We are grateful to the Pacific Northwest National Laboratory (PNNL) Nuclear Process Science Initiative (NPSI)-Laboratory Directed Research and Development (LDRD) fund for support. Dr. Sayandev Chatterjee provided the synthesized boehmite particles. 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. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.

1. Prepare AlOOH Liquid Sample
NOTE: Do not touch the specimen or anything inside the SEM chamber with bare hands. Powder free gloves should be worn at all times when handling the SALVI device and mounting it onto the SEM stage in order to avoid potential contamination during surface analysis.
<ol>
	<li>Make an AlOOH stock solution (1 mg/mL)
	<ol>
		<li>Dissolve 10 mg of AlOOH powder in 10 mL DI water to make the 1 mg/mL AlOOH stock solution.</li>
		<li>Ultrasonicate the stock solution for 5 min. 
		NOTE: The pH of the stock solution is approximately 4.6 measured by a pH meter. The solution of pH is not adjusted and used as it is in this work.</li>
	</ol>
	</li>
	<li>Make a diluted solution of 10 &#181;g/mL
	<ol>
		<li>Dilute the 1 mg/mL AlOOH stock solution to 10 &#181;g/mL by dispensing 1 mL into 99 mL DI water via pipette.</li>
		<li>Ultrasonicate the solution for 5 min. 
		NOTE: The pH of the stock solution is approximately 5.8 measured by a pH meter after dilution.</li>
	</ol>
	</li>
</ol>
2. Sputter Coat the SALVI SiN Membrane Window with Carbon
<ol>
	<li>Insert the carbon rod into the rod holder. 
	NOTE: The rod holder can be identified as the piece that is attached to the hinged lid.</li>
	<li>Use a pair of tweezers and carefully remove the tape on the SALVI&#39;s SiN membrane window frame. 
	NOTE: The tape is used to protect the SiN membrane prior to surface analysis.</li>
	<li>Secure the SALVI device upright inside the carbon coater chamber using carbon tape to fix the polytetrafluoroethylene tubing of the SALVI device on the stage of the coater. Close the lid.</li>
	<li>Press the &#34;POWER&#34; button to initiate the vacuum pump.</li>
	<li>Press the &#34;VOLTAGE&#34; button at the front panel of the carbon coater and set the value to 4.6 V by adjusting the up (&#9650;) and down (&#9660;) buttons for this operation. 
	NOTE: The voltage setting may vary due to various carbon coaters.</li>
	<li>Turn on the coating thickness monitor by switching on its &#34;POWER&#34; button. Clear the reading displayed in the screen of &#34;THICKNESS (nm)&#34; to zero by pressing the button &#34;ZERO&#34; if the reading is not zero. Press the &#34;TIMER&#34; at the carbon coater&#39;s front panel to set the deposition time to 30 s by adjusting the up (&#9650;) and down (&#9660;) buttons.</li>
	<li>Keep the carbon coater&#39;s operation mode to auto by switching the button &#34;AUTO &#9668;&#9658;MANUAL&#34; to &#34;AUTO&#34;. Switch the &#34;START/STOP&#34; button to &#34;START&#34; when the vacuum reaches about 4 &#215; 10-4 mbar as measured by the vacuum gauge at the carbon coater&#39;s front panel.</li>
	<li>Once the thickness monitor indicates that the carbon coating has reached 20 nm, press the &#34;stop&#34; button to terminate the coating process and to vent the vacuum seal.</li>
	<li>Open the lid and take out the carbon coated SALVI device using vinyl gloves when handling the device. 
