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.

<ol>
	<li>Renner, L. D., Weibel, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+regulation+of+biofilm+formation.">Physicochemical regulation of biofilm formation.</a> MRS Bull. 36 (5), 347-355 (2011).</li><li>Flemming, H. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nat Rev Microbiol. 8 (9), 623-633 (2010).</li><li>Aldeek, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+hydrophobic+domains+in+the+exopolymer+matrix+of+Shewanella+oneidensis+MR-1+biofilms.">Patterned hydrophobic domains in the exopolymer matrix of Shewanella oneidensis MR-1 biofilms.</a> Appl Environ Microbiol. 79 (4), 1400-1402 (2013).</li><li>Hua, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+molecular+imaging+of+a+hydrated+biofilm+in+a+microfluidic+reactor+by+ToF-SIMS.">In situ molecular imaging of a hydrated biofilm in a microfluidic reactor by ToF-SIMS.</a> Analyst. 139 (7), 1609-1613 (2014).</li><li>Yu, X. Y., Yang, L., Cowin, J. P., Iedema, M., Zhu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+and+methods+for+analyzing+liquids+under+vacuum.">Systems and methods for analyzing liquids under vacuum.</a> US Patent. , (2013).</li><li>Yu, X. Y., Liu, B., Yang, L., Zhu, Z., Marshall, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+electrochemical+device+and+process+for+chemical+imaging+and+electrochemical+analysis+at+the+electrode-liquid+interface+in+situ.">Microfluidic electrochemical device and process for chemical imaging and electrochemical analysis at the electrode-liquid interface in situ.</a> US Patent. , (2014).</li><li>Yang, L., Yu, X. Y., Zhu, Z. H., Thevuthasan, T., Cowin, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+a+hybrid+microfluidic+platform+compatible+for+in+situ+imaging+by+vacuum-based+techniques.">Making a hybrid microfluidic platform compatible for in situ imaging by vacuum-based techniques.</a> J Vac Sci Technol A. 29 (6), (2011).</li><li>Yang, L., Yu, X. Y., Zhu, Z. H., Iedema, M. J., Cowin, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+liquid+surfaces+under+vacuum+using+SEM+and+ToF-SIMS.">Probing liquid surfaces under vacuum using SEM and ToF-SIMS.</a> Lab Chip. 11 (15), 2481-2484 (2011).</li><li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Molecular+Imaging+of+the+Biofilm+and+Its+Matrix.">In Situ Molecular Imaging of the Biofilm and Its Matrix.</a> Anal Chem. , (2016).</li><li>Yang, J., Ghobadian, S., Montazami, R., Hashemi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the Asme 11th Fuel Cell Science, Engineering, and Technology Conference, 2013. , (2013).</li><li>Yu, F., Wang, C. X., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Graphene-Modified+Electrodes+in+Microbial+Fuel+Cells.">Applications of Graphene-Modified Electrodes in Microbial Fuel Cells.</a> Materials. 9 (10), (2016).</li><li>Yu, J., Zhou, Y., Hua, X., Zhu, Z., Yu, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Characterization+of+Hydrated+Proteins+in+Water+by+SALVI+and+ToF-SIMS.">In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS.</a> J Vis Exp. (108), e53708 (2016).</li><li>Hill, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Effects of Electron-Transport-System Impairment on Hydrogen Gas Production by the Bacterium Shewanella oneidensis MR-1. , (2007).</li><li>McCormick, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophotovoltaics:+oxygenic+photosynthetic+organisms+in+the+world+of+bioelectrochemical+systems.">Biophotovoltaics: oxygenic photosynthetic organisms in the world of bioelectrochemical systems.</a> Energy Environ Sci. 8 (4), 1092-1109 (2015).</li><li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+chemical+probing+of+the+electrode-electrolyte+interface+by+ToF-SIMS.">In situ chemical probing of the electrode-electrolyte interface by ToF-SIMS.</a> Lab Chip. 14, 855-859 (2014).</li><li>Keune, K., Hoogland, F., Boon, J. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+and+Depth+Profiling+by+Mass+Spectrometry--Sims,+MALDI+or+DESI.">Molecular Imaging and Depth Profiling by Mass Spectrometry--Sims, MALDI or DESI.</a> Analyst. 136 (11), (2011).</li><li>Weng, L. T., Bertrand, P., Stonemasui, J. H., Stone, W. E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tof+Sims+Study+of+the+Desorption+of+Emulsifiers+from+Polystyrene+Latexes.">Tof Sims Study of the Desorption of Emulsifiers from Polystyrene Latexes.</a> Surf Interface Anal. 21 (6-7), 387-394 (1994).</li><li>Pe&#241;uelas-Urquides, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+of+Mycobacterium+tuberculosis+crowth.+A+correlation+of+the+optical+measurements+with+colony+forming+units.">Measuring of Mycobacterium tuberculosis crowth. A correlation of the optical measurements with colony forming units.</a> Braz J Microbiol. 44 (1), 287-289 (2013).</li><li>Dohnalkova, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+hydrated+microbial+extracellular+polymers:+comparative+analysis+by+electron+microscopy.">Imaging hydrated microbial extracellular polymers: comparative analysis by electron microscopy.</a> Appl Environ Microbiol. 77 (4), 1254-1262 (2011).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+SEM+and+ToF-SIMS+analysis+of+IgG+conjugated+gold+nanoparticles+at+aqueous+surfaces.">In situ SEM and ToF-SIMS analysis of IgG conjugated gold nanoparticles at aqueous surfaces.</a> Surf Interface Anal. 46, 224-228 (2013).</li><li>Yu, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+the+transient+species+at+the+electrode-electrolyte+interface+by+in+situ+dynamic+molecular+imaging.">Capturing the transient species at the electrode-electrolyte interface by in situ dynamic molecular imaging.</a> Chem Commun. 52 (73), 10952-10955 (2016).</li></ol>

