走査透過電子顕微鏡における弾性・非弾性散乱信号のオンライン・ディジタル演算処理

1.電子検出法の改善 STEM(走査透過電子顕微鏡)における電子検出器として従来使われているP46粉末蛍光体について、100kVの加速圧電圧において最高感度を与える厚さを調べた結果、0.07mm厚さの蛍光体が最適であり、高いDQEが得られることを示した。また、1mm厚さのYAG単結晶を使用すれば、約4倍の感度が得られ、感度の均一性もすぐれていることを明らかにした。非弾性散乱および非散乱電子の検出については、信号電子偏向用マグネットおよび絞り板の設置により、X線および漂遊電子線を除いた高精度検出ができることを示した。 2.像信号のディジタル処理システム 3種類までの電子信号をディジタル的に高精度で取り込み、処理のできるシステムを開発した。サンプリングによる高周波の折り返しの誤差に特に注意を払い、バタ-ワ-ス型アンチェイリアシングフィルタの使用によって、ディジタル化の誤差を0.2%以下に抑えることができた。 3.ディジタル演算処理ロ-パスフィルタと併用したデコンボリュ-ションにより、擬似像の影響のない解像度向上のための、ロ-パスフィルタ-の窓の形状を明らかにし、適切な条件のもとでは約40%の解像度の改善ができることを明らかにした。また、入射電子による信号電子の規格化による像SN比の改善の定量的評価を行った結果、理論から期待されるSN比の改善ができることが明らかとなった。さらに、弾性散乱および非弾性散乱電子を用いた演算処理を行い、比の演算像においては局所厚さに依存しない原子番号に比例したコントラストの像が得られ、減算像においては特定原子番号の元素の構造のコントラストを消去、あるいは強調できることを実証した。科学研究費補助金 研究種目:試験研究 課題番号:63850078 研究代表者:日比野 倫夫 研究期間:1988-1989年度research repor

Observation of the Decay B0→D±D*∓

journal articl

Evaluation of Hydrogen Spillover in Diffusion Processes*

application/pdfThe contribution of the spillover of hydrogen adsorbed on a silica gel supported platinum catalyst to gas phase mass transport has been studied under atmospheric pressure at 30?300℃. The amount of spiltover hydrogen was evaluated by the hydrogenation of ethylene, and the spiltover hydrogen was characterised by the temperature programmed desorption spectrum. The spillover of hydrogen from platinum to the SiO_2 surface enhanced gas phase diffusion more than 60％ at 90℃. The higher percentage of platinum supported on SiO_2 increased the temperature dependency of the diffusion coefficient.departmental bulletin pape

Comprehensive and accurate genetic variant identification from contaminated and low-coverage Mycobacterium tuberculosis whole genome sequencing data

Supplementary Material for ‘Comprehensive and accurate genetic variant identification from contaminated and low coverage Mycobacterium tuberculosis whole genome sequencing data’, as published in Microbial Genomics

Rv1460 truncation mutants are impaired for growth in vitro under standard culture conditions.

(A–C) Growth of H37Rv (wild-type), three truncation (ΔRv1460stop) mutants and their complemented strains under standard conditions in 7H9 OADC. The results shown are the mean and standard deviation of three experiments.</p

Rv1460 represses its own expression.

(A) Schematic representation of the β-galactosidase assay procedure. Each reporter vector (pJEM15, pJEM139pro or pJEM314pro) (containing the 139 bp or 314 bp regions upstream of Rv1460 fused to a lacZ reporter gene) was co-transformed into M. smegmatis with a protein expression vector either encoding no protein (pSE100) or encoding Rv1460 (indicated by the grey oval). (B) β-galactosidase activity from the 139 bp and 314 bp promoter fragments with (pSE1460) and without (pSE100) co-expression of Rv1460 in M. smegmatis. Transcriptional repression by wild-type or variants of Rv1460 results in a decrease in β-galactosidase activity, which is expressed in Miller units calculated as follows: 200 × (change in OD450nm) per mg protein per min. The results shown are the mean and standard deviation for three experiments. Statistical analysis compared the mean activity for each plasmid with or without co-expression of Rv1460 e.g. pJEM15 (pSE100) vs. pJEM15 (pSE1460) using an unpaired t-test (*p ≤0.05). (C) β-galactosidase activity from the 314 bp promoter (pJEM314pro) when wild-type (pSE1460) or mutated forms of Rv1460 (pSE_C203S or pSE_C216S or pSE_C242S or pSE_C244S or pSE_C203S/C216S/C244S) are expressed in M. smegmatis. The results shown are the mean and standard deviation for three experiments. Statistical analysis compared the mean activity for wild-type with each variant using an unpaired t-test.</p

Rv1460 binds an Fe-S cluster.

