bluuebunny/crossref_metadata_2025 huggingface dataset:
Low-temperature field-emission scanning electron microscopy of high-pressure frozen samples reveals structural information below 10 nm
Authors: Walther Paul , Müller Martin
Published: June 2020

High-pressure-frozen samples can be imaged in the frozen hydrated state with a low temperature scanning electron microscope (LTSEM) thereby preventing many of the fixation and drying artifacts related to chemical fixation and dehydration. One limiting factor of high resolution LTSEM, however, is mass loss of the hydrated specimen due to electron beam irradiation. This effect is significantly reduced by using double layer coating and backscattered electron imaging (Walther et al., 1995). Thereby the sample is first coated with a contrast forming heavy metal layer (1 - 3 nm thick) and afterwards with a stabilising carbon coat (5 - 10 nm; Walther and Hentschel, 1989). The heavy metal layer is imaged with backscattered electrons that penetrate the carbon layer. Using this method structural information comparable to the TEM-replica (“freeze-etch”) technique can be achieved, without the need of replica cleaning. The method has been especially useful for samples where replica cleaning is difficult; e. g. investigation of new materials for filling cavities in dentistry, or the effects of drug application to human skin, or investigation of new materials in polymer science.
Emerging applications of fluorescence spectroscopy to cellular imaging: Long-lifetime metal-ligand probes, multi-photon excitation, light quenching, and lifetime imaging
Authors: Lakowicz J. R.
Published: June 2020

The applications of advanced concepts in fluorescence spectroscopy are increasing rapidly due to developments in probe chemistry and optical technology. In this talk we describe recent developments and their applications to cellular imaging.

While there exists thousands of fluorescent probes, almost all display lifetimes from 1 to 10 ns. These ns decay times are comparable to that found for cellular and tissue autofluorescence, which typically limits the sensitivity of most measurements. The lanthanides display long decay times, but their emission is unpolarized and cellular labeling is difficult. To circumvent these difficulties we are developing metal-ligand complexes as typified by Ru(bpy)3 and its conjugtable derivatives (Figure 1). The asymmetrical complexes display high anisotropy. The high anisotropy, combined with decay times of 400 ns or longer (Figure 2) results in probes capable of measuring microsecond macromolecular hydrodynamics. The use of long lived probes allows gated detection to avoid the prompt autofluorescence. Additionally, the metal-ligand probes are highly photostable.
Large-cluster and combined fluorescent and gold immunoprobes
Authors: Powell Richard D. , Hainfeld James F. , Halsey Carol M. R. , Spector David L. , Kaurin Shelley , McCann Jennifer , Craig Roger , Fay Fredric S. , McNamarat Kathryn E.
Published: June 2020

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.
Green fluorescent protein (GFP) as a tracer dye for cell movements in developing zebrafish embryos
Authors: Murakami T. , Doerre O.G. , Peachey L.D. , Weinberg E.S.
Published: June 2020

Fluorescent dyes have been extremely useful in determining cell lineages in the developing zebrafish embryo. For example, injection of FITC-dextran into blastomeres has allowed the determination of a tissue fate map at late blastula and a description of cell movements during gastrulation. Here we describe the use of green fluorescent protein (GFP) as a tracer dye for cell movements in developing early zebrafish(Danio rerio)embryos. Since GFP was purified and cloned from the jellyfishAequorea victoria,it has attracted great attention from biologists as an in vivo tracer and a reporter gene. The protein requires no cofactors or substrates for its fluorescence. This property allows one to inject GFP DNA constructs or in vitro synthesized GFP mRNA into individual cells which can then be expected to continuously synthesize the protein, thus obviating the dilution and bleaching effects often observed when fixed amounts of fluorescent tracer dyes are injected into dividing cells.
CCD cameras facilitate the imaging of small gold particles in immunogold-labelled ultrathin cryosections
Authors: Ackerley C.A. , Becker L.E. , Tilups A. , Rutka J.T. , Mancuso J.F.
Published: June 2020

Immunogold labelled ultrathin cryosections have proven themselves invaluable in subcellular localizations of many epitopes. As there are no embeddents to block an antibody‘s access to an epitope beyond physical size or chemical alteration by primary fixation, immunogold labelling density is increased dramatically with smaller (>5nm) gold-ligand complexes. Takizawa and Robinson have elegantly demonstrated penetrations in excess of 1μm using 1.4nm gold-Fab conjugates, while 5nm IgG gold particles only partially penetrated the surface and l0nm IgG gold particles labelled the surface. In order to visualize the 1.4nm gold-Fab particles silver enhancement was required leading one to doubt the viability of doing any quantitation.

Modern CCD cameras provide an effective alternative method of data collection to photographic plates with their ability to record 1000 × 1000 pixel digital images. Using a simple sharpen filter and contrast enhancement, 1.4nm gold Fab particles are easily resolved on thin films. Problems arise when using this small a particle on conventionally stained immunogold labelled cryosections.
Development of a cryosection EM autoradiography technique and its application for the subcellular localization of receptors
Authors: Na C.-L. , Hagler H. K. , Muntz K. H.
Published: June 2020

Recent progress in immunocytochemistry and cryo-techniques has made it possible to study receptor localization at the subcellular level. For many receptor-ligand systems suitable antibodies are not available and it would be more appropriate to use radioligands to study these receptors. Although fresh frozen sections have been widely used in light microscopy (LM) autoradiography studies, to our knowledge, no one has established a technique using electron microscope (EM) autoradiography with ultrathin frozen sections.

