太平洋の北西国立研究所 (なり) 地球と生物科学 (EBD) ミッション種監督研究と開発 (LDRD) 基金へのサポート感謝しております。インストゥルメンタル アクセスが w. r. ワイリー環境分子科学研究所 (EMSL) 一般ユーザーの提案書を提供されました。EMSL はオフィスの生物と環境研究 (BER) なりで主催国立科学ユーザー施設です。著者は博士 『 鼎の証拠、原稿を読んで、有用なフィードバックを提供することをありがちましょう。バテル契約 DE AC05 76RL01830 下 DOE のなりが運営しています。

<ol>
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J., Peggie, D., Higgitt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+"added+value"+of+SIMS:+A+mass+spectrometric+and+spectroscopic+study+of+an+unusual+Naples+yellow+oil+paint+reconstruction.">Evaluation of the "added value" of SIMS: A mass spectrometric and spectroscopic study of an unusual Naples yellow oil paint reconstruction.</a> Int J mass Spectrom. 284 (1-3), 22-34 (2009).</li><li>Lee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mass Spectrometry Handbook. , 988 (2012).</li><li>Petrovic, M., Barcelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+anionic+and+nonionic+surfactants,+their+degradation+products,+and+endocrine-disrupting+compounds+in+sewage+sludge+by+liquid+chromatography/mass+spectrometry.">Determination of anionic and nonionic surfactants, their degradation products, and endocrine-disrupting compounds in sewage sludge by liquid chromatography/mass spectrometry.</a> Anal Chem. 72 (19), 4560-4567 (2000).</li><li>Vickerman, J. 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著者が明らかに何もありません。

ログ段階で接種後数日とバイオ フィルムが 3.1 の手順で説明するよう健康とイメージングのため十分な厚さだ前に拡大する温度をテストすることが重要です。S. oneidensis 室温; MR1 バイオ フィルムを養殖具体的手順します。しかし別の部屋の温度の成長率に影響を与えます。したがって、バイオ フィルムは TOF-SIMS 解析に進む前に準備ができているかどうかを理解する光イメージング技術を使?...

