fig. 3), demonstrating that they are deployed under tension. SEM images of shells reconstructed with Sepharose show compressed particle profiles (Fig. 4).
Induction of Transfer Cell Formation in a Plant Infected by a Biotrophic Fungal Parasite
Authors: Mims Charles W , Richardson Elizabeth A
Published: July 2020

In this study a combination of scanning (SEM) and transmission electron microscopy (TEM) was used to examine leaf cells of the common weed Indian strawberry (Duchesnea indica) infected by the biotrophic rust fungus Frommeëla mexicana var. indicae. For SEM, free-hand sections of infected leaves cut with a razor blade were placed in a 5% (wt/vol) aqueous solutions of Ariel (Proctor and Gamble, Cincinnati, OH), a washing powder containing a bacterial protease. This treatment removed exposed host cell cytoplasm so as to reveal details of fungal haustoria and host cell wall responses to infection (Figs. 1, 2). Samples then were processed for study according to procedures described previously. For TEM, samples were prepared for study using high pressure freezing followed by freeze substitution. This fixation procedure has been used with excellent results to study hostpathogen interactions in rust-infected plants.

Frommeëla mexicana var. indicae produced specialized hyphal branches known as haustoria that extended through the walls of infected cells. Each haustorium possessed a long, slender neck region (Fig. 3) and an expanded body (Figs. 1, 4) that was separated from the host cell cytoplasm by the invaginated host cell plasma membrane. At maturity the neck region was surrounded by a extensive labyrinth cell wall in-growth or elaboration (Figs. 1-3) that appeared to be identical to wall elaborations that characterize plant transfer cells. Such cells are thought to be involved in intensive solute transfer over short distances. While transfer cells have been reported within cells of the haustoria of various plant parasitic higher plants and in plant giant cells induced by certain plant parasitic nematodes, this appears to be the first report of their formation in response to infection by a plant pathogenic fungus. Not only did these elaborations form in the immediate vicinity of haustoria, they also arose apart from haustoria in infected cells. However, such elaborations were not found in cells of uninfected leaves.
Napsin is Localized in the Lysosomal Compartments of Specialized Epithelial Cells in the Lungs and in the Kidney
Authors: Na C-L , Falconieri M W , Liu L , Melton K , Weaver T E
Published: July 2020

Napsin is an aspartic proteinase which has two essential asparagine residues in the active peptide (Tatnell et al., 1997). Napsin may participate in the processing of pulmonary surfactant protein B (Weaver et al., 1992; Chuman et al., 1999). in addition, changes in napsin expression have been linked to primary adenocarcinoma as well as kidney dysfunction (Hirano et al., 2000; Schauer-Vukasinovic et al., 2000). Localization studies by light microscope immunocytochemistry and in situ hybridization detected napsin in the lungs as well as in the kidney (Tatnell et al., 1997; Chuman et al, 1999; Schauer- Vukasinovic et al., 1999). However, these studies did not resolve the ultrastructure localization of napsin. in this study, EM cryoimmunogold labeling was used to characterize the subcellular distribution of napsin in lung and kidney cells.

A truncated murine napsin propeptide construct (1212 b.p.) without the signal peptide sequence (47 b.p.) was cloned into pProEX™HTb plasmid vector for the generation of recombinant protein. Polyclonal antibodies were generated against purified recombinant protein. The specificity of polyclonal antibodies was determined by immunoprecipitation and Western blotting using isolated mouse alveolar type 2 epithelial cells and kidney homogenates. The napsin antibodies detected a single protein with Mr= 43 kD.

Localization of napsin by EM cryoimmunogold labeling was performed using protein A gold probes as previously described (Na et al, 2000). in the lungs, napsin was localized in both alveolar type 2 epithelial cells and in alveolar macrophages. in alveolar type 2 epithelial cells, napsin was detected in multivesicular bodies and in lamellar bodies; internalized BSA gold colocalized with napsin in the lysosomes of alveolar macrophages (Figure 1A and 1B), consistent with the observation that napsin is active at low pH (Schauer-Vukasinovic et al., 1999). Although Schauer-Vukasinovic et al. (1999) reported that napsin was expressed by some bronchiolar cells, we did not observe any significant napsin labeling in Clara cells or in ciliated epithelial cells.