Unlike conventional EM approaches which can extract many biological molecules during dehydration and plastic embedding steps, we have adopted the method of Tokuyasu combined with LM autoradiography protocol for frozen sections to develop a new EM autoradiography technique using ultrathin frozen sections. Heart blocks were fixed in 2% periodate-lysine-paraformaldehyde (PLP) and 0.1% glutaraldehyde (GA), sucrose infused, and frozen in liquid nitrogen. They were sectioned in a Reichert Ultracut S cryo-microtome equipped with a Reichert FCS cryo-unit.
Effects of substrate orientation on growth of epitaxial layers
Authors: Hsu Y. , Kuan T. S. , Wang W. I.
Published: June 2020

Semiconductor superlattices or epitaxial overlayers have so far been grown mostly on (100)-oriented substrates. It has long been suspected that growth on surfaces such as (m11) could produce equally good or better epitaxial layers. In this work, we have systematically tested the growth of multi- or single-layered structures, including the GaAs/AlAs, AlAs/Ge, and GaAs/Si systems, on surfaces of different orientations. The crystallinity and defect mechanisms in the layers grown on (100), (311), and (511) surfaces at different temperatures were compared. In most cases we found that (311) surfaces can produce epitaxial layers as good as on (100) surfaces.

Perfect lattice-matched GaAs/AIAs superlattices were grown on (311)A-, (311)B-, and (100)-oriented GaAs substrates. Superlattices with good layer morphology were achieved on all three surfaces at 600°C. Fig. 1(a) shows an image of a typical GaAs/AIAs superlattice grown on a (311)B surface. This superlattice exhibits sharp interface abruptness as indicated by the high-order satellites observed in the diffraction pattern in Fig. 1(b).
Determination of arsenic atom distribution arsenic doped silicon using HOLZ-line analysis
Authors: Kikuchi Y. , Hashikawa N. , Uesugi F. , Wakai E. , Watanabe K. , Hashimoto I.
Published: June 2020

In order to measure the concentration of arsenic atoms in nanometer regions of arsenic doped silicon, the HOLZ analysis is carried out underthe exact [011] zone axis observation. In previous papers, it is revealed that the position of two bright lines in the outer SOLZ structures on the[011] zone axis is little influenced by the crystal thickness and the background intensity caused by inelastic scattering electrons, but is sensitive to the concentration of As atoms substitutbnal for Siatomic site.As the result, it becomes possible to determine the concentration of electrically activated As atoms in silicon within an observed area by means of the simple fitting between experimental result and dynamical simulatioan. In the present work, in order to investigate the distribution of electrically activated As in silicon, the outer HOLZ analysis is applied using a nanometer sized probe of TEM equipped with a FEG.

Czodiralsld-gown<100>orientated p-type Si wafers with a resistivity of 10 Ώ cm are used for the experiments.TheAs+ implantation is performed at a dose of 5.0X1015cm-2at 25keV.
Time-resolved high-resolution electron microscopy of nanometer-scale electron beam processing of PbTe films
Authors: Kizuka T. , Tanaka N.
Published: June 2020

Various kinds of nanometer scale processings are required to produce advanced materials, for example, nano-structured electric devices. Electron beam processing at nanometer scale using STEM and TEM, such as drilling and line-writing, is recently interested as a most useful method. Details of structural change during the processing should be elucidated at atomic resolution in order to establish the processing. In the present work we have processed lead telluride (PbTe) films with nanometer electron beam in a high-resolution transmission electron microscope and in-situ observed the variation of atomic arrangements during the processing.

PbTe of 99.99% was vacuum-deposited on air-cleaved (001) surfaces of sodium chloride at room temperature. Time-resolved high-resolution electron microscopy was carried out at room temperature using a 200-kV electron microscope (JEOL, JEM2010) equipped with a high sensitive TV camera and a video tape recorder. The spatial resolution of thesystem was 0.2 nm at 200 kV and the time resolution was 1/60 s. Electron beam irradiation density was 120 A/cm2 at the processing and the observation.
Characterization of precipitates of Ti3Al in a titanium alloy
Authors: Zhang X.D. , Wiezorek J.M.K. , Evanst D.J. , Fraser H.L.
Published: June 2020