細菌バイオ フィルム細菌前記細胞が接続し、多くの可能な環境の中で生き残ることができる有害な物理的・機械的刺激の変化を生き残るためにための防衛として時間をかけて進化してきた表面関連のコミュニティであります。1,2バイオ フィルム大幅に調査した、生体臨床医学、医用生体工学、農業、産業の研究・開発など多くの分野のアプリケーションがあります。1,2セルフ プロデュースに細胞外高分子物質 (EPS) と、ローカル水クラスター環境などを含むこれらの複雑な微生物の化学マッピングを理解する正確を得る不可欠ですな彼らの生物学的活動の描写です。2
バイオ フィルムは存在し、高含水状態の内で育ちます。これは、揮発性液体の真空の勉強の難しさのため飛行時間二次イオン質量分析 (TOF-SIMS) など真空表面解析テクニックを使用して偉大な課題を提示します。その結果、真空ベースの表面分析技術は、乾燥状態だけでバイオ フィルム サンプルを勉強にほとんど専ら限られています。しかし、乾燥状態でバイオ フィルムを勉強して、真の生物学的微小環境の正確な調査を阻害します。それはしばしば、EPS の整合性とバイオ フィルム形態、その場で液体研究にバイオ フィルムの乾燥質量スペクトル結果を比較した後示されている抜本的な変更が発生します。3,4この記事分析液体真空インターフェイス (サルヴィ)、5,6マイクロ流体リアクターでの我々 のシステムの使用を採用することにより自然な水和状態内のバイオ フィルムを勉強するためのソリューションを提示します。したがって真空内液体マトリックスの構造の整合性を維持しながら二次イオンビーム プローブへの直接アクセスを提供するマイクロ ポリジメチルシロキサン (PMDS) の薄いシリコン窒化 (罪) 膜の下で液体が入っています。商工会議所。7,8
S. oneidensis氏 1 緑色蛍光タンパク質 (GFP) を表現する変異は、その代謝の多様性と環境、応用微生物学、基づいていたで一般的な使用のためこのバイオ フィルムの手順図のモデル生物として選ばれました。大きく上金属還元剤や細胞外の電子転送のための独自の機能。9,10,11また、GFP の存在は簡単連続バイオ フィルム厚蛍光顕微鏡による監視、フルオレセイン イソチオ シアン酸 (FITC) フィルターを使用しての許可。私たちの以前の研究では、最大 100 μ m の厚さにバイオ フィルムの成長のためoperando の蛍光イメージングを使用して罪ウィンドウに添付ファイルを好むこの細菌の証拠を示しています。4,12本稿が蛍光顕微鏡によるバイオ フィルムの存在の確認を説明するだけのサルヴィは超解像などその他の光学撮像方法と互換性のある蛍光イメージング (すなわち、構造化照明顕微鏡 (SIM)9) および共焦点レーザー走査顕微鏡 (CLSM) 画像4)。光イメージングは、バイオ フィルムの厚さを測定する機能し、それは、その厚さと罪ウィンドウへの付着を確認する表示されます、バイオ フィルムの形状の 3 D イメージを取得できます。9 GFP は、SIMS 分析で使用された、GFP なしS の oneidensisを成長曲線光学密度のこの唯一の必要な測定として使用され、任意の蛍光イメージングを必要としません。一般的に、タグ付きの GFP の違いと成長曲線のタグの付いていない種が重要ではありません。さらに、このプロトコルは、手順を記述するモデル生物としてs. oneidensis氏 1 GFP を使用して、この手順はサルヴィ内栽培のために必要とされるすべての菌株の設計されています。とはいえ、必要な細菌株の知識を与え、時間、温度、酸素環境など生育条件が使用される細菌の歪みに合わせて変更する必要があります。成長媒体のためこの手順は養殖で、ブドウ糖とブドウ糖なしトリプシン大豆寒天 (TSA) なし「ナノワイヤー」中, トリプシン大豆スープ (TSB) を使用します。成長のため、膜の拡張を監視するため、「ナノワイヤ」中の組成は、特別策定されていますと、細いワイヤと培地成分形を取るように見えるs. oneidensisのペリプラズムがされている以前の研究で確立。13,14
その場で当社の以前の議定書液体 TOF-SIMS サルヴィは、タンパク質の固定化と TOF-SIMS 解析およびデータ圧縮に関する詳細なプロトコルと同様に、罪に添付ファイルを提供している利点について説明しました。12データ削減の手順を繰り返しよりもむしろ本稿になる代わりにセットアップおよびバイオ フィルムの存在と事前の厚みを検出する画像処理の手順と同様に、私たちのサルヴィのマイクロ チャネル内のバイオ フィルムを育成のユニークなアプローチに焦点を当てるTOF-SIMS 解析。バイオ フィルムが以前のみ真空ベース表面分析技術のチャンバ内でサンプルを乾燥する限定されていました、ライブ バイオ フィルムの詳細な EPS とバイオ化学マッピング今入手できますその場でこの新しい機能であるためです。

