X-ray micrography and imaging of Escherichia coli cell shape using laser plasma pulsed point x-ray sources.

High-resolution x-ray microscopy is a relatively new technique and is performed mostly at a few large synchrotron x-ray sources that use exposure times of seconds. We utilized a bench-top source of single-shot laser (ns) plasma to generate x-rays similar to synchrotron facilities. A 5 microlitres suspension of Escherichia coli ATCC 25922 in 0.9% phosphate buffered saline was placed on polymethylmethyacrylate coated photoresist, covered with a thin (100 nm) SiN window and positioned in a vacuum chamber close to the x-ray source. The emission spectrum was tuned for optimal absorption by carbon-rich material. Atomic force microscope scans provided a surface and topographical image of differential x-ray absorption corresponding to specimen properties. By using this technique we observed a distinct layer around whole cells, possibly representing the Gram-negative envelope, darker stained areas inside the cell corresponding to chromosomal DNA as seen by thin section electron microscopy, and dent(s) midway through one cell, and 1/3- and 2/3-lengths in another cell, possibly representing one or more division septa. This quick and high resolution with depth-of-field microscopy technique is unmatched to image live hydrated ultrastructure, and has much potential for application in the study of fragile biological specimens.     

纳米二次离子质谱技术(NanoSIMS)在微生物生态学研究中的应用

超高分辨率显微镜成像技术与同位素示踪技术相结合的纳米二次离子质谱技术(NanoSIMS)具有较高的灵敏度和离子传输效率、极高的质量分辨率和空间分辨率(＜ 50 nm),代表着当今离子探针成像技术的最高水平.利用稳定性或者放射性同位素在原位或者微宇宙条件下示踪目标微生物,然后将样品进行固定、脱水、树脂包埋或者导电镀膜处理,制备成可供二次离子质谱分析的薄片,进一步通过NanoSIMS成像分析,不仅能够在单细胞水平上提供微生物的生理生态特征信息,而且能够准确识别复杂环境样品中的代谢活跃的微生物细胞及其系统分类信息,对于认识微生物介导的元素生物地球化学循环机制具有重要意义.介绍了纳米二次离子质谱技术的工作原理和技术路线,及其与同位素示踪技术、透射电子显微镜(TEM)、扫描电子显微镜(SEM)、荧光原位杂交技术(FISH)、催化报告沉积荧光原位杂交技术(CARD-FISH)、卤素原位杂交技术(Halogen In Situ Hybridization,HISH)等联合使用在微生物生态学研究方面的应用.     

A focused scanning vertical beam for charged particle irradiation of living cells with single counted particles

The Surrey vertical beam is a new facility for targeted irradiation of cells in medium with singly counted ions. A duo-plasmatron ion source and a 2 MV Tandem? accelerator supply a range of ions from protons to calcium for this beamline and microscope endstation, with energy ranges from 0.5 to 12 MeV. A magnetic quadrupole triplet lens is used to focus the beam of ions. We present the design of this beamline, and early results showing the capability to count single ions with 98% certainty on CR-39 track etch. We also show that the beam targeting accuracy is within 5 μm and selectively target human fibroblasts with a <5 μm carbon beam, using γ-H2AX immunofluorescence to demonstrate which cell nuclei were irradiated. We discuss future commissioning steps necessary to achieve submicron targeting accuracy with this beamline.     

Microirradiation of cells with energetic heavy ions

The ion microprobe SNAKE at the Munich 14 MV tandem accelerator achieves beam focussing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, from 20 MeV protons to 200 MeV gold ions. Because of these properties, SNAKE is particularly attractive for biological microbeam experiments. Here we describe the adaptation of SNAKE for microirradiation of cell samples. This includes enlarging of the focal distance in order to adjust the focal plane to the specimen stage of a microscope, construction of a beam exit window in a flexible nozzle and of a suitable cell containment, as well as development of procedures for on-line focussing of the beam, preparation of single ions and scanning by electrostatic deflection of the beam. When irradiating with single 100 MeV ¹⁶O ions, the adapted set-up permits an irradiation accuracy of 0.91 µm (full width at half maximum) in the x-direction and 1.60 µm in the y-direction, as demonstrated by retrospective track etching of polycarbonate foils. Accumulation of the repair protein Rad51, as detected by immunofluorescence, was used as a biological track detector after irradiation of HeLa cells with geometric patterns of counted ions. Observed patterns of fluorescence foci agreed reasonably well with irradiation patterns, indicating successful adaptation of SNAKE. In spite of single ion irradiation, we frequently observed split fluorescence foci which might be explained by small-scale chromatin movements.     

Low-dose studies of bystander cell killing with targeted soft X rays

The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic CK X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 microm at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a CK X-ray beam was measured in the 0-2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (<200 mGy) and then reached a plateau (>200 mGy). In the low-dose region (<200 mGy), the response after irradiation of a single cell was not significantly different from that when all cells were exposed to radiation. Damaged cells were distributed uniformly over the area of the dish scanned (approximately 25 mm2). However, critical analysis of the distance of the damaged, unirradiated cells from other damaged cells revealed the presence of clusters of damaged cells produced under bystander conditions.     