A two phase alpha-beta titanium alloy, Ti-6Al-2Mo-2Cr-2Sn-2Zr-0.2Si (Ti-6-22-22S), has recently been reconsidered as a high temperature material for aircraft engine applications. This alloy exhibits specific strength and fracture toughness superior to those of Ti-6A1-4V. However, similar to other alpha-beta titanium alloys, microstructural stability is one of the major concerns regarding industrial application of Ti-6-22-22S, since changes of the microstructure during long term high temperature exposure significantly affect the performance of components. Two types of precipitates have been observed in Ti-6-22-22S alloys, namely silicides and alpha 2-Ti3Al. The presence of intermetallic precipitates, such as alpha 2-Ti3Al, in the parent alpha matrix has been reported to result in brittle behaviour of the alpha-beta alloys due to the formation of intense planar slip bands. The present paper presents results of the characterization of intermetallic alpha2-Ti3Al precipitates in the alpha phase by methods of scanning and transmission electron microscopy (SEM and TEM respectively).
Microstructure of aged Ni-Mo (Ni/Mo = 4) alloys containing Al
Authors: Shen Elaine , Brooks Charlie R. , Kenikt E. A.
Published: June 2020

Ni-rich Ni-Mo alloys containing around 20 at. % Mo exist as a face-centered cubic, short-range ordered (SRO), solid solution (α) above around 868 °C, and transform to a long-range ordered (LRO), body-centered tetragonal structure (β) upon cooling slowly to below this temperature. Upon quenching, the α is retained, which can then be converted to β upon aging below 868 °C. The formation of the β structure induces considerable strengthening (e.g.. the hardness doubles), but is accompanied by embrittlement.

A commercial alloy based on this composition is Hastelloy B2, and precautions are exercised in its use to prevent ordering and hence embrittlement. In a continuing effort to develop Ni-Mo alloys strengthened by ordering but with usable toughness, we have been examining the addition of Al to the base Ni-25 at. % Mo composition. Specimens containing 2. 5, 7 and 9 at. % Al, with Ni/Mo = 4.0, were made by arc melting the appropriate amounts of a Hastelloy B2 alloy and Al. The buttons were melted several times under an argon atmosphere.
In situ reversed deformation of prefatigued aluminum single crystals in the HVEM
Authors: Wall M. A. , Kassner M. E.
Published: June 2020

Utilizing a technique describe earlier by Wall et al., prefatigued aluminum single crystals oriented for single slip, were cyclically deformed in the HVEM operating at 1.5Mev by deforming in mutually perpendicular “X and Y” directions. Deforming in X and Y avoids foil buckling and allows for shear reversal (fatigue). Initial observations are reported here and will aid in the understanding of the micro-mechanisms of cyclic deformation.

In situ specimens extracted from prefatigued single crystals (560 cycles, strain amplitude of 1.2 X 10−3at 77K) were oriented in such a manner as to facilitate imaging primary dislocations, b=[0,l,-1], on the primary (111) slip plane. The (111) plane is inclined 25° about the [0,1,-1] direction to increase the projected length of these dislocations for viewing on the TEM screen. The [0,1,-1] direction is also contained in the plane of the specimen foil and is 45° to the tensile axes. The specimens are then tilted to a [1,1,-1] diffracting condition. Nonperforated specimens were prepared by a technique described by Kassner Specimens were then alternately deformed along mutually perpendicular tensile axis in the HVEM.
Applications of laboratory-based X-ray microbeam diffraction and fluorescence analysis
Authors: York Brian R.
Published: June 2020

The quantitative structural and chemical analysis of bulk materials is generally done using x-ray diffraction and fluorescence techniques. This is due, in large part, to the vast body of work which has gone into characterizing the x-ray scattering process and the ease with which this interaction canbe kinematically modeled to high precision. Other salient features of x-ray analytical techniques aretheir use as subsurface probes, they require minimal specimen preparation, and ambient conditions are generally adequate for analysis. However, as microanalytical tools, these techniques were typically abandoned because of the difficulty encountered in confining the x-rays to dimensions <<.1 mm with sufficient intensity for a timely analysis. Recently, there has been a rekindled interestin x-ray microbeam analysis because of the reintroduction of tapered capillaries as total reflection x-ray optics. Tapered capillaries can essentially capture larger solid angles nearer the x-ray sourceand act as x-ray wave guides to transport the x-rays to the specimen with high efficiency. It is now possible using laboratory x-ray sources, to produce x-ray beams suitable for diffraction or fluorescence analysis, with diameters in the range of 3-12 μm. X-ray diffraction patterns have been acquired from diffraction volumes as small as 2 μm and fluorescence maps with 5 μm spatialresolution have been demonstrated.
Image analysis of fish stained specimens: A new diagnostic tool in genetics and oncology
Authors: Tanke H. J. , Vrolijk J. , Raap A. K.
Published: June 2020

In situ hybridization allows the detection of specific nucleic acid sequences in morphologically intact cells and chromosomes. Presently, fluorescence in situ hybridization (FISH) has reached a high detection sensitivity (defined as the smallest DNA target detectable, high DNA resolution (defined as the smallest distance in kilobasepairs between two DNA targets that can be resolved microscopically and a high multiplicity (defined as the number of different probes that can be identified simultaneously. The current status of FISH methodology:

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several direct and indirect nucleic acid modifications

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sensitivity: unique DNA sequences 1-5 kb

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DNA resolution: metaphase ~ > 1-3 Mbp (light microscopy) interphase ~50 kbp DNA halos ~ 1 kbp