	NOTE: Coating the sample with carbon creates a conductive layer on the sample to inhibit the charging effect and improve the SE signal required for the SEM imaging. Securely store the coated device in a clean Petri dish with a cover until the device is ready to be installed in the SEM stage. In order to ensure the SiN membrane is sufficiently coated, it is recommended to check the device visually after the coating. If the coating is not thick enough, a second time of sputter coating can be applied with the thickness measured until 10 nm.</li>
</ol>
3. Mount the Device and Use SEM/Focused Ion Beam (FIB) to Make Apertures on the SALVI SiN Membrane Using FIB
<ol>
	<li>Open the SEM specimen chamber
	<ol>
		<li>Open the associated microscope control software on the SEM instrument control computer. 
		NOTE: The control software may vary due to various SEMs.</li>
		<li>Vent the specimen chamber by clicking &#34;Vent&#34; on the graphic user interface (GUI) of the associated microscope control software under &#34;Beam Control&#34; tab in order to open the chamber door.</li>
		<li>Open the chamber door carefully (once venting has completed).</li>
	</ol>
	</li>
	<li>Mount the SALVI device onto the SEM stage 
	NOTE: Check the surface of the SiN membrane to see if it is intact either visually or using a light microscope before mounting. The SALVI device mounted on the SEM stage must not touch the Everhart Thornley Detector (ETD) detector inside the specimen chamber.
	<ol>
		<li>Select the standard SEM sample holder stub. Fix the stub onto the center of the stage using the appropriate bolt and hex wrench.</li>
		<li>Place two strips of double-sided carbon tape on the stub.</li>
		<li>Stick the SALVI device onto the carbon tape placed on the stub with the SiN membrane side facing up.</li>
		<li>Securely immobilize the SALVI on the stub using an additional two strips of single-sided copper tape to bind the SALVI PDMS block to the SEM metal stub. In addition, use the copper tapes to connect the SiN frame and the metal stub. Make sure that the tape does not completely cover the SiN membrane. 
		NOTE: The use of carbon and copper tapes help ensure a continuous grounding path for the removal of charge from the SiN membrane during the SEM measurement. The position of the tape on the edge of the SiN frame is quite important, because it ensures grounding and reduces charging during analysis. The bottom of the device must also have full contact with the SEM stub via double-sided carbon tape. The tape must not cover the SiN membrane to avoid possible damage in handling.</li>
	</ol>
	</li>
	<li>Pump down the specimen chamber
	<ol>
		<li>Close the specimen chamber door. Select the &#34;High Vacuum&#34; mode on the SEM software GUI under the &#34;Beam Control&#34; page.</li>
		<li>Click the &#34;Pump&#34; button on the &#34;Beam Control&#34; page to start vacuuming and apply pressure by hand to the chamber door until the desired vacuum is established. 
		NOTE: The chamber pressure must reach at least 1.0 &#215; 10-5 Torr and must steadily remain at or below this value before imaging. This is an important step to enable the highly-resolved resolution for imaging. The pressure setting can be monitored from the right corner of the GUI.</li>
	</ol>
	</li>
	<li>Make apertures in the SiN membrane using FIB
	<ol>
		<li>Activate the electron beam imaging area by clicking the &#34;Pause&#34; icon on the toolbar. Turn on the electron beam by clicking the &#34;Beam On&#34; button on the &#34;Beam Control&#34; page. Select the ETD detector and the SE mode for imaging from the &#34;Detectors&#34; drop down menu. 
		NOTE: The detector may vary due to different configuration of the SEMs. The in-lens detector is also applicable for liquid SEM analysis.</li>
		<li>Link the Z coordinate value to the actual Free Working Distance (FWD) value by clicking the &#34;Link&#34; icon on the tool bar. Set the working distance (WD) as 10 mm by typing the number 10 into the text box of coordinate &#34;Z&#34; on the &#34;Navigation&#34; page when the &#34;Actual&#34; distance is selected. 
		NOTE: The WD may vary due to various SEMs.</li>
		<li>Set the electron beam current to 0.47 nA, the accelerating voltage to 8 keV, and the resolution to 1,024 &#215; 884 from the corresponding list boxes displayed on the toolbar at the electron beam imaging area. 
