We are grateful to the Pacific Northwest National Laboratory (PNNL) Earth and Biological Sciences (EBD) mission seed Laboratory Directed Research and Development (LDRD) fund for support. Instrumental access was provided through a W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) General User Proposal. EMSL is a national scientific user facility sponsored by the Office of Biological and Environmental Research (BER) at PNNL. The authors thank Dr. Yuanzhao Ding for proof reading the manuscript and providing useful feedback. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.

<ol>
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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. 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The authors have nothing to disclose.

After inoculating at log-phase, it is important to test the number of days and temperature at which the biofilm should grow before it is healthy and thick enough for imaging, as described in step 3.1. This procedure specifically covers culturing a S. oneidensis MR1 biofilm at room temperature; however different room temperatures can influence the rate of growth. Therefore, it is critical to use optical imaging to understand whether the biofilm is ready before proceeding to ToF-SIMS analysis. Similarly, different strains ...

Bacterial biofilms are surface-associated communities which have evolved over time as a defense for bacteria to survive varying adverse physical and mechanical stimuli, wherein cells are able to attach and survive in many possible environments.1,2 Biofilms are vastly investigated and have applications in many fields such as biomedicine, biomedical engineering, agriculture, and industrial research and development.1,2 Understanding the chemical mapping of these complex microbial communities, including their self-produced extracellular polymeric substances (EPS) and their local water-cluster environment, is essential to gaining an accurate and detailed depiction of their biological activities.2
Biofilms exist and grow within a highly hydrated state. This presents a great challenge in using vacuum-based surface analysis techniques such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) due to the difficulty in studying volatile liquids in vacuum. As a result, vacuum-based surface analysis techniques have been limited almost exclusively to studying biofilm samples at only their dried state. However, studying a biofilm in its dried state inhibits the accurate investigation of its true biological microenvironment. It often causes drastic changes to the EPS integrity and biofilm morphology, which has been demonstrated after comparing dry biofilm mass spectral results to in situ liquid studies.3,4 This article presents a solution for studying biofilms within their natural hydrated state by employing the use of our System for Analysis at the Liquid Vacuum Interface (SALVI),5,6 a microfluidic reactor that contains liquid under its thin silicon nitride (SiN) membrane in a microchannel made of polydimethylsiloxane (PMDS), thus providing direct access to the secondary ion probe beam while still maintaining the structural integrity of the liquid matrix within a vacuum chamber.7,8
S. oneidensis MR-1 mutated to express green fluorescence protein (GFP) was chosen as a model organism for this biofilm procedure illustration due to its metabolic versatility and common use in environmental and applied microbiology, which was based heavily on its unique capability for metal reduction and extracellular electron transfer.9,10,11 Additionally, the presence of GFP allowed for easy continuous biofilm-thickness monitoring through fluorescence microscopy, using a fluorescein isothiocyanate (FITC) filter. Our previous studies have shown evidence of this bacteria favoring attachment to the SiN window using in operando fluorescence imaging for biofilm growth to a thickness of up to one hundred micrometers.4,12 While this paper will only discuss the confirmation of biofilm&#39;s presence through fluorescence microscopy, the SALVI is compatible with other optical imaging methods such as super-resolution fluorescence imaging (i.e., structured illumination microscopy (SIM)9) and confocal laser scanning microscopy (CLSM) imaging4). Optical imaging can serve to measure the biofilm thickness, and obtain a 3D image of, the shape of the biofilm as it appears, confirming its thickness and its attachment to the SiN window.9 While GFP was used in the SIMS analysis, S. oneidensis without GFP was used for the growth curve, as this only required measurement of optical density and did not require any fluorescent imaging. Generally, the difference between the GFP tagged and untagged species in the growth curve is insignificant. Additionally, while this protocol uses S. oneidensis MR-1 GFP as a model organism to describe the procedure, this procedure is designed for any bacterial strain that may be needed for cultivation within SALVI. Although, given knowledge of the bacterial strain needed, some growth conditions such as time, temperature, and oxygen environment may need to be modified to accommodate the strain of bacteria to be used. For growth medium, this procedure uses &#34;nanowires&#34; medium, tryptic soy broth (TSB) without dextrose, and tryptic soy agar (TSA) without dextrose for culturing. The composition of &#34;nanowires&#34; medium has been specially formulated for the growth and for monitoring of extensions of the membrane and periplasm of S. oneidensis that appear to take the shape of small wires, and the medium composition has been established within previous research.13,14
Our previous protocol on in situ liquid ToF-SIMS has illustrated the benefit that SALVI has to offer for protein immobilization and attachment to the SiN, as well as a detailed protocol on ToF-SIMS analysis and data reduction.12 Rather than reiterate data reduction steps, this paper will serve to instead focus on the unique approach of setting up and cultivating biofilms within our SALVI microchannel, as well as the imaging steps to detect biofilm presence and thickness prior to ToF-SIMS analysis. While biofilms have been previously limited to only dried samples within the chamber of vacuum-based surface analytical techniques, detailed EPS and biofilm chemical mapping of live biofilms can now be obtained in situ because of this new capability.

<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

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

These representative results serve to show how the chemical profile of the attached biofilm can be identified and interpreted, as obtained through ToF-SIMS. After plotting mass spectra from ToF-SIMS data acquisition, highlighted briefly in the procedures section, peak identification should be conducted in order to assign identities to each respective m/z value. This can be done through extensive literature review on mass spectrometry on bacteria and specific chemical fragments that are ex...

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

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and 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

8.9K Views.  Pacific Northwest National Laboratory. This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

Video: 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.

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

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.

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.

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.

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.

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.

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 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.

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.

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.

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