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multiplicity: at least 12

Progress in in situ hybridization and related technology has caused molecular cytogenetics to become an established. In particular the ability of ISH techniques to detect chromosomes and/or chromosome parts in interphase nuclei (interphase cytogenetics) has significantly contributed to this acceptance of ISH, since this technique provides statistically reliable means to study the genetic composition of cells that can not, or only by complicated techniques, be brought in mitosis.
DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization
Authors: Trask Barbara , Yokota Hiroki , Esposito Richard , Friedman Cynthia , Massa Hillary , van den Engh Ger
Published: June 2020

By using FISH, the positions of DNA sequences can be discretely marked with a fluorescent spot. For this reason, FISH has proven useful in gene mapping, studying large-scale variation in the human genome, detecting chromosome rearrangements associated with disease, and in understanding the organization of the cell nucleus.

The applications of FISH in gene mapping and cytogenetics requires a basic understanding of the organization of the interphase nucleus. These applications are constrained by the efficiency of hybridization, the spatial resolution of hybridization sites in nuclei, and the correlation (if any) between the separation of sequences in interphase chromatin and their separation on the linear DNA molecule.

The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ∼0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.
Digital imaging microscopy for molecular cytogenetics
Authors: Gray J. , Lockett S. , Mascio L. , Mullikin J. , Pinkel D. , Piper J. , Sudar D. , Thompson C.
Published: June 2020

Digital imaging microscopy is an essential tool in molecular cytogenetics. We describe here, hardware and software for sensitive multi-color image acquisition and display, gene mapping, generation ofcopy number karyotypes using comparative genomic hybridization (CGH), and correlation between genotype and histology in three dimensions.

(a) QUantitative Image Processing System (QUIPS):

Hardware We have developed a QUIPS that is now in routine use. This system is built around a commercial fluorescence microscope equipped with computer-controlled stage, focus and fluorescence excitation filter selection. A high resolution cooled CCD camera and a video frame rate intensified CCD (ICCD) are attached to different ports on the microscope and light is split between them. The real time images of the ICCD are continuously displayed on a monitor and are used for visual scanning and focusing. The high resolution CCD is used for image acquisition. Images from the CCD are digitized at 12 bits per pixel resulting in high dynamic range and high precision. QUIPS employs a fluorescence filter package containing a beam splitter and emission filter that pass light in bands centered at approximately 440 nm (blue), 525 nm (green) and 600 nm (red).
Comparison of human normal and neoplastic prostate epithelial cells by TEM and quantitative image analysis and morphometry (QIAM)
Authors: Gau J. T. , Grove M. L. , Strom S. , Orsini R. , Song K. C. , Bahnson R. , Becich M. J.
Published: June 2020

Prostate cancer is the most common tumor in men in the U.S.A. The regulation of growth control in normal prostate cells and its contribution to tumor progression is a subject of intense investigation.

Recently our group developed culture techniques that allowed us to study normal and neoplastic human prostate epithelial cells from human prostatectomy specimens. Since quantitative evaluation of intracellular compartments will be critical to defining the role of membrane trafficking in the growth regulation, we used TEM and QIAM to characterize normal and neoplastic prostate epithelial cells invivo, as well as in vitro. This work will serve as a baseline to subsequent studies using perturbants of cell trafficking and conditions ;hat promote or block cellular growth, to investigate the role of growth factors and stromal/epithelial nteraction in prostate growth regulation.

Normal and neoplastic tissues were sampled for ultrastructural examination from grid mapped radical prostatectomy specimens that were processed for whole mounts. The SV40 immortalized "normal" prostate cell line (P69) was provided by Dr. Strom.
Structure of frozen hydrated deoxy-sickle hemoglobin fibers
Authors: Lewis Michael R. , Josephs Robert
Published: June 2020

Three-dimensional reconstruction of a helical particle can be accomplished from a single lateral view, but in practice the reconstruction procedure must be optimized to make the best use of the available projection data. This is especially true for unstained frozen hydrated (ice embedded) specimens, whose micrographs have a characteristically low signal to noise ratio. Deoxy-sickle hemoglobin (HbS) fibers present an additional difficulty as well because these particles have a variable pitch and may also have variation in their unit cell structure. We have carried out a three-dimensional reconstruction of an unstained frozen hydrated HbS fiber (Figs. 1 & 2) by convolution back projection. The procedures used to facilitate this three-dimensional reconstruction are described below.

In this work especially close attention was paid to determining the image magnification in order that the reconstruction of this HbS fiber could be compared with previous HbS fiber models (Table 1) which are based on reconstructions of negatively stained HbS fibers. In order to determine the HbS fiber's dimensions accurately the particle (Fig. 1a) was reconstructed twice.
Bacteriophage HK97: Structural transitions along the capsid assembly pathway
Authors: Conway J. F. , Duda R. L. , Cheng N. , Hendrix R. W. , Steven A. C.
Published: June 2020

The assembly pathway of the HK97 capsid passes through four stages, starting with the first complete precursor and ending with the mature head. The earliest particle, Prohead I, contains 415-420 copies of the major capsid protein (42kDa) arrayed in a T=7 icosahedral lattice. Proteolytic cleavage (42kDa → 31kDa) converts Prohead I into Prohead II. Expansion of Prohead II into Head I involves a major conformational change that is thought to be triggered in vivo by DNA packaging. The final transition to Head II involves the formation of covalent crosslinks between capsid protein subunits that confer additional stability. We have been examining the structural basis of these transitionsand describe here the Prohead II and Head II states.

