Name: in situ characterization of shewanella oneidensis mr1 biofilms by salvi and tof-sims
Uploaded: 2017-08-18T13:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS at JoVE.com

Wir sind dankbar für Unterstützung des Pacific Northwest National Laboratory (PNNL) Erde und biologischen Wissenschaften (EBD) Mission Samen Labor leitete Forschung und Entwicklung (LDRD) Fonds. Instrumental wurde durch W. R. Wiley Umwelt Molecular Sciences Laboratory (EMSL) allgemeine Benutzer Vorschlag zur Verfügung. EMSL ist eine nationale wissenschaftliche Benutzer Einrichtung Büro der biologischen und ökologischen Forschung (BER) auf PNNL gesponsert. Die Autoren danken Dr. Yuanzhao Ding zum Beweis das Manuskript zu lesen und nützliche Rückmeldungen. Battelle für DOE unter Vertrag DE AC05 76RL01830 PNNL gesteuert.

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Nach dem impfen bei Log-Phase, ist es wichtig, die Anzahl der Tage und die Temperatur bei dem der Biofilm wachsen sollte, bevor es gesund und dick genug für die Bildgebung, wie unter Punkt 3.1 beschrieben testen. Dieses Verfahren umfasst speziell Kultivierung einer S. Oneidensis MR1 Biofilm bei Raumtemperatur; jedoch können unterschiedliche Raumtemperaturen die Wachstumsrate beeinflussen. Daher ist es wichtig um zu verstehen, ob der Biofilm bereit, bevor Sie fortfahren, ToF-SIMS Analyse ist verwenden optische Bildgebun...

Bakterielle Biofilme sind Oberfläche-assoziierte Gemeinschaften, die im Laufe der Zeit als eine Verteidigung gegen Bakterien überleben unterschiedlicher ungünstige physikalische und mechanische reizen, wobei Zellen in der Lage sind zu befestigen und in vielen möglichen Umgebungen überleben entwickelt haben. 1 , 2 Biofilme werden erheblich untersucht und finden Anwendung in vielen Bereichen wie Biomedizin, biomedizinische Technik, Landwirtschaft und industrielle Forschung und Entwicklung. 1 , 2 die chemische Zuordnung dieser komplexe mikrobielle Gemeinschaften, einschließlich ihre selbst produzierten extrazellulären Polymeren Substanzen (EPS) und ihre lokalen Wasser-Cluster-Umgebung zu verstehen gilt, gewinnt eine genaue und detaillierte Darstellung ihrer biologischen Aktivitäten. 2
Biofilme bestehen und wachsen innerhalb eines stark hydratisiert Staates. Dies stellt eine große Herausforderung im Umgang mit Vakuum-basierten Oberflächenanalyse Techniken wie Time-of-Flight secondary Ion mass Spectrometry (ToF-SIMS) aufgrund der Schwierigkeiten bei der Untersuchung der flüchtiger Flüssigkeiten im Vakuum. Infolgedessen wurden Vakuum-basierten Oberflächenanalyse Techniken beschränkt sich fast ausschließlich auf Biofilm Proben im getrockneten Zustand zu studieren. Studium einen Biofilm im getrockneten Zustand hemmt jedoch die genaue Untersuchung der seine wahre biologische Mikroumgebung. Es führt oft zu drastische Veränderungen der EPS Integrität und Biofilm-Morphologie, die nach einem Vergleich trocken Biofilm Masse spektrale Ergebnisse in Situ flüssige Studien nachgewiesen wurde. 3 , 4 dieser Artikel stellt eine Lösung für das Studium von Biofilmen innerhalb ihrer natürlichen hydratisierten Zustand durch die Verwendung unseres Systems zur Analyse an flüssigen Vakuum Schnittstelle (SALVI),5,6 mikrofluidischen Reaktor, enthält Flüssigkeit unter seine dünne Silizium-Nitrid (SiN) Membran in eine Microchannel gemacht von Polydimethylsiloxan (PMDS), so bietet direkten Zugang zu den sekundären Ionenstrahl Sonde unter Beibehaltung der strukturellen Integritätdes der flüssigen Matrix in einem luftleeren Raum Kammer. 7 , 8
S. Oneidensis MR-1 mutiert auszudrücken, grüne Fluoreszenz-Protein (GFP) wurde gewählt, als Modellorganismus für dieser Biofilm Verfahren Abbildung aufgrund seiner metabolischen Vielseitigkeit und gemeinsame Nutzung in Umwelt- und angewandte Mikrobiologie, basiert stark auf seine einzigartige Fähigkeit zur Verringerung der Metall und extrazelluläre Elektronentransfer. 9 , 10 , 11 darüber hinaus ermöglichte das Vorhandensein von GFP einfach kontinuierliche Biofilm-Dicke Überwachung durch Fluoreszenz-Mikroskopie, mithilfe eines Filters Fluorescein erfolgt (FITC). Unsere frühere Studien haben Hinweise auf diese Bakterien begünstigt Anlage zum Fenster SiN mit in Operando Fluoreszenz-Bildgebung für Biofilm Wachstum bis zu einer Dicke von bis zu hundert Mikrometern gezeigt. 4 , 12 während dieses Papier nur die Bestätigung der Biofilm Präsenz durch Fluoreszenzmikroskopie besprechen, die SALVI ist kompatibel mit anderen optischen bildgebenden Verfahren wie Höchstauflösung Fluoreszenz-Bildgebung (d. h. die strukturierte Beleuchtung Mikroskopie (SIM)9) und konfokale Laser-scanning-Mikroskopie (CLSM) imaging-4). Optische Bildgebung kann dienen zur Messung der Dicke von Biofilm und ein 3D-Bild der Form des Biofilms erhalten, wie es scheint, seine Stärke und seine Verbundenheit mit der Sünde-Fenster bestätigt. 9 während GLP diente in der SIMS-Analyse, S. Oneidensis , ohne GFP diente für die Wachstumskurve, als diese nur erforderliche Messung der optischen Dichte und Fluoreszenz-Bildgebung nicht erforderlich. In der Regel die Differenz zwischen der GFP markiert und untagged Arten in der Wachstumskurve ist bedeutungslos. Darüber hinaus während dieses Protokoll S. Oneidensis MR-1 GFP als Modellorganismus verwendet, um das Verfahren zu beschreiben, ist dieses Verfahren für jeden Bakterienstamm ausgelegt, die für den Anbau in SALVI erforderlich sein kann. Obwohl Wissen der bakteriellen Belastung benötigt gegeben, müssen einige Bedingungen wie Zeit, Temperatur und Sauerstoff-Umgebung den Bakterienstamm zu verwendende angepasst werden. Wachstumsmedium verwendet dieses Verfahren "Nanodrähte" Mittel, tryptic Soy Bouillon (TSB) ohne Traubenzucker und tryptic Soy Agar (TSA) ohne Traubenzucker zur Kultivierung. Die Zusammensetzung des "Nanodrähte" Medium speziell formuliert für das Wachstum und für die Überwachung von Erweiterungen der Membran und Periplasma S. Oneidensis , die angezeigt werden, nehmen die Form von kleinen Drähten und die mittlere Zusammensetzung wurde in früheren Untersuchungen niedergelassen. 13 , 14
Unsere vorherigen Protokoll auf in Situ illustrierte flüssige ToF-SIMS nutzen SALVI für Protein Immobilisierung und Verbundenheit mit der Sünde, sowie ein detailliertes Protokoll auf ToF-SIMS Analyse und Datenreduktion zu bieten hat. 12 statt Daten Reduzierung Schritte wiederholen, wird dieses Papier dienen sich stattdessen auf den einzigartigen Ansatz der Einrichtung und Pflege von Biofilmen innerhalb unserer SALVI Microchannel sowie die bildgebenden Schritte zur Erkennung von Biofilm Präsenz und Stärke vor ToF-SIMS-Analyse. Während Biofilme zuvor begrenzt worden, haben nur getrocknet Proben innerhalb der Kammer Vakuum-basierten analytische Oberflächentechniken, detaillierte EPS und Biofilm chemische Zuordnung von Biofilmen Leben jetzt in Situ durch diese neue Funktion erhalten.