Yeast importin-β is required for nuclear import of the Mig2 repressor

Background

In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade.

Results

This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain.

Conclusions

It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.     

On the fine structure of the cerebral ganglion of Sagitta (Chaetognatha)

Summary In this study the fine structure of the cerebral ganglion of Sagitta setosa (Chaetognatha) is investigated. The ganglion is flat and superficially positioned dorsally, below the basal lamina of the cephalic epidermis. It is surrounded by a specifically differentiated sheath. This sheath is made up of cells, which are interpreted as representing glial cells, and can be divided into an outer and an inner zone. The outer zone is composed of flat sheath cells with pale nuclei and few organelles. The inner zone consists of densely packed, extremely thin lamellar cellular processes. These attenuated lamellae, which still contain cytoplasm, resemble the myelin sheath of vertebrate axons. The intercellular space between the lamellae contains electron-dense material. In the sheath specialized intercellular contacts occur. The inner zone of this sheath extends at definite points into the centre of the ganglion and separates a zone of perikarya from the neuropil, as well as the single perikarya from each other. The perikarya are relatively uniform and do not form a cortex, but are concentrated mainly in lateral parts of the cerebral ganglion. Within the neuropil are axonal endings which have synaptic contacts with several postsynaptic elements. These anatomical findings are discussed with respect to their functional significance.     

Trichoplax adhaerens (Phylum Placozoa) has Cells that React with Antibodies Against the Neuropeptide RFamide

Abstract The phylum Placozoa contains the most simply organized multicellular animals: they have a thick lower epithelium and a thin upper epithelium with some single cells sandwiched between these layers. Placozoans are generally considered to have no nerve cells or specialized sensory cells. However, the results of this investigation demonstrate the existence of a specific set of cells which react with antibodies against the neuropeptide family RFamide. The RFamide-positive cells of Trichoplax adhaerens are positioned at some distance along the margin of the animal.     

Liposomes targeting tumour stromal cells

Liposomes have found clinical application in cancer therapy in the delivery of cytostatic agents. As a result of the targeted delivery of these toxic molecules to the tumour cells coupled to avoidance of toxicity-sensitive tissues, the therapeutic window is widened. Over the past years the focus of cancer therapy has shifted towards the stromal cells that are present in the tumour. It appears that clinically relevant tumours have acquired the ability to modulate the microenvironment in such a way that a chronic pro-inflammatory and pro-angiogenic state is achieved that contributes to invasion and metastasis and continued proliferation. Over the past years, liposomal formulations have been designed that target key stromal cell types that contribute to tumour growth. At the same time, many promising cell types have not been targeted yet and most of the studies employ drugs that aim at depleting stromal cells rather than modulating their activity towards an anti-tumour phenotype. In this review these target cell types will be addressed. Complementing these targeted formulations with the appropriate drugs to optimally suppress tumour-promoting signals while preserving anti-tumour action will be the challenge for the future.     

Full-term development of rat after transfer of nuclei from two-cell stage embryos

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.     

Single-cell electroporation

Electroporation is a widely used method for the introduction of polar and charged agents such as dyes, drugs, DNA, RNA, proteins, peptides, and amino acids into cells. Traditionally, electroporation is performed with large electrodes in a batch mode for treatment of a large number of cells in suspension. Recently, microelectrodes that can produce extremely localized electric fields, such as solid carbon fiber microelectrodes, electrolyte-filled capillaries and micropipettes as well as chip-based microfabricated electrode arrays, have proven useful to electroporate single cells and subcellular structures. Single-cell electroporation opens up a new window of opportunities in manipulating the genetic, metabolic, and synthetic contents of single targeted cells in tissue slices, cell cultures, in microfluidic channels or at specific loci on a chip-based device.     

Liposomes targeting tumour stromal cells

Abstract

Liposomes have found clinical application in cancer therapy in the delivery of cytostatic agents. As a result of the targeted delivery of these toxic molecules to the tumour cells coupled to avoidance of toxicity-sensitive tissues, the therapeutic window is widened. Over the past years the focus of cancer therapy has shifted towards the stromal cells that are present in the tumour. It appears that clinically relevant tumours have acquired the ability to modulate the microenvironment in such a way that a chronic pro-inflammatory and pro-angiogenic state is achieved that contributes to invasion and metastasis and continued proliferation. Over the past years, liposomal formulations have been designed that target key stromal cell types that contribute to tumour growth. At the same time, many promising cell types have not been targeted yet and most of the studies employ drugs that aim at depleting stromal cells rather than modulating their activity towards an anti-tumour phenotype. In this review these target cell types will be addressed. Complementing these targeted formulations with the appropriate drugs to optimally suppress tumour-promoting signals while preserving anti-tumour action will be the challenge for the future.     