Highly purified preparations of capsids were prepared either from expression vectors of capsid proteins or from growing phage mutants in E. coli cells. Samples were prepared for electron microscopy in the frozen, hydrated, state, and imaged in a Philips EM400T operating at 100kV, using a Gatan 626 cryo-holder. Micrographs were digitized and three-dimensional density maps were calculated as described.
Fluorescence lifetime-resolved two-photon microscope
Authors: Gratton Enrico , So Peter , French Todd
Published: June 2020

Two photon fluorescence microscopy was first proposed by Denk et al. in 1990. There are a number of advantages which this new kind of microscopy can offer, including the high spatial resolution, rejection of out of plane fluorescence and reducing photobleaching. We have constructed a fluorescence microscope similar in concept to that of Denk et al. with the addition of lifetime fluorescence capability. The microscope system is built around a Zeiss Axiovert unit. The light source is a Coherent Mira 900 Ti:Sapphire laser with about 150 fs pulse width at a repetition of 80 MHz. The average laser power in the 720 nm-960 nm range varies between 0.5 W to about 1.5 W. The laser scanning system is a digital scanner from Cambridge Research with a resolution of 16 bits. A digital controller interfaced with the data acquisition unit permits positioning and scanning in an arbitrary area of the field of view. Two channels of light emission are available. Typical frame acquisition times are less than a second for the intensity only acquisition mode and about 2-3 seconds for the intensity and lifetime acquisition mode. In a typical run about 15 to 20 frames at a resolution of 257 x 256 are acquired and stored at different z-positions for off-line 3-D reconstruction. The lifetime mode of operation is based on the frequency-domain technique.
The automated interactive microscope (AIM) for investigating the functional dynamics of living cells: Status and vision
Authors: Harris Lowell D. , Deerfield David W. , Fahlman Scott E. , Gough Albert H. , Lanni Frederick , Farkas Daniel L. , Taylor D. Lansing
Published: June 2020

The current status and future development plans for the Automated Interactive Microscope (AIM), to better define the mechanisms of the function of living cells, is described. The development of AIM is fueled by biologists need to spatially and temporally correlate biochemical, molecular and genetic patterns to study cell functions such as division, locomotion and endocytosis. AIM will allow the investigator to use the cell as a “living microcuvette”.

AIM redefines the meaning of the term “microscope” to include the functional capability of the microscope system provided by computerization, in addition to the more classical definition which emphasizes the opto-mechanical hardware. AIM is an next generation electronic light microscope imaging system which has grown out of our work in the development of the multimode light microscope. New functionality includes new experiment control facilities as well as related multidimensional image processing, image analysis and interactive data visualization. AIM is built around microscope hardware with high precision motion controls and a powerful host workstation computer linked, via a very high speed data gateway, with the high performance computing and communication (HPCC) facilities of the Pittsburgh Supercomputing Center (PSC).
Progression of foam-cell formation in vitro in elicited macrophages after incubation with ox-LDL
Authors: Klinkner A.M. , Waites R. , Bugelski P. , Kerns W. D.
Published: June 2020

Thioglycollate elicited mouse peritoneal macrophages incubated with copper-oxidized LDL(ox-LDL) developed foam cell morphology as observed by Nile red fluorescent staining for neutral lipids and by electron microscopic observations. Total cellular cholesterol content rose steadily over 168 hrs and esterified vs free cholesterol ratios also increased.

Cultured macrophages were incubated with ox-LDL, fixed, and stained with Nile red or processed for electron microscopy. Macrophages stained with Nile red exhibited progressive accumulation of lipid over 24, 72 and 168 hrs (Fig. 1-3). The percentage of cells with lipid inclusions and the size and number of inclusions per cell increased progressively over 168 hrs. Control cells incubated without ox-LDL did not have staining indicative of foam cell formation.

Transmission electron microscopy of cultured cells at 24, 72, and 168 hrs showed distinct ultrastructural changes (Fig. 4-6). Macrophages at 24 hrs possessed non-membrane bound lipid droplets. Following 72 hrs, lysosomes containing multilamellar lipoid structures were also present. Cells at 168 hrs had characteristics similar to those at 24 and 72 hrs with the addition of cholesterol crystal profiles.
High-speed brightfield 3-D imaging and 3-D image analysis of thick and overlapped cell clusters in cytological preparations
Authors: Mackin Robert W.
Published: June 2020

This paper presents two advances towards the automated three-dimensional (3-D) analysis of thick and heavily-overlapped regions in cytological preparations such as cervical/vaginal smears. First, a high speed 3-D brightfield microscope has been developed, allowing the acquisition of image data at speeds approaching 30 optical slices per second. Second, algorithms have been developed to detect and segment nuclei in spite of the extremely high image variability and low contrast typical of such regions. The analysis of such regions is inherently a 3-D problem that cannot be solved reliably with conventional 2-D imaging and image analysis methods.