<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

Bakterielle Biofilme sind Oberfläche-assoziierte Gemeinschaften, die stark untersucht werden, um ihre selbst produzierten extrazellulären Polymeren Substanzen (EPS) und ihre Rolle im Umweltmikrobiologie zu verstehen. Diese Studie beschreibt eine Methode zum Biofilm Bindung an das System für die Analyse bei flüssigen Vakuum Schnittstelle (SALVI) zu kultivieren und in Situ chemische Zuordnung der lebende Biofilm durch Time-of-Flight secondary Ion mass Spectrometry (ToF-SIMS) zu erreichen. Dies geschieht durch die Kultivierung von Bakterien sowohl außerhalb als auch innerhalb des Kanals SALVI mit unseren spezialisierten Einrichtung sowie durch optische bildgebende Verfahren zur Erkennung von Biofilm Präsenz und Stärke vor der ToF-SIMS-Analyse. Unsere Ergebnisse zeigen die charakteristischen Gipfel des Biofilms Shewanella in seinem Naturzustand hydratisiert dehydriert Hervorhebung auf seine lokalisierte Wasser-Cluster-Umgebung, sowie EPS Fragmente, die sich drastisch von des gleichen Biofilms unterscheiden Zustand. Diese Ergebnisse zeigen die Durchbruch-Fähigkeit von SALVI, der in Situ Biofilm Bildgebung mit einem Vakuum-basierten chemischen Bildgebung Instrument ermöglicht.

Diese repräsentative Ergebnisse dienen, wie das chemische Profil des angehängten Biofilms kann erkannt und interpretiert, zu zeigen, wie durch ToF-SIMS erhalten. Nach dem Plotten Massenspektren von ToF-SIMS Datenerfassung, kurz im Abschnitt "Vorgehensweise" hervorgehoben sollte Peak Identifikation durchgeführt werden, um Identitäten zu jeder jeweiligen m/Z-Wert zuweisen. Dies kann erfolgen durch umfangreiche Literaturrecherche auf Massenspektrometrie auf Bakterien und bestimmte chemis...

