Phase Contrast Transmission Electron Microscopy Research Articles

Growth Process and Molecular Packing of a Self-assembled Lipid Nanotube: Phase-Contrast Transmission Electron Microscopy and XRD Analyses

Phase-contrast transmission electron microscopy (PC-TEM) and quick freezing method have been combined to study the initial growing process of a self-assembled lipid nanotube in water. The PC-TEM enabled us to detect thin lamellar edge structure and the very fast growth of the newborn edge to a thin tube with high contrast. The thin tube acts as a core structure for further growth into thick complete lipid nanotube. The initially formed nanotube structure is denoted as a "core tube". The core tube has uniform wall structure that consists of five lamellar layers and the inner and outer diameters of the core tube are 130 and 180 nm, respectively. The evaluated lamellar spacing of 4.6 nm is well compatible with that measured by X-ray diffraction. We also discussed the molecular packing of the nanotube from the pitch angle determined by the PC-TEM images, X-ray diffraction pattern in wide-angle region, and IR spectroscopy. The subcell structure of the nanotube is assigned to an orthorhombic type. The twisting angle between the neighboring lipid molecules is determined as ca. 0.26 degrees for the first time; it is a crucial parameter for the formation of a lipid nanotube in chiral packing but has not been elucidated before.

Evaluation of Asbestos Exposures during Firewood-Harvesting Simulations in Libby, MT, USA—Preliminary Data

Research was conducted in order to assess potential exposure to asbestos while harvesting firewood from amphibole-contaminated trees near Libby, MT, USA. Three firewood-harvesting simulations took place in the summer and fall of 2006 in the Kootenai Forest inside the US Environmental Protection Agency (EPA) restricted zone surrounding the former W.R. Grace vermiculite mine. Another simulation was conducted near Missoula, MT, USA, which served as the control. The work practices following each simulation were consistent throughout each trial. Personal breathing zone (PBZ) asbestos concentrations were measured by phase contrast microscopy (PCM) and transmission electron microscopy (TEM). Surface wipe samples of personal protective clothing were measured by TEM. The mean (n = 12) PBZ PCM sample time-weighted average (TWA) concentration was 0.29 fibers per milliliter, standard deviation (SD = 0.54). A substantial portion (more than five fibers per sample) of non-asbestos fibers (cellulose) was reported on all PBZ samples (excluding field blanks) when analyzed by TEM. The mean (n = 12) PBZ TEM sample TWA concentration for amphibole fibers <5-microm long was 0.15 fibers per milliliter (SD = 0.21) and the mean (n = 12) PBZ TEM concentration for amphibole fibers >5-microm long was 0.07 fibers per milliliter (SD = 0.08). Substantial amphibole fiber concentrations were revealed on Tyvek clothing wipe samples. The mean concentration (n = 12) was 29 826 fibers per square centimeter (SD = 37 555), with 91% (27 192 fibers per square centimeter) comprised fibers <5-microm long. There were no significant differences in PBZ and wipe sample concentrations among the tasks performed by four investigators. Each of these three simulations were consistent in demonstrating that amphibole fibers are released from tree reservoirs during firewood-harvesting activities in asbestos-contaminated areas and that the potential for exposure exists during such activities.

Single particle analysis based on Zernike phase contrast transmission electron microscopy

We present the first application of Zernike phase-contrast transmission electron microscopy to single-particle 3D reconstruction of a protein, using GroEL chaperonin as the test specimen. We evaluated the performance of the technique by comparing 3D models derived from Zernike phase contrast imaging, with models from conventional underfocus phase contrast imaging. The same resolution, about 12A, was achieved by both imaging methods. The reconstruction based on Zernike phase contrast data required about 30% fewer particles. The advantages and prospects of each technique are discussed.