High-Speed 3-D imaging of the specimen is accomplished by moving the specimen axially relative to the objective lens of a standard microscope (Zeiss) at a speed of 30 steps per second, where the stepsize is adjustable from 0.2 - 5μm. The specimen is mounted on a computer-controlled, piezoelectric microstage (Burleigh PZS-100, 68/μm displacement). At each step, an optical slice is acquired using a CCD camera (SONY XC-11/71 IP, Dalsa CA-D1-0256, and CA-D2-0512 have been used) connected to a 4-node array processor system based on the Intel i860 chip.
Low-light-level three-dimensional video microscope with long working distance objective lenses
Authors: Lin W. , Gregorio J. , Holmes T.J. , Szarowski D. H. , Turner J.N.
Published: June 2020

A low-light level video microscope with long working distance objective lenses has been built as part of our integrated three-dimensional (3-D) light microscopy workstation (Fig. 1). It allows the observation of living specimens under sufficiently low light illumination that no significant photobleaching or alternation of specimen physiology is produced. The improved image quality, depth discrimination and 3-D reconstruction provides a versatile intermediate resolution system that replaces the commonly used dissection microscope for initial image recording and positioning of microelectrodes for neurobiology. A 3-D image is displayed on-line to guide the execution of complex experiments. An image composed of 40 optical sections requires 7 minutes to process and display a stereo pair.

The low-light level video microscope utilizes long working distance objective lenses from Mitutoyo (10X, 0.28NA, 37 mm working distance; 20X, 0.42NA, 20 mm working distance; 50X, 0.42NA, 20 mm working distance). They provide enough working distance to allow the placement of microelectrodes in the specimen.
Real-time scanning-slit in vivo confocal microscope based on bilateral scanning which solves the problem of specimen motion blur: Applications to ocular neuroimaging
Authors: Masters Barry R. , Thaer Andreas A.
Published: June 2020

The standard method to observe nerves in tissue is to use some agent such as silver or gold compounds which are localized in the nerve fibers and increase their contrast. These were the techniques developed by Camillo Golgi and Santiago Ramón y Cajal. Cajal’s work was published in a two-volume edition in French in 1909 with the title Histologie du Systéme Nerveux de l’ Homme et des Vertébrés. This work was republished in 1952-1955 as Histologie du Systéme Nerveux by L. Azoulay, Instituto Ramón y Cajal.

Alternatively a fluorescent compound may be introduced near the cell bodies of the neurons and the compound diffuses along the lipid membranes of the nerve. Subsequent observations of the specimen in a fluorescent microscope provides the observer with a bright, high contrast image of the nerve and its branches and bifurcations.

The standard instrument for the observation of the cornea is the slit-lamp. The slit-lamp was invented by Allvar Gullstrand. Gullstrand made major contributions to the theory of astigmatism and to the mechanism of accommodation of the eye.
Paracrystalline inclusions within the cisternae of rough endoplasmic reticulum in a pleomorphic sarcoma: A marker of fibroblastic differentiation?
Authors: Gunning W.T. , Hameed K. , Calomeni E.P.
Published: June 2020

Many pleomorphic sarcomas with a storiform growth pattern have been classified as malignant fibrous histiocytomas (MFH) for the last 20 years. The diagnosis is debatable and has received much criticism of late since MFH is a diagnosis of exclusion; it has no definable, reproducible criteria for its diagnosis. The advent of immunocytochemistry has established that nonspecific antibodies to alpha-1-antitrypsin and alpha-1-chymotrypsin will commonly react with these neoplasms. However, these antibodies are not reliable markers as they are 1) nonspecific and 2) not always reactive. It has been proposed that with sufficient effort in investigating these sometimes bizarre sarcomas, a more precise differentiation may be determined. We have recently examined a primitive sarcoma which could have been classified as MFH on the basis of histologic presentation (Figure 1), however, the tumor tissue reacted in an unusual manner with antibodies to high molecular weight keratin, vimentin, alpha-1-antitrypsin and alpha-1-chymotrypsin. Furthermore, significant accumulations of paracrystalline arrays (Figure 2) were found within the cisternae of the rough endoplasmic reticulum (RER). These findings led to a literature search in which we found that similar observations have been described in pediatric fibroblastic neoplasms.
Does a nuclear localizing signal always assure protein transport into the nucleus in transfected cells?
Authors: Cui C. , Cardell E.L. , Stambrook P.J. , Cardell R.R.
Published: June 2020