<table><tbody><tr><td>ToF-SIMS</td><td>IONTOF</td><td>TOF.SIMS 5</td><td>Resolution:&gt;10,000 m/&amp;Delta;m for mass resolution;&gt;4,000 m/&amp;Delta;m for high spatial resolution</td></tr><tr><td>System for Analysis at the Liquid Vacuum Interface (SALVI)</td><td>Pacific Northwest National Laboratory</td><td>N/A</td><td>SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level.</td></tr><tr><td>-80&amp;deg;C Freezer</td><td>New Brunswick Scientific</td><td>N/A</td><td>U410 Premium Energy Efficient Ultra-Low Temperature Freezer</td></tr><tr><td>4&amp;deg;C Refrigerator</td><td>BioCold Scientific</td><td>N/A</td><td>COLDBOX1</td></tr><tr><td>Orbital Shaker</td><td>New Brunswick Scientific</td><td>N/A</td><td>Innova 4900 Multi-Tier Environmental Shaker, set at 30 degrees Celsius for serum bottle and flask culturing, set at 150rpm.</td></tr><tr><td>Syringe Pump</td><td>Cole-Parmer</td><td>EW-74905-02</td><td>Cole-Parmer Syringe Pump, Infusion Only, Touchscreen Control 74905-02, used for injecting liquid into the tubing system and SALVI at a constant flowrate.</td></tr><tr><td>Incubator</td><td>Barnstead International</td><td>LT1465X3</td><td>Lab-Line incubator, set at 30 degrees Celsius for plate culturing.</td></tr><tr><td>Autoclave</td><td>Getinge</td><td>533LS</td><td>Used to sterilize PEEK fittings, tubing systems, serum vials, and medium. Model 533LS Vacuum Steam Sterilizer</td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>4001-000</td><td>GENESYS 20 spectrophotometer for OD600 readings of cuvettes for growth curves.</td></tr><tr><td>Biological Safety Cabinet</td><td>Thermo Fisher Scientific</td><td>1385</td><td>1300 Series AZ Biological Safety Cabinet</td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>N/A</td><td>Nikon OPTIPHOT-2 fluorescence microscope with camera and super high pressure mercury lamp power supply.</td></tr><tr><td>pH Meter</td><td>Mettler Toledo</td><td>51302803</td><td>Used to test the pH of the &amp;ldquo;nanowires&amp;rdquo; medium after finished and before autoclaving.</td></tr><tr><td>PEEK Union</td><td>Valco</td><td>ZU1TPK</td><td>For connecting the inlet and outlet of SALVI, the syringe to the tubing system, and the inlet of the SALVI to the drip chamber of the tubing system.</td></tr><tr><td>5 Axes Sample Stage</td><td>IONTOF</td><td>N/A</td><td>Stage is self-made for mounting SALVI in ToF-SIMS.</td></tr><tr><td>Barnstead Nanopure Water Purification System</td><td>Thermo Fisher Scientific</td><td>D11921</td><td>ROpure LP Reverse Osmosis filtration module (D2716)</td></tr><tr><td>Pipette</td><td>Thermo Fisher Scientific</td><td>21-377-821</td><td>Range: 100 to 1,000 &amp;micro;L.</td></tr><tr><td>Pipette Tip</td><td>Neptune</td><td>2112.96.BS</td><td>1,000 &amp;micro;L pipette tips</td></tr><tr><td>Razor Blade Handle</td><td>Stanley</td><td>N/A</td><td>Stanley Bostitch Razor Blade Scraper with 5 Single-Edge Blades, used for cutting PTFE tubing</td></tr><tr><td>Syringe</td><td>BD</td><td>309659</td><td>1 mL</td></tr><tr><td>Syringe</td><td>BD</td><td>309657</td><td>3 mL</td></tr><tr><td>Syringe</td><td>BD</td><td>309646</td><td>5 mL; Used for making the drip chamber</td></tr><tr><td>Syringe</td><td>BD</td><td>309604</td><td>10 mL</td></tr><tr><td>Syringe</td><td>BD</td><td>302830</td><td>20 mL</td></tr><tr><td>Disposable Pipette</td><td>Thermo Fisher Scientific</td><td>13-678-11</td><td>25 mL Fisherbrand&amp;trade; Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe, for filling serum bottles.</td></tr><tr><td>Electric Pipette Filler</td><td>Pipet-aid</td><td>P-57260</td><td>Vacuum pressure electric serological pipette filler</td></tr><tr><td>Serum Bottle</td><td>Sigma</td><td>33109-U</td><td>Holds approximately 69 mL of liquid for culture growth, optimum for use of 20mL culture per bottle.</td></tr><tr><td>Anaerobic Culture Tube</td><td>VWR</td><td>89167-178</td><td>Anaerobic Tubes, 18 x 150 mm, Supplied with 20 mm Blue Butyl Rubber Stopper and Aluminum Seal.</td></tr><tr><td>Rubber Stopper</td><td>Sigma</td><td>27235-U</td><td>Silicone stopper, used for sealing serum bottles and for creating the tubing system/drip chamber.</td></tr><tr><td>Aluminum Crimp Seal (without septum)</td><td>Sigma</td><td>27227-U</td><td>Aluminum seal for top of serum bottle for use with serum bottle crimper.</td></tr><tr><td>Serum Bottle Aluminum Seal Crimper</td><td>Wheaton</td><td>224307</td><td>30 mm crimper with standard seal.</td></tr><tr><td>PTFE Tubing</td><td>Supelco</td><td>58697-U</td><td>1.58 mm OD x 0.5 mm ID 50 ft. PTFE Teflon tubing, used for creating the tubing system.</td></tr><tr><td>Disposable Cuvettes</td><td>GMBH</td><td>759085D</td><td>1.5 Ml for use with spectrophotometer.</td></tr><tr><td>Needle</td><td>BD</td><td>303015</td><td>22G; used for serum bottle injection.</td></tr><tr><td>Needle</td><td>BD</td><td>305120</td><td>23G; used for punching-through rubber stopper to create drip tubing system.</td></tr><tr><td>Shewanella oneidensis MR-1 with GFP</td><td>N/A</td><td>N/A</td><td>Matthysse AG, Stretton S, Dandie C, McClure NC, &amp;amp; Goodman AE (1996) Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiol Lett 145(1):87-94.&amp;nbsp;</td></tr><tr><td>Ethanol</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;S25310A</td><td>95% Denatured</td></tr><tr><td>TSA</td><td>BD</td><td>212305</td><td>Tryptic soy agar for culturing the model organism (S. oneidensis) used in this protocol</td></tr><tr><td>PIPES Buffer</td><td>Sigma</td><td>P-1851</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>S-5881</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Ammonium Chloride</td><td>Sigma</td><td>A-5666</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P-4504</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma</td><td>S-9638</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Chloride</td><td>Thermo Fisher Scientific</td><td>S271-3</td><td>Used for &amp;ldquo;nanowires&amp;rdquo; medium, and used to make mineral solution used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium lactate</td><td>Sigma</td><td>L-1375</td><td>60%(w/w) syrup @ 98% pure, d=1.3 g/mL, 7M, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S-5761</td><td>Used to make ferric NTA solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Nitrilotriacetic Acid Trisodium Salt</td><td>Sigma</td><td>N-0253</td><td>Used to make ferric NTA solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Iron (III) Chloride</td><td>Sigma</td><td>451649</td><td>Used to make ferric NTA solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma</td><td>208094</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Manganese (II) Sulfate Monohydrate</td><td>Sigma</td><td>M-7634</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Iron(II) Sulfate Heptahydrate</td><td>Sigma</td><td>215422</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma</td><td>223506</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Cobalt(II) Chloride</td><td>Sigma</td><td>60818</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Zinc Chloride</td><td>Sigma</td><td>229997</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Copper(II) Sulfate Pentahydrate</td><td>Sigma</td><td>C-8027</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Aluminum Potassium Sulfate Dodecahydrate</td><td>Sigma</td><td>237086</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Boric Acid</td><td>Sigma</td><td>B-6768</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Molybdate Dihydrate</td><td>Sigma</td><td>331058</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Nickel(II) Chloride</td><td>Sigma</td><td>339350</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Sodium Tungstate Dihydrate</td><td>Sigma</td><td>14304</td><td>Used to make minerals solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>D-Biotin</td><td>Sigma</td><td>47868</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Folic Acid</td><td>Sigma</td><td>F-7876</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Pyridoxine Hydrochloride</td><td>Sigma</td><td>P-9755</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Riboflavin (B2)</td><td>Sigma</td><td>47861</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Thiamine Hydrochloride</td><td>Sigma</td><td>T-4625</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Nicotinic Acid</td><td>Sigma</td><td>N4126</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>D-Pantothenic Acid Hemicalcium Salt</td><td>Sigma</td><td>21210</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Vitamin B12</td><td>Sigma</td><td>V-2876</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>4-Aminobenzoic Acid</td><td>Sigma</td><td>A-9878</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr><tr><td>Thioctic Acid</td><td>Sigma</td><td>T-1395</td><td>Used to make vitamin solution, used for &amp;ldquo;nanowires&amp;rdquo; medium {Hill, E.A. 2007}</td></tr></tbody></table>