		NOTE: The current and voltage setting may vary due to different SEMs.</li>
		<li>Locate the microchannel (0.2 mm x 1.5 mm) by twisting the &#34;X&#34; and &#34;Y&#34; shift knobs on the Manual User Interface (MUI) board to observe the live image on the control monitor. Draw a line paralleled to the microchannel from one end to the other using the mouse. Click &#34;xT Align Feature&#34; from the drop down menu of &#34;Stage&#34; tab on the toolbar and select &#34;horizontal&#34; to align the microchannel.</li>
		<li>Set the stage tilt to 0 &#176; by selecting the value from the &#34;T&#34; list box on the &#34;Navigation&#34; page. Locate a distinct particle feature near the microchannel and center it under the yellow cross by moving the stage using the &#34;X&#34; and &#34;Y&#34; shift knobs. Magnify the feature to 1,000X and twist the &#34;Contrast&#34;, &#34;Brightness&#34;, &#34;Coarse&#34;, and &#34;Fine&#34; knobs on the MUI to optimize the image of the particle feature.</li>
		<li>Tilt the stage to 15 &#176; by selecting the value from the &#34;T&#34; list box on the &#34;Navigation&#34; page. Use &#34;Z-control&#34; by pressing down the wheel on the mouse and drag the feature back under the yellow cross on the screen of the electron beam imaging area after the stage is tilted.
		<ol>
			<li>Tilt the stage again to 30 &#176; and bring the feature back under the cross using the &#34;Z-control&#34;. Tilt the stage back to 0 &#176; and observe the location of the feature; the eucentric height is confirmed if the feature does not shift significantly. 
			NOTE: Locating the eucentric height is performed to keep the ion beam and electron beam focused at the same position to achieve good FIB milling precision. Repeat the processes in step 3.4.6 if the feature shifts significantly after tilting the stage back to 0 &#176;. The eucentric height should be adjusted for each new mounted sample for the greatest accuracy.</li>
		</ol>
		</li>
		<li>Tilt the stage to 52&#176; by selecting the value from the &#34;T&#34; list box on the &#34;Navigation&#34; page. 
		NOTE: The tilting degree may vary due to various SEMs.</li>
		<li>Deactivate the &#34;Pause&#34; icon on the toolbar by clicking the button to ensure that the Ion beam imaging area is on. Turn on the gallium source ion beam by clicking on the &#34;Beam On&#34; button under the &#34;Beam Control&#34; page.
		<ol>
			<li>Set the accelerating voltage of the ion beam to 30 keV and beam current to 0.3 nA by selecting these values from the corresponding voltage and current list boxes located at the toolbar. Bring the microchannel to the center of this imaging area.</li>
		</ol>
		</li>
		<li>Choose the circle as the pattern by selecting this feature from the list box of &#34;Pattern&#34; on the &#34;Patterning&#34; page. Set the &#34;Outer Diameter&#34; to 1 &#181;m, the &#34;Inner diameter&#34; to 0 &#181;m, the &#34;Z size&#34; to 500 nm, and the &#34;Dwell Time&#34; to 1 &#181;s in the corresponding text box.
		<ol>
			<li>Type &#34;Si&#34; in the &#34;Application&#34; text box because the main component of the about-to-be-milled detection window is silicon nitride. Then click the &#34;Patterning menu/Start patterning&#34; button to begin milling the holes on the detection window that covered the microchannel. Repeat the milling process multiple times to obtain a series of round holes. In an experiment, several holes may be made. 