A decade ago Lanford and Butel demonstrated that proteins required a specific amino acid sequence in order to be routed from the cell cytosome to the nucleus. Using recombinant DNA and gene transfection techniques, Kalderon et al and Lanford and colleagues found that the addition of a specific amino acid sequence, the nuclear localizing signal (NLS), to a protein normally secreted or localized within the cytosome resulted in its transport from its cytosomal site of synthesis to the nucleus. In such studies, immunofluorescence microscopy is routinely used to evaluate the intracellular localization of the protein encoded by the transgene in intact transfected cells. The nucleus is brightly fluorescent in cells transfected with genes for a protein with an associated NLS. In contrast, proteins without an NLS remain in the cytosome. Generally, in transfected cells, the nuclear association of the fluorescent-labeled antibody directed against a protein of interest is interpreted to mean that this protein is localized within the nucleus.
Centrosomes are phosphorylated in sea urchin eggs throughout the cell cycle, during artificial activation, and when microtubule growth is inhibited
Authors: Schatten Heide , Chakrabarti Amitabha
Published: June 2020

In most animal systems, microtubules are nucleated and organized by centrosomes which undergo considerable modifications during the cell cycle. Typically, centrosomes are phoshorylated at the transition from interphase to mitosis as shown with MPM2, an antibody directed against phosphoproteins. Using the MPM2 antibody, we show in this paper with transmission electron microscopy (TEM) and immunofluorescence microscopy (IFM) that in the sea urchin system centrosomes are phosphorylated in the sperm before fertilization and at every stage of the first cell cycle. MPM2 exhibits identical staining patterns as Ah-6 and 5051 previously shown to reliably identify centrosomal material in sea urchin cells. After centrosomal material is brought into the egg by the sperm, it spreads around the zygote nucleus where it gets distributed and becomes bipolar and compacted to form the mitotic apparatus. Typically, at these mitotic stages, centrosomal material exhibits the brightest staining with MPM2, Ah-6 and 5051 Since in this system phoshorylated centrosomal material is contributed by the sperm, the egg's competence for centrosome phosphorylation was analyzed by activating centrosomal material in the unfertilized egg by treatment with either A23187, ammonia, D2O, or taxol.
Electron-Microscopic localization of biotinylated gastrin bound to eosinophils in the rat stomach
Authors: Miller Caroline A. , Nichols David H. , Murphy Richard F.
Published: June 2020

Gastrin is a small peptide capable of both stimulating gastric acid secretion and acting as an enteric growth factor. Known functions of eosinophils in the rat stomach are related to immunological defense. Here we demonstrate the binding of biotinylated gastrin to rat stomach eosinophils in the electron microscope. Small pieces of stomach were fixed by immersion in 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 for 1 hour. The tissue was then cryoprotected in 30% sucrose/0.1 M phosphate buffer, transferred to Tissue Tek OCT compound and frozen in isopentane cooled with liquid nitrogen. Transverse cryostat sections were cut at 25 μm, thawed in PBS and free floating sections exposed to 10−5 M biotinylated 1-17 gastrin (human sequence; Peninsula Labs) for 1 hour. Controls omitted the biotinylated gastrin from this step. Sections were then rinsed 3X in PBS and exposed to either:

1).

a 1:50 dilution of 10 nm Extravidin colloidal gold (Sigma) for 2 hours, or

2).

an avidin-biotin-alkaline phosphatase complex (ABC-AP;

Vector) for 1 hour. A substrate solution containing cerium chloride was used to generate an electron dense reaction product.

Sections from both procedures were postfixed in 1% OsO4 in 0.1 M phosphate buffer, rinsed and dehydrated. These were then flat embedded in EMbed 812 between two microscope slides coated with Liquid Release (both from Electron Microscopy Sciences).

Polymerized sections were adhered to resin blocks using super glue, cut at 70-90 nm, stained with uranyl acetate/lead citrate and observed in a Philips CM-10 electron microscope.
A rapid phosphotungstic acid staining method on ultra-thin sections
Authors: Chien K. , Van de Velde R. L. , Heusser R.C. , Shiroishi H. , Cohen A.H.
Published: June 2020

The Nail-Patella Syndrome (NPS) is an autosomal dominant hereditary disease in which about 60% of the patients have glomerular abnormalities. Phosphotungstic acid (PTA) has been used successfully to demonstrate the specific collagen fibrils of NPS within the glomerular basement membrane and/or mesangial matrix in ultra-thin sections. However, in our laboratory we had difficulty staining Eponate 12 sections with PTA. A modified staining procedure which makes use of the microwave has been developed as follows:

1. Glutaraldehyde/osmium fixed kidney specimens were embedded in Eponate 12 and ultra-thin sections collected on Gilder 400 mesh copper grids. Grids were stained using a RTV 700 silicone rubber staining pad. This pad contains shallow, round wells (5mm in diameter and 2mm deep) with slots in the center bottom of each well. By bending the pad, the rim of a grid can be inserted into the slot. On release, the grids adhere securely to the wall of the slot.