in situ characterization of shewanella oneidensis mr1 biofilms by salvi and tof-sims

細菌バイオ フィルムの自主制作に細胞外高分子物質 (EPS) と環境微生物学での自分の役割を理解する研究が大幅に表面関連のコミュニティであります。本研究は、バイオ フィルムの添付ファイル分析液体真空インターフェイス (サルヴィ) でのシステムを開拓し、飛行時間二次イオン質量分析 (TOF-SIMS) によってその場で生きているバイオ フィルムの化学マッピングを実現する方法をについて説明します。これは外側と専門のセットアップでは、サルヴィ チャネル内の両方の細菌の培養と同様、バイオ フィルムの存在と TOF-SIMS 解析の前に厚さを検出する光イメージング技術です。我々 の結果は自然な水和状態でシェワネラバイオ フィルムの特徴的なピークを表示によってローカライズされた水クラスター環境、EPS の強調表示のフラグメントは、同じバイオ フィルムから大幅に異なって脱水状態です。これらの結果は、真空ベースの化学イメージング装置とその場観察バイオ イメージングは、サルヴィの画期的な機能を示しています。

これらの代表的な結果は、TOF-SIMS を通じて得られた添付のバイオ フィルムの化学物質のプロファイルを識別して、解釈方法を示すのに役立ちます。質量スペクトル TOF-SIMS データ集録、簡潔に手順セクションで強調表示を印刷後各それぞれ m/z 値識別情報を割り当てるためにピークの同定を実施しなければなりません。これは、細菌や様々 な水のクラスター、脂肪酸?...