Structural fingerprinting in the transmission electron microscope: overview and opportunities to implement enhanced strategies for nanocrystal identification

This paper illustrates the prospective need for structural fingerprinting methods for nanocrystals. A review of the existing fingerprinting methods for crystal structures by means of transmission electron microscopy (TEM) which work for a single setting of the specimen goniometer is given. Suggestions are made on how some of these methods could be enhanced when nanocrystals and novel instrumentation are involved, i.e. when either the kinematic or quasi-kinematic scattering approximations are sufficiently well satisfied. A novel strategy for lattice-fringe fingerprinting of nanocrystals from Fourier transforms of high-resolution phase contrast transmission electron microscopy (HRTEM) images is briefly outlined. Nanocrystal structure specific limitations to the application of this strategy are discussed.

3-D reconstruction of the atomic positions in a simulated gold nanocrystal based on discrete tomography: Prospects of atomic resolution electron tomography

A novel reconstruction procedure is proposed to achieve atomic resolution in electron tomography. The method exploits the fact that crystals are discrete assemblies of atoms (atomicity). This constraint enables us to obtain a three-dimensional (3-D) reconstruction of test structures from less than 10 projections even in the presence of noise and defects. Phase contrast transmission electron microscopy (TEM) images of a gold nanocrystal were simulated in six different zone axes. The discrete number of atoms in every column is determined by application of the channelling theory to reconstructed electron exit waves. The procedure is experimentally validated by experiments with gold samples. Our results show that discrete tomography recovers the shape of the particle as well as the position of its 309 atoms from only three projections. Experiments on a nanocrystal that contains several missing atoms, both on the surface and in the core of the nanocrystal, while considering a high noise level in each simulated image were performed to prove the stability of the approach to reconstruct defects. The algorithm is well capable of handling structural defects in a highly noisy environment, even if this causes atom count “errors” in the projection data.

Morphologic, cytometric and functional characterisation of abalone ( Haliotis tuberculata) haemocytes

This work presents the first detailed microscopic and functional analysis of the haemocytes of an abalone; the European Haliotis tuberculata. It is shown that in contrast to the situation in bivalves, only very few basophilic “granulocytes” could be found and exclusively with a histological stain. Neither flow cytometry, phase contrast observation nor transmission electron microscopy were able to detect any granular cells. The large majority of cells was constituted of “hyalinocytes”, which could be sorted by flow cytometry, for the first time, into small (blast-like) and large cells. This permits a detailed analysis of haemocytes and especially of the lowly represented blast-like cells. The differences in haemolymph cell composition between bivalves and gastropods is reviewed in depth and discussed in view of the new data we present. Most of the abalone haemocytes analysed harbour many vacuoles, large glycogen deposits, lipid inclusions and acidic compartments. However, although the number of these “inclusions” was rather variable in between individual hyalinocytes, these experiments did not allow to discern subpopulations using these criteria, and the population appears more as a “differentiation continuum”. Haemocytes adhere very rapidly and are immunologically active as they quickly phagocytose latex beads and zymozan particles. This study is the first step towards understanding the H. tuberculata immune system by adapting new tools to gastropods and in providing a first detailed morpho-functional study of their haemocytes.

3P285 位相差電子顕微鏡を用いた凍結切片の微細構造観察(バイオイメージング、生命の起源・進化,口頭発表,第45回日本生物物理学会年会)

3P285 位相差電子顕微鏡を用いた凍結切片の微細構造観察(バイオイメージング、生命の起源・進化,口頭発表,第45回日本生物物理学会年会)

Species diversity of heterotrophic flagellates in White Sea littoral sites

The species diversity and distribution of benthic heterotrophic flagellates in sediment samples from along the salinity gradient in the Chernaya River Estuary and from Velikaya Salma Strait (Kandalaksha Bay, the White Sea) were investigated during August 2004. One hundred and six taxa have been identified by means of phase and interference contrast light microscopy and transmission electron microscopy. The majority of observed flagellates were bacterivores. The species diversity of the following groups: choanoflagellates, euglenids, kinetoplastids, bicosoecids, chrysomonads, thaumatomonads and flagellates Incertae sedis was the highest. Ancyromonas sigmoides and Petalomonas pusilla were the most common species. The species richness was lowest in the brackish water estuarine part with salinity levels between 5‰ and 8‰. The distribution of heterotrophic flagellates conforms to the so-called “rule of critical salinity”, possessing, apparently, the same universal character for organisms of different size levels. Heterotrophic flagellate communities in these littoral sites were highly heterogeneous. The curve of “cumulative species number vs. sampling effort” is well fitted by equation S=21.17 N 0.50 and unsaturated, which indicates that more intensive investigations of the heterotrophic flagellates in the White Sea should be expected to reveal more species.