2. 10% aqueous phosphotungstic acid was dropped into the staining wells around each grid. The staining pad was then placed in a moisture chamber consisting of a pre-heated Petri dish containing wetted filter paper and placed in Pelco 3400 laboratory microwave oven for 110 seconds at 80% power. A Tripour beaker containing 200ml room temperature tap water was placed in the microwave oven as a heat sink.
SEM evaluation of acid-etched human dentin: Alternative techniques to critical-point drying
Authors: Perdigao Jorge , Lambrechts Paul , Van Meerbeek Bart , Vanherle Guido , Lopes Augusto L. B.
Published: June 2020

Scanning electron microscopy (SEM) has become a useful tool in dentistry. During the late 1950's and yearly 1960's the primary use of SEM in dentistry was to study dentin and enamel ultrastructure. After the introduction of the acid-etch technique, SEM has also been extensively used to study the mechanisms of enamel bonding and dentin bonding.

The mechanism of bonding of resin to enamel is the formation of resin tags into the microporesities created by the acid. Bonding to dentin is not as simple as bonding to enamel. Acidic agents demineralize dentin, expose lie collagen and increase the microporosity of the intertubular dentin. If this collagen is air-dried, it collapses over lie underlying unaffected dentin. If this collagen is air-dried, it collapses over the underlying unaffected dentin. In electron microscopy, this is called die “drying down” phenomenon. Criticalpoint drying (CPD) has been described as being essential for etched-dentin preservation and for resin-dentin interface observation.
New image-modeling software used for 3-D analysis of biological EM data on silicon graphics computers
Authors: Kremer James R. , Furcinitti Paul S. , Mcintosh J. Richard
Published: June 2020

Since the early 1980’s, there have been several software packages available for no or low cost that have been developed for image processing of 3-dimensional (3-D) biological images as well as several viewing packages that can be used for viewing 3-D data sets. Work has been done to develop a modeling program that can make interactive 3-D models in stereo, but there is a present a gap between the image processing and model viewing steps for complex 3-D biological data. Although packages available for no or low cost have only rudimentary model building tools the biologist would like to have as accurate a 3-D model as possible. Here we describe a new image modeling software package for the 3-D analyses of biological EM data.

We have developed a useful software package (IMOD, Image Modeler) that allows the user to draw two dimensional contours in 3-dimensional image data. The power of IMOD is the ability to see the 3-dimensional image data in several different views at the same time.
Corneal ultrastructure after excimer laser keratectomy
Authors: Nguyen H. , Eiferman R. S. , Nordquist R. E.
Published: June 2020

The Argon fluoride excimer laser emits light at 193nm. At a fluence of 500mJ/CM2, one micron of organic tissue should be removed. This removal occurs by the disruption of chemical bonds rather than the normal heat vaporization seen with other types of lasers. These characteristics should allow the use of this laser in performing precise removal of superficial corneal scars and opacities as well as inducing changes in corneal curvature for refractive corrections. In our studies we will report the ultrastructural changes induced in both primates and man following excimer laser treatment.

Following excimer laser treatment, the primates were followed for two years and the corneas removed for morphological studies. The human specimens were taken at various times after excimer treatment for clinical reasons, all having to do with unfavorable outcomes. All human specimens were taken at the time of transplantation of a new cornea to these eyes. The specimens were fixed with 2% paraformaldehyde and 2% gluderaldehyde in cacodylate buffer at pH 7.3. After fixation, these specimens were dehydrated, imbedded in Spurr epoxy resin, then sectioned and examined ultrastructurally.
EDXS in the electron microscope: The hardware
Authors: Garratt-Reed Anthony J.
Published: June 2020

The hardware employed for microanalysis by energy-dispersive x-ray analysis in the electron microscope consists, logically enough, of two principal parts, i.e. the electron microscope and the x-ray detector/analyzer combination. There are a number of excellent sources which discuss in depth how these work and interact to allow an analysis to be performed, and how to obtain the best possible results.

The basic principle of operation of an energy-dispersive x-ray detector is as follows: a small piece of single-crystal intrinsic semiconductor (typically silicon doped with lithium for compensation, although high-purity germanium detectors are for some applications an attractive alternative) about 10-30 mm2 in area by 2-3 mm thick is subject to a moderate electric field, generated by applying a bias of about 500V between electrodes plated on the two opposite faces of the crystal. When an x-ray enters the crystal, it ionizes one of the atoms of semiconductor, generating a photoelectron of well-defined energy (i.e. the energy of the incoming x-ray less the ionization energy of the excited energy level). The photoelectron travels through the crystal, ionizing further atoms as it goes, each ionization requiring on average 3.86 eV (in the case of silicon detectors).
Recent advances in light microscopy
Authors: Herman Brian
Published: June 2020

The optical microscope has been an essential tool for generations of biologists, allowing the examination of details of individual cells and organisms impossible with the naked eye. The development of a large number of fluorescence dyes, allowing one to study the chemical and molecular dynamics of intact cells, has been of major importance in broadening the usefulness of optical microscopy. Attaching fluorescent dyes to specific molecular cell constituents allows real-time observation and quantitation of the activities of these constituents in cells and tissue. Beginning in the early 1950's, a number of improvements have been made in light microscope technology including advances in optics, digital computers, digitizers and image processing, and low-light-level (intensified) photodetectors. The ability of this instrumentation to intensify weakly fluorescent signals at the level of single living cells, without injuring the cell, has led to major advances in the understanding of the physiology and pathophysiology of living cells.