Watch this Scientific Journal Video about In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS at JoVE.com

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

マイクロ流体リアクター、液体真空界面解析システムにより水和状態の化学マッピングのための in situ飛行時間二次イオン質量分析のためのバイオ フィルムの成長のための手法を提案します。緑色蛍光タンパク質を持つシェワネラ oneidensis氏 1 は、モデルとして使用されました。

その場でサルヴィ、TOF-SIMSシェワネラ oneidensis MR1 バイオ フィルムの特性

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

in-situ-characterization-shewanella-oneidensis-mr1-biofilms-salvi-tof

Research

JoVE Journal

Chemistry

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

8.9K ビュー.  Pacific Northwest National Laboratory. マイクロ流体リアクター、液体真空界面解析システムにより水和状態の化学マッピングのための in situ飛行時間二次イオン質量分析のためのバイオ フィルムの成長のための手法を提案します。緑色蛍光タンパク質を持つシェワネラ oneidensis氏 1 は、モデルとして使用されました。

ビデオ: In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

尿サンプル中の無細胞DNAインテグリティ解析

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

がん研究

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

その場 SIMSおよび質量選択されたイオンのソフトランディングで調製し明確に定義された表面の赤外分光法における

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

化学

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

血清およびリアルタイム PCR を用いたプラズマ コピー数検出

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

チップを使ったデジタル PCR による新鮮凍結胃癌組織内CDH1まれなトラン スクリプト変種の検出

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

ホウ素ドープダイヤモンド電極品質とアプリケーションの評価<em&gt;その場で</em&gt;水電解による局所のpHの変更

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

高速液体クロマトグラフィー結合フローシンチレーションにより放射性標識およびホスホイノシチドの細胞レベルの定量化

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

サルヴィとTOF-SIMSによる水中で水和タンパク質のその場キャラクタリゼーションで

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

超分子コロイドの合成とキャラクタリゼーション

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

免疫ブロットやプロテオミクスによるタンパク質局在の解析シロイヌナズナから葉緑体小班の準備

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

生化学

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

その場でサルヴィ、TOF-SIMSシェワネラ oneidensis MR1 バイオフィルムの特性

要約

さらに動画を探す

この動画の章

尿サンプル中の無細胞DNAインテグリティ解析

その場 SIMSおよび質量選択されたイオンのソフトランディングで調製し明確に定義された表面の赤外分光法における

血清およびリアルタイム PCR を用いたプラズマコピー数検出

チップを使ったデジタル PCR による新鮮凍結胃癌組織内CDH1まれなトランスクリプト変種の検出

ホウ素ドープダイヤモンド電極品質とアプリケーションの評価

高速液体クロマトグラフィー結合フローシンチレーションにより放射性標識およびホスホイノシチドの細胞レベルの定量化

サルヴィとTOF-SIMSによる水中で水和タンパク質のその場キャラクタリゼーションで

超分子コロイドの合成とキャラクタリゼーション

免疫ブロットやプロテオミクスによるタンパク質局在の解析シロイヌナズナから葉緑体小班の準備

ウシガエルの球形嚢（からの有毛細胞の生理的な準備

その場でサルヴィ、TOF-SIMSシェワネラ oneidensis MR1 バイオ フィルムの特性

要約

さらに動画を探す

この動画の章

尿サンプル中の無細胞DNAインテグリティ解析

その場 SIMSおよび質量選択されたイオンのソフトランディングで調製し明確に定義された表面の赤外分光法における

血清およびリアルタイム PCR を用いたプラズマ コピー数検出

チップを使ったデジタル PCR による新鮮凍結胃癌組織内CDH1まれなトラン スクリプト変種の検出

ホウ素ドープダイヤモンド電極品質とアプリケーションの評価

高速液体クロマトグラフィー結合フローシンチレーションにより放射性標識およびホスホイノシチドの細胞レベルの定量化

サルヴィとTOF-SIMSによる水中で水和タンパク質のその場キャラクタリゼーションで

超分子コロイドの合成とキャラクタリゼーション

免疫ブロットやプロテオミクスによるタンパク質局在の解析シロイヌナズナから葉緑体小班の準備

ウシガエルの球形嚢（からの有毛細胞の生理的な準備

その場でサルヴィ、TOF-SIMSシェワネラ oneidensis MR1 バイオフィルムの特性

血清およびリアルタイム PCR を用いたプラズマコピー数検出

チップを使ったデジタル PCR による新鮮凍結胃癌組織内CDH1まれなトランスクリプト変種の検出