2P540 Single Particle Analysis of Image Data Acquired by Zernike Phase Contrast Transmission Electron Microscope(52. Bio-imaging,Poster Session,Abstract,Meeting Program of EABS &amp; BSJ 2006)

2P540 Single Particle Analysis of Image Data Acquired by Zernike Phase Contrast Transmission Electron Microscope(52. Bio-imaging,Poster Session,Abstract,Meeting Program of EABS &amp; BSJ 2006)

Epitaxial and endotaxial semiconductor quantum dots: A brief review on atomic ordering and the void-mediated formation mechanism

On the basis of experimental evidence that was presented in earlier original works and reviews, the suggestion is made that atomically ordered quantum dots which are grown by either epitaxy or endotaxy may in addition to their larger quantum confinement potentials possess superior long-term structural stability. Such atomically ordered quantum dots should, therefore, be superior to random alloy quantum dots as far as prospective device applications are concerned. The theoretical basis for this suggestion is simple thermodynamic calculations. The predictions from our simple model are in agreement with high resolution electron phase contrast transmission electron microscopy (HRTEM) and atomic resolution Z-contrast scanning transmission electron microscopy (Z-STEM) observations on epitaxially grown atomically ordered In(Sb,As), (In,Ga)Sb, (Cd,Zn)Se, (Cd,Mn,Zn)Se quantum dots, epitaxial Pb(Se,Te) quantum dot predecessor islands, and endotaxially grown (In,Si,As) quantum dots in Si matrix. Endotaxially grown (Sn,Si) quantum dots in Si matrix are also discussed. It is suggested that the void-mediated formation mechanism of such (Sn,Si) quantum dots in Si may be employed for other endotaxially grown QDs systems.

Image-based nanocrystallography by means of transmission electron goniometry

Since the crystallographic phase and morphology of many materials changes with the crystal size in the 1–100 nm range and the potential technological applications of nanoparticles are enormous, a need arises to determine the crystallography of nanoparticles individually. Direct space high-resolution phase-contrast transmission electron microscopy (TEM) and atomic resolution Z-contrast scanning TEM when combined with goniometry of direct and/or reciprocal lattice vectors offer the possibility of developing dedicated nanocrystallography characterization methods for such small nanoparticles. Although experimentally feasible for cubic nanocrystals with lattice constants larger than 0.4 nm in contemporary high-resolution TEMs with modest tilt range, image-based nanocrystallography by means of transmission electron goniometry has so far only been employed by a few specialists worldwide. This is likely to change in the future with the availability of aberration-corrected TEMs.

3P025 Single Particle Analysis of Image Data Acquired by Zernike Phase Contrast Transmission Electron Microscope

3P025 Single Particle Analysis of Image Data Acquired by Zernike Phase Contrast Transmission Electron Microscope

Complex Observation in Electron Microscopy: IV. Reconstruction of Complex Object Wave from Conventional and Half Plane Phase Plate Image Pair

An extension of the Complex Observation series based on a novel imaging mode of the transmission electron microscope (TEM) is presented. A half plane phase plate is used to produce images that exhibit topographic-like appearances and have spectral modulation similar to the modulation of Zernike phase contrast TEM (ZPC-TEM) images. The Differential Interference Contrast (DIC) technique in light microscopy produces a similar contrast. The main difference between the two techniques is the asymmetry in one direction of the spectral modulation of the half plane phase plate images, which causes the topographic-like appearance of these images. The complex form of the object wave can be numerically rebuilt using a properly demodulated half plane phase plate image and a conventional TEM image. Experimental results demonstrating the success of the complex reconstruction scheme are presented.