Plasmon‐Active Nano‐Aperture Window Electrodes for Organic Photovoltaics

A lithography free approach to fabricating optically thin (～10 nm) noble metal electrodes with a dense array of sub‐wavelength apertures is reported. These nano‐structured electrodes support surface plasmon resonances which couple strongly with visible light concentrating it near to the electrode surface. They are also remarkably robust and can be fabricated on glass and plastic substrates with a sheet resistance of <15 Ω sq^?1. As the window electrode in solution processed and vacuum deposited organic photovoltaics (OPV) the photocurrent is increased by as much as 28% as compared to identical devices without apertures, demonstrating that the apertures do not need to have a tight size and/or shape distribution to be effective. As a drop‐in replacement for the indium‐tin oxide electrode in flexible OPV these plasmon‐active electrodes offer superior performance; 5.1% vs. 4.6%, demonstrating that this class of electrode is a truly viable alternative to conducting oxide window electrodes for OPV.     

Influence of econazole and 5-fluorocytosine on the ultrastructure of Aspergillus fumigatus and the cytochemical localization of calcium ions as measured by laser microprobe mass analysis

The ultrastructural changes and the localization of Ca++ ions have been investigated in Aspergillus fumigatus after exposure in vitro to econazole (50 micrograms ml-1) and to 5-fluorocytosine (100 micrograms ml-1) for 24 h. The changes obtained with econazole concerned the cell periphery, necrotization of mitochondria and hyperactivity of the central vacuole. After cytochemical Ca++ localization a marked increase in precipitate was observed on mitochondria, vacuoles and collapsed membranes, compared to the control. Laser microprobe mass analysis confirmed that the measured amount of Ca++ ions corresponded to the degree of precipitate formation in the different cellular compartments. After exposure to 5-fluorocytosine, abnormal behaviour of the nuclei and internal lipidification of the mitochondria and of the cytoplasm were seen. No discernible Ca++ activity was present on the cellular structures by cytochemical localization. Assay by laser microprobe mass analysis, however, showed a slight increase in Ca++ which points to a structural bonding of the Ca++.     

Infrared mass spectrometric imaging below the diffraction limit

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)1 is an established technique for the analysis of biological macromolecules. Its relative insensitivity to pollutants makes MALDI-MS very suitable for the direct analysis of biological samples. As such, it has facilitated great advances in the field of biomolecular imaging mass spectrometry. Traditionally, MALDI-MS imaging is performed in a scanning microprobe methodology.(2-4) However, in a recent study we have demonstrated an alternative methodology; the so-called microscope mode,(5) where the requirement for a highly focused ionization beam is removed. Spatial details from within the desorption area are conserved during the flight of the ions through the mass analyzer, and a magnified ion image is projected onto a 2D-detector. In this paper, we demonstrate how imaging mass spectrometry benefits from the microscope mode approach. For the first time, high-lateral resolution ion images were recorded using infrared MALDI at 2.94 microm wavelength. The ion optical resolution achieved was well below the theoretical limit of (light-) diffraction for the setup used, which is impossible to achieve in the conventional scanning microprobe approach.     

DNA strand breaks induced by low-energy heavy ions

The DNA unwinding method was used to estimate DNA breakage in Chinese hamster cells exposed to heavy ions with LET in the range of 750-5000 keV/micron. Comparison of the primary induced unwinding rate per dose unit for ions with various track diameters but similar LET showed a pronounced influence on the track diameter. Low-energy ions, producing thin tracks with diameters (penumbra) in the submicrometer region, were almost two orders of magnitude less efficient than more energetic ions producing tracks with diameters of several micrometers and about three orders of magnitude less efficient than X-rays. For the thin tracks, clustering of breaks was indicated by comparison of the DNA unwinding rates in two different alkaline solutions. The results indicate that the unwinding rate cannot be used as a good measurement for DNA breaks in this case. The residual unwinding remaining after 4 h of repair at 37 degrees C correlated well with the ability of the various ions to produce cell-killing.     

Nuclear movement in filamentous fungi

One of the most striking features of eukaryotic cells is the organization of specific functions into organelles such as nuclei, mitochondria, chloroplasts, the endoplasmic reticulum, vacuoles, peroxisomes or the Golgi apparatus. These membrane-surrounded compartments are not synthesized de novo but are bequeathed to daughter cells during cell division. The successful transmittance of organelles to daughter cells requires the growth, division and separation of these compartments and involves a complex machinery consisting of cytoskeletal components, mechanochemical motor proteins and regulatory factors. Organelles such as nuclei, which are present in most cells in a single copy, must be precisely positioned prior to cytokinesis. In many eukaryotic cells the cleavage plane for cell division is defined by the location of the nucleus prior to mitosis. Nuclear positioning is thus absolutely crucial in the unequal cell divisions that occur during development and embryogenesis. Yeast and filamentous fungi are excellent organisms for the molecular analysis of nuclear migration because of their amenability to a broad variety of powerful analytical methods unavailable in higher eukaryotes. Filamentous fungi are especially attractive models because the longitudinally elongated cells grow by apical tip extension and the organelles are often required to migrate long distances. This review describes nuclear migration in filamentous fungi, the approaches used for and the results of its molecular analysis and the projection of the results to other organisms.