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

Dieser Artikel stellt eine Methode für den Anbau eines Biofilms für in Situ Time-of-Flight secondary Ion Massenspektrometrie für chemische Zuordnung im hydratisierten Zustand durch einen mikrofluidischen Reaktor, System zur Analyse an der flüssigen Vakuum-Schnittstelle aktiviert. Die Shewanella Oneidensis MR-1 mit grünen Fluoreszenz-Protein wurde als Modell verwendet.

In Situ Charakterisierung von Shewanella Oneidensis MR1 Biofilmen von SALVI und ToF-SIMS

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 Aufrufe.  Pacific Northwest National Laboratory. Dieser Artikel stellt eine Methode für den Anbau eines Biofilms für in Situ Time-of-Flight secondary Ion Massenspektrometrie für chemische Zuordnung im hydratisierten Zustand durch einen mikrofluidischen Reaktor, System zur Analyse an der flüssigen Vakuum-Schnittstelle aktiviert. Die Shewanella Oneidensis MR-1 mit grünen Fluoreszenz-Protein wurde als Modell verwendet.

Serie: 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.

Zellfreie DNA-Integritätsanalyse in Urinproben

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 ...

Forschung

Krebsforschung

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.

In-situ-SIMS und IR-Spektroskopie von gut definierten Oberflächen durch weiche Landung der Messe-Ionen ausgewählt Vorbereitet

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

Chemie

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.

Serum und Plasma Kopie Nummer Erkennung mittels 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, ...

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.

Erkennung einer CDH1 selten Transkript Variante in Tiefkühlfrische Magenkrebs Geweben durch Chip-basierte digitale 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 ...

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.

Beurteilung der bordotierten Diamantelektrode Qualität und Anwendung auf<em&gt; In-situ-</em&gt; Änderung des Local pH durch Elektrolyse von Wasser

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.

Radiomarkierung und Quantifizierung von Cellular Ebenen Phosphoinositide von High Performance Liquid Chromatography-gekoppelten Durchflussscintilla

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.

In-situ-Charakterisierung von Hydrated Proteine ​​in Wasser durch SALVI und TOF-SIMS

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.

Synthese und Charakterisierung von supramolekularen Kolloid-

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.

Vorbereitung der Chloroplasten Sub Fächer von Arabidopsis für die Analyse von Protein-Lokalisierung durch Immunoblotting oder Proteomics

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

Biochemie

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.

In Situ Charakterisierung von Shewanella Oneidensis MR1 Biofilmen von SALVI und ToF-SIMS

Zusammenfassung

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In-situ-SIMS und IR-Spektroskopie von gut definierten Oberflächen durch weiche Landung der Messe-Ionen ausgewählt Vorbereitet

Serum und Plasma Kopie Nummer Erkennung mittels Real-Time PCR

Erkennung einer CDH1 selten Transkript Variante in Tiefkühlfrische Magenkrebs Geweben durch Chip-basierte digitale PCR

Beurteilung der bordotierten Diamantelektrode Qualität und Anwendung auf

Radiomarkierung und Quantifizierung von Cellular Ebenen Phosphoinositide von High Performance Liquid Chromatography-gekoppelten Durchflussscintilla

In-situ-Charakterisierung von Hydrated Proteine in Wasser durch SALVI und TOF-SIMS

Synthese und Charakterisierung von supramolekularen Kolloid-

Vorbereitung der Chloroplasten Sub Fächer von Arabidopsis für die Analyse von Protein-Lokalisierung durch Immunoblotting oder Proteomics

Physiologische Herstellung von Haarzellen aus dem Sacculus der American Bullfrog (

In Situ Charakterisierung von Shewanella Oneidensis MR1 Biofilmen von SALVI und ToF-SIMS

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Zellfreie DNA-Integritätsanalyse in Urinproben

In-situ-SIMS und IR-Spektroskopie von gut definierten Oberflächen durch weiche Landung der Messe-Ionen ausgewählt Vorbereitet

Serum und Plasma Kopie Nummer Erkennung mittels Real-Time PCR

Erkennung einer CDH1 selten Transkript Variante in Tiefkühlfrische Magenkrebs Geweben durch Chip-basierte digitale PCR

Beurteilung der bordotierten Diamantelektrode Qualität und Anwendung auf

Radiomarkierung und Quantifizierung von Cellular Ebenen Phosphoinositide von High Performance Liquid Chromatography-gekoppelten Durchflussscintilla

In-situ-Charakterisierung von Hydrated Proteine ​​in Wasser durch SALVI und TOF-SIMS

Synthese und Charakterisierung von supramolekularen Kolloid-

Vorbereitung der Chloroplasten Sub Fächer von Arabidopsis für die Analyse von Protein-Lokalisierung durch Immunoblotting oder Proteomics

Physiologische Herstellung von Haarzellen aus dem Sacculus der American Bullfrog (

In-situ-Charakterisierung von Hydrated Proteine in Wasser durch SALVI und TOF-SIMS