Towards 3D image-based nanocrystallography by means of transmission electron goniometry

ABSTRACTIt is well known that the crystallographic phase and morphology of many materials changes with the crystal size in the tens of nanometer range and that many nanocrystals possess structural defects in excess of their equilibrium levels. A need to determine the ideal and real structure of individual nanoparticles, therefore, arises. High-resolution phase-contrast transmission electron microscopy (TEM) and atomic resolution Z-contrast scanning TEM (STEM) when combined with transmission electron goniometry offer the opportunity of develop dedicated methods for the crystallographic characterization of nanoparticles in three dimensions. This paper describes tilt strategies for taking data from individual nanocrystals “as found”, so as to provide information on their lattice structure and orientation, as well as on the structure and orientation of their surfaces and structural defects. Internet based java applets that facilitate the application of this technique for cubic crystals with calibrated tilt-rotation and double-tilt holders are mentioned briefly. The enhanced viability of image-based nanocrystallography in future aberration-corrected TEMs and STEMs is illustrated on a nanocrystal model system.

Image-Based Nanocrystallography in Future Aberration-Corrected Transmission Electron Microscopes

AbstractSince the crystallographic phase and morphology of many materials changes with the crystal size in the one to hundred nanometer range and the potential technological applications of nanoparticles are enormous, a need arises to determine the crystallography of nanoparticles individually. Direct space high- resolution phase-contrast transmission electron microscopy (TEM) and atomic resolution Z-contrast scanning TEM when combined with goniometry of direct and/or reciprocal lattice vectors offer the possibility of developing dedicated nanocrystallography characterization methods for such small nanoparticles. Although experimentally feasible for cubic nanocrystals with lattice constants larger than 0.4 nm in contemporary high-resolution TEMs with modest tilt range, image-based nanocrystallography by means of transmission electron goniometry has so far only been employed by a few specialists worldwide. This is likely to change in the future with the availability of aberration-corrected TEMs. The reasons why this change is likely to happen are outlined in this paper.

Comparison of phase contrast transmission electron microscopy with optimized scanning transmission annular dark field imaging for protein imaging

Henderson has already shown that electron microscopy should be superior to X-ray and neutron diffraction for determining protein structure with minimum radiation damage. Since the contrast for a molecule embedded in vitreous ice is very low, it is conceivable that dark field imaging would be superior to bright field phase contrast microscopy. A detailed analysis of contrast and signal/noise for both imaging modes is presented. Annular dark field scanning transmission microscopy gives improved contrast and equivalent signal/noise to phase contrast TEM when the molecule is the same thickness as a vitreous ice embedding medium. For a constant embedding medium thickness of 200 Å the contrast is equivalent to phase contrast TEM but the signal/noise is 5 times worse. Even with an efficient detector that only excludes scattering less than 5 mrad there is insufficient signal at a dose of 5 electrons/Å 2 to produce an image with more than 1 electron/per pixel. For larger molecules (>100 Å thick which corresponds to 420 kDa for spherical molecules) the weak phase object approximation used to analyse a phase contrast image no longer applies at 100 kV. This limit could be extended to about 200 Å (about 3 MDa) if a 400 kV microscope were used.

Effects of exogenous nitric oxide on oral squamous cell carcinoma: An in vitro study