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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 width="2446">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">ToF-SIMS</td>
			<td style="width:260px;">IONTOF</td>
			<td style="width:123px;">TOF.SIMS 5</td>
			<td style="width:1675px;">Resolution:&#62;10,000 m/&#916;m for mass resolution;&#62;4,000 m/&#916;m for high spatial resolution</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">System for Analysis at the Liquid Vacuum Interface (SALVI)</td>
			<td style="width:260px;">Pacific Northwest National Laboratory</td>
			<td style="width:123px;">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 height="20">
			<td height="20" style="width:389px;height:20px;">-80&#176;C Freezer</td>
			<td style="width:260px;">New Brunswick Scientific</td>
			<td style="width:123px;">N/A</td>
			<td>U410 Premium Energy Efficient Ultra-Low Temperature Freezer</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">4&#176;C Refrigerator</td>
			<td style="width:260px;">BioCold Scientific</td>
			<td style="width:123px;">N/A</td>
			<td>COLDBOX1</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Orbital Shaker</td>
			<td style="width:260px;">New Brunswick Scientific</td>
			<td style="width:123px;">N/A</td>
			<td style="width:1675px;">Innova 4900 Multi-Tier Environmental Shaker, set at 30 degrees Celsius for serum bottle and flask culturing, set at 150rpm.</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Syringe Pump</td>
			<td style="width:260px;">Cole-Parmer</td>
			<td style="width:123px;">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 height="20">
			<td height="20" style="width:389px;height:20px;">Incubator</td>
			<td style="width:260px;">Barnstead International</td>
			<td style="width:123px;">LT1465X3</td>
			<td>Lab-Line incubator, set at 30 degrees Celsius for plate culturing.</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Autoclave</td>
			<td style="width:260px;">Getinge</td>
			<td style="width:123px;">533LS</td>
			<td>Used to sterilize PEEK fittings, tubing systems, serum vials, and medium. Model 533LS Vacuum Steam Sterilizer</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Spectrophotometer</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">4001-000</td>
			<td>GENESYS 20 spectrophotometer for OD600 readings of cuvettes for growth curves.</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Biological Safety Cabinet</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">1385</td>
			<td>1300 Series AZ Biological Safety Cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Fluorescence Microscope</td>
			<td style="width:260px;">Nikon</td>
			<td style="width:123px;">N/A</td>
			<td>Nikon OPTIPHOT-2 fluorescence microscope with camera and super high pressure mercury lamp power supply.</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">pH Meter</td>
			<td style="width:260px;">Mettler Toledo</td>
			<td style="width:123px;">51302803</td>
			<td>Used to test the pH of the &#8220;nanowires&#8221; medium after finished and before autoclaving.</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">PEEK Union</td>
			<td style="width:260px;">Valco</td>
			<td style="width:123px;">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 height="20">
			<td height="20" style="width:389px;height:20px;">5 Axes Sample Stage</td>
			<td style="width:260px;">IONTOF</td>
			<td style="width:123px;">N/A</td>
			<td>Stage is self-made for mounting SALVI in ToF-SIMS.</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Barnstead Nanopure Water Purification System</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">D11921</td>
			<td>ROpure LP Reverse Osmosis filtration module (D2716)</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Pipette</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">21-377-821</td>
			<td>Range: 100 to 1,000 &#181;L.</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Pipette Tip</td>
			<td style="width:260px;">Neptune</td>
			<td style="width:123px;">2112.96.BS</td>
			<td>1,000 &#181;L pipette tips</td>
		</tr>
		<tr height="20">
			<td height="20" style="width:389px;height:20px;">Razor Blade Handle</td>
			<td style="width:260px;">Stanley</td>
			<td style="width:123px;">N/A</td>
			<td>Stanley Bostitch Razor Blade Scraper with 5 Single-Edge Blades, used for cutting PTFE tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Syringe</td>
			<td style="width:260px;">BD</td>
			<td style="width:123px;">309659</td>
			<td>1 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Syringe</td>
			<td style="width:260px;">BD</td>
			<td style="width:123px;">309657</td>
			<td>3 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Syringe</td>
			<td style="width:260px;">BD</td>
			<td style="width:123px;">309646</td>
			<td>5 mL; Used for making the drip chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Syringe</td>
			<td style="width:260px;">BD</td>
			<td style="width:123px;">309604</td>
			<td>10 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Syringe</td>
			<td style="width:260px;">BD</td>
			<td style="width:123px;">302830</td>
			<td>20 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Disposable Pipette</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">13-678-11</td>
			<td>25 mL Fisherbrand&#8482; Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe, for filling serum bottles.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Electric Pipette Filler</td>
			<td style="width:260px;">Pipet-aid</td>
			<td style="width:123px;">P-57260</td>
			<td>Vacuum pressure electric serological pipette filler</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Serum Bottle</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">33109-U</td>
			<td>Holds approximately 69 mL of liquid for culture growth, optimum for use of 20mL culture per bottle.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Anaerobic Culture Tube</td>
			<td style="width:260px;">VWR</td>
			<td style="width:123px;">89167-178</td>
			<td>Anaerobic Tubes, 18 x 150 mm, Supplied with 20 mm Blue Butyl Rubber Stopper and Aluminum Seal.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Rubber Stopper</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">27235-U</td>
			<td>Silicone stopper, used for sealing serum bottles and for creating the tubing system/drip chamber.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Aluminum Crimp Seal (without septum)</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">27227-U</td>
			<td>Aluminum seal for top of serum bottle for use with serum bottle crimper.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Serum Bottle Aluminum Seal Crimper</td>