(A) UV-visible spectrum of NifS reconstitution reaction (at 1 and 20 hours) containing Rv1460 (50 μM) (black) or without Rv1460 (grey). The absorbance at 2 minutes was used as a blank for the reaction. (B) Near-UV CD spectrum of NifS reconstitution reaction containing Rv1460 (50 μM). Insert shows the near-UV CD spectrum (Ellipticity in millidegrees) of reactions with (solid line) and without Rv1460 (dashed line). (C) UV-visible spectrum of lithium sulphide reconstitution reaction with (black) and without Rv1460 (50 μM) over time. The absorbance at 2 minutes was used as a blank for the reaction. UV-visible spectrum of the reactions after buffer exchange are indicated in Figure K. in S1 File (D) Near-UV CD spectrum of lithium sulphide reconstitution reaction after buffer exchange containing (23 μM) Rv1460. Insert shows the near-UV CD (Ellipticity in millidegrees) of reactions with (solid line) and without Rv1460 (dashed line) after buffer exchange.</p

Fe-S cluster assembly is regulated by multiple mechanisms in <i>M</i>. <i>tuberculosis</i>.

Rv1460 is transcribed independently from the operon allowing differential expression and regulation by the repressor. Initiation of translation is leaderless upstream of Rv1460, while a 5’-UTR containing a ribosome binding site regulates translation of Rv1461. The intein within Rv1461 must be resolved before it is functional and may represent an additional level of regulation of the system. Rv1460 is predicted to bind within the promoter region upstream of Rv1460 as well as within Rv1461 (as indicated by the dashed line boxes). Binding sites for other regulators within the Rv1460 promoter region and within the operon are also predicted, providing another level of regulation. Binding of an Fe-S cluster may change Rv1460’s affinity for DNA. Genes are not drawn to scale. The association of the Fe-S cluster machinery is inferred from other bacterial SUF systems, but has not been experimentally validated in mycobacteria. Cysteine is provided by the cysteine desulphurase, while the source of iron is unknown. The oligomeric state of Rv1460 is not indicated since it has not been confirmed, and the ratio of Fe-S cluster to Rv1460 protein is unknown.</p

Rv1460, a SufR homologue, is a repressor of the <i>suf </i>operon in <i>Mycobacterium tuberculosis</i>

<div><p>Iron–sulphur (Fe-S) clusters are ubiquitous co-factors which require multi-protein systems for their synthesis. In <i>Mycobacterium tuberculosis</i>, the <i>Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466</i> operon (<i>suf</i> operon) encodes the primary Fe-S cluster biogenesis system. The first gene in this operon, <i>Rv1460</i>, shares homology with the cyanobacterial SufR, which functions as a transcriptional repressor of the <i>sufBCDS</i> operon. Rv1460’s function in <i>M</i>. <i>tuberculosis</i> has however not been determined. In this study, we demonstrate that <i>M</i>. <i>tuberculosis</i> mutants lacking a functional Rv1460 protein are impaired for growth under standard culture conditions. Elevated expression of <i>Rv1460</i> and <i>Rv1461</i> was observed in the mutant, implicating Rv1460 in the regulation of the <i>suf</i> operon. Binding of an Fe-S cluster to purified recombinant Rv1460 was confirmed by UV-visible spectroscopy and circular dichroism. Furthermore, three conserved cysteine residues, C203, C216 and C244, proposed to provide ligands for the coordination of an Fe-S cluster, were shown to be required for the function of Rv1460 in <i>M</i>. <i>tuberculosis</i>. Rv1460 therefore seems to be functionally analogous to cyanobacterial SufR.</p></div

Succinate dehydrogenase and aconitase activity is not impaired in Rv1460 truncation mutants.

<p>(A) Succinate dehydrogenase activity and (B) aconitase activity in the H37Rv (wild-type), Δ<i>Rv1460</i>stop_5.20 and complemented strains cultured in 7H9 OADC. Activity was standardised relative to total protein. The results shown are the mean and standard deviation of five and three experiments respectively.</p