Purpose: Nitric oxide (NO) is a newly found unstable free radical gas, serving as an important mediator, messenger, and signal transduction molecule and involved in a variety of pathophysiologic processes. Recently, NO has been reported to have cytotoxic effects on several tumor cells as an effector molecule of activated macrophage. The objective of this study was to investigate the effects of exogenous NO on oral squamous cell carcinoma cell line and to try to clarify the possible mechanisms by which it kills tumor cells. Methods: TSCCa cell line, established from a patient with oral squamous cell carcinoma of the tongue, was exposed to various concentrations of exogenous NO that were released from an NO donor, sodium nitroprusside (SNP), for 48 hours. Nitrite/nitrate levels in the culture supernatant were determined with a commercial available NO kit. Both morphologic and ultrastructural changes were evaluated by reverse phase contrast microscopy or transmission electron microscopy. The DNA was harvested from SNP-treated or untreated TSCCa cells and assessed by agarose gel electrophoresis. Results: SNP released NO into medium in a dose-dependent manner. NO had a concentration-dependent cytotoxicity against TSCCa cells. NO induced tumor cell death through apoptosis, which was characterized by incompleteness of nuclear membrane, disappearance of nucleole and nuclear condensation, chromatin margination, or chromatin homogenization. Agarose gel electrophoresis showed a typical internucleosomal DNA cleavage pattern (DNA ladder), a reliable indicator of apoptosis. Conclusions: Our results suggested that NO had a tumoricidal potential against oral cancer cells. NO might exert its cyotoxicity as an effector molecule of activated microphage through at least apoptosis. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:905-910, 2002

Quantitative phase-amplitude microscopy II: differential interference contrast imaging for biological TEM.

Although phase contrast microscopy is widespread in optical microscopy, it has not been as widely adopted in transmission electron microscopy (TEM), which has therefore to a large extent relied on staining techniques to yield sufficient contrast. Those methods of phase contrast that are used in biological electron microscopy have been limited by factors such as the need for small phase shifts in very thin samples, the requirement for difficult experimental conditions, or the use of complex data analysis methods. We here demonstrate a simple method for quantitative TEM phase microscopy that is suitable for large phase shifts and requires only two images. We present a TEM phase image of unstained Radula sp. (liverwort spore). We show how the image may be transformed into the differential interference contrast image format familiar from optical microscopy. The phase images contain features not visible with the other imaging modalities. The resulting technique should permit phase contrast TEM to be performed almost as readily as phase contrast optical microscopy.

Fiber release during the removal of asbestos-containing gaskets: a work practice simulation.

Work practice studies were conducted involving the removal of asbestos-containing sheet gaskets from steam flanges. These studies were performed to determine potential exposure levels to individuals who have worked with these types of materials in the past and may still work with these products today. The work practices were conducted inside an exposure characterization laboratory (ECL) and were performed by scraping and wire brushing, chrysotile-containing (65% to 85%) sheet gaskets from a number of used steam flanges. Airborne asbestos levels were measured by phase contrast microscopy (PCM) and transmission electron microscopy (TEM) for the personnel and area air samples collected during the study. These workplace simulations showed substantial asbestos fiber release using scraping, hand wire brushing, and power wire brushing techniques during the gasket removal process. The range of concentration was 2.1 to 31.0 fibers/cc greater than 5 micrometers when measured by PCM. These results contrasted with the few reported results in the published literature where lower airborne asbestos levels were reported. In these studies the airborne asbestos fiber levels measured in many of the samples exceeded all current and historical Occupational Safety and Health Administration (OSHA) excursion limits (15-30 minutes) and some previous permissible exposure limits (PEL) based on eight-hour time-weighted average (TWA) standards. Also, individuals who performed this type of work in the past may have had exposures higher than previously suspected. The results demonstrated that employees who remove dry asbestos-containing gaskets with no localized ventilation should wear a full face supplied air respirator with a HEPA escape canister and the work area should be designated a regulated area.

A Novel Phase-contrast Transmission Electron Microscopy Producing High-contrast Topographic Images of Weak objects.

We report a novel class of transmission electron microscope (TEM), the difference-contrast electron microscope (DTEM), which displays nanostructures of thin specimen objects in a topographical manner. Topography obtained by the difference-contrast develops shadowgraphs in pseudo three-dimension, namely volume-like representation of projected objects as if things are illuminated by light from one direction. The specific optical device tomanipulate electron waves for DTEM is the hemicircular π phase-plate, which appears to be quite distinguishable from the Zernike phase plate utilized in Zernike phase-contrast TEM, while both have to be placed onto the back-focal plane of the objective lens. The topographic images obtained with DTEM for ultrathin sections of kidney cells were compared with those obtained with conventional TEM. DTEM confirmed the experimental advantage of high contrast topography by visualizing ultrastructural details inside the cells.