			<td style="width:260px;">Wheaton</td>
			<td style="width:123px;">224307</td>
			<td>30 mm crimper with standard seal.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">PTFE Tubing</td>
			<td style="width:260px;">Supelco</td>
			<td style="width:123px;">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 height="21">
			<td height="21" style="width:389px;height:21px;">Disposable Cuvettes</td>
			<td style="width:260px;">GMBH</td>
			<td style="width:123px;">759085D</td>
			<td>1.5 Ml for use with spectrophotometer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Needle</td>
			<td style="width:260px;">BD</td>
			<td style="width:123px;">303015</td>
			<td>22G; used for serum bottle injection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Needle</td>
			<td style="width:260px;">BD</td>
			<td style="width:123px;">305120</td>
			<td>23G; used for punching-through rubber stopper to create drip tubing system.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Shewanella oneidensis MR-1 with GFP</td>
			<td style="width:260px;">N/A</td>
			<td style="width:123px;">N/A</td>
			<td>Matthysse AG, Stretton S, Dandie C, McClure NC, &#38; Goodman AE (1996) Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiol Lett 145(1):87-94.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Ethanol</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">&#160;S25310A</td>
			<td>95% Denatured</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">TSA</td>
			<td style="width:260px;">BD</td>
			<td style="width:123px;">212305</td>
			<td>Tryptic soy agar for culturing the model organism (S. oneidensis) used in this protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">PIPES Buffer</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">P-1851</td>
			<td>Used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Sodium Hydroxide</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">S-5881</td>
			<td>Used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Ammonium Chloride</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">A-5666</td>
			<td>Used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Potassium Chloride</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">P-4504</td>
			<td>Used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Sodium Phosphate Monobasic</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">S-9638</td>
			<td>Used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Sodium Chloride</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">S271-3</td>
			<td>Used for &#8220;nanowires&#8221; medium, and used to make mineral solution used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Sodium lactate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">L-1375</td>
			<td>60%(w/w) syrup @ 98% pure, d=1.3 g/mL, 7M, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Sodium Bicarbonate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">S-5761</td>
			<td>Used to make ferric NTA solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Nitrilotriacetic Acid Trisodium Salt</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">N-0253</td>
			<td>Used to make ferric NTA solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Iron (III) Chloride</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">451649</td>
			<td>Used to make ferric NTA solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Magnesium Sulfate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">208094</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Manganese (II) Sulfate Monohydrate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">M-7634</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Iron(II) Sulfate Heptahydrate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">215422</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Calcium Chloride Dihydrate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">223506</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Cobalt(II) Chloride</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">60818</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Zinc Chloride</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">229997</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Copper(II) Sulfate Pentahydrate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">C-8027</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Aluminum Potassium Sulfate Dodecahydrate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">237086</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Boric Acid</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">B-6768</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Sodium Molybdate Dihydrate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">331058</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Nickel(II) Chloride</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">339350</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Sodium Tungstate Dihydrate</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">14304</td>
			<td>Used to make minerals solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">D-Biotin</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">47868</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Folic Acid</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">F-7876</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Pyridoxine Hydrochloride</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">P-9755</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Riboflavin (B2)</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">47861</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Thiamine Hydrochloride</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">T-4625</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Nicotinic Acid</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">N4126</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">D-Pantothenic Acid Hemicalcium Salt</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">21210</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Vitamin B12</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">V-2876</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">4-Aminobenzoic Acid</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">A-9878</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; medium {Hill, E.A. 2007}</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:389px;height:21px;">Thioctic Acid</td>
			<td style="width:260px;">Sigma</td>
			<td style="width:123px;">T-1395</td>
			<td>Used to make vitamin solution, used for &#8220;nanowires&#8221; 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

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 ...

Forschung

Chemie

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 ...

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

Charakterisierung von Elektronentransport durch lebende Biofilme

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface (SALVI) and Scanning Electron Microscopy (SEM). This paper describes the method and key steps in integrating the vacuum compatible SAVLI to SEM and obtaining secondary electron (SE) images of particles in liquid in high vacuum. Energy dispersive x-ray spectroscopy (EDX) is used to obtain elemental analysis of particles in liquid and control samples including deionized (DI) water only and an empty channel as well. Synthesized boehmite (AlOOH) particles suspended in liquid are used as a model in the liquid SEM illustration. The results demonstrate that the particles can be imaged in the SE mode with good resolution (i.e., 400 nm). The AlOOH EDX spectrum shows significant signal from the aluminum (Al) when compared with the DI water and the empty channel control. In situ liquid SEM is a powerful technique to study particles in liquid with many exciting applications. This procedure aims to provide technical know-how in order to conduct liquid SEM imaging and EDX analysis using SALVI and to reduce potential pitfalls when using this approach.

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

We present a procedure for real-time imaging and elemental composition analysis of boehmite particles in deionized water by in situ liquid Scanning Electron Microscopy.

In Situ Charakterisierung von Böhmit Teilchen im Wasser mit flüssigem SEM

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

DNA-Magnetpartikel verbindlich Analyse durch dynamische und elektrophoretische Lichtstreuung

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

Mikrofluidische Chips für In Situ Crystal-Röntgenbeugung und In Situ dynamische Lichtstreuung für serielle Kristallographie

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

Schnelle Nanoprobe Signal-Verbesserung durch In Situ -Gold-Nanopartikel-Synthese

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...