			NOTE: The holes are 100 &#181;m apart, from one side of the microchannel to the other. Move quickly to minimize the beam damage on the SiN membrane. The SEM FIB milling process usually starts from either the very left or right side of the microchannel in order to track and number the apertures easily. The operator may choose to go from the bottom or top depending on the orientation of the channel and personal preference. Ensure that the SEM FIB milling is completed and adequate so the specimen can be probed within the apertures.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Vent the chamber after the SEM/FIB
	<ol>
		<li>Tilt the stage back to 0 &#176; by selecting 0 from the &#34;T&#34; list box on the &#34;Navigation&#34; page. Turn off both the electron beam and ion beam by clicking &#34;Beam On&#34; when the corresponding beam imaging area is activated. Click &#34;Vent&#34; on the &#34;Beam Control&#34; page to vent the specimen chamber.</li>
	</ol>
	</li>
</ol>
4. Load SALVI with Liquid Samples
<ol>
	<li>Clean the SALVI using DI water
	<ol>
		<li>Carefully open the SEM chamber door after it is fully vented, and leave the SALVI device as it is on the stage. 
		NOTE: To save time on mounting the device and focusing, it is strongly recommended to keep the device on the stage when loading the sample.</li>
		<li>Draw 1 mL DI water into a sterile syringe, connect the syringe with the inlet of the microfluidic device using a polytetrafluoroethylene tubing adaptor fitting, and slowly inject the liquid for 3 - 5 min. 
		NOTE: A syringe pump is recommended for all steps where injecting solutions into the SALVI is required. This can be done in this step by setting a 1 mL sterile syringe containing the solution to a flow-rate of 100 - 250 &#181;L/min. Utilizing a syringe pump at a constant flow-rate can decrease the likelihood of damage to the SiN membrane.</li>
		<li>Repeat step 4.1.2 three times using 1 mL of 10 &#181;g/mL AlOOH, prepared in step 1, to ensure the concentration of the sample is not diluted by the preloaded DI water.</li>
		<li>After the injection, remove the syringe. Connect the inlet and outlet of SALVI using the polyether ether ketone union. Dry any liquid outside the SALVI with lab wipes. If there are any bubbles within the polytetrafluoroethylene tubing or microchannel, redo the AlOOH sample injection until no bubbles are seen within the polytetrafluoroethylene tubing. 
		NOTE: Finger-tighten the polyether ether ketone union. Do not use too much strength when tightening the union to avoid creating a significant internal pressure increase inside the SALVI device, which could result in damage to the SiN membrane. 
		NOTE: The bubbles inside the microchannel may affect the scanning and cause image shift. Any liquid outside of the device will affect the vacuum status, therefore the outside of the SALVI and polytetrafluoroethylene tubing should be thoroughly dried prior to inserting it to the vacuum chamber. In addition, the device should not have physical damage (e.g., cut on the tubing, broken SiN membrane window) that leads to leaking. Otherwise, the chamber pressure may not reach the desired high vacuum, bubbles may form in the tubing, and the liquid sample will be lost during vacuuming.</li>
	</ol>
	</li>
</ol>
5. Conduct Liquid SEM Imaging and Elemental Analysis
<ol>
	<li>Take images using the ETD detector and SE mode
	<ol>
		<li>Close the specimen chamber door. Select the &#34;High Vacuum&#34; mode on the SEM software GUI under the &#34;Beam Control&#34; page. Click the &#34;Pump&#34; button on the &#34;Beam Control&#34; page to start vacuuming and apply hand pressure to the chamber door until the desired vacuum is established.</li>
		<li>Activate the electron beam imaging area by clicking the &#34;Pause&#34; icon on the toolbar. Turn on the electron beam by clicking the &#34;Beam On&#34; button on the &#34;Beam Control&#34; page. Select the ETD detector and the SE mode for imaging from the &#34;Detectors&#34; drop down menu.
		<ol>
			<li>Set the accelerating voltage to 8 keV and beam current to 0.47 nA from the corresponding list boxes displayed on the GUI toolbar at the electron beam imaging area. Set the WD as 7 mm by typing the number &#34;7&#34; into the text box of the coordinate &#34;Z&#34; on the &#34;Navigation&#34; page when the &#34;Actual&#34; distance is selected. 
			NOTE: The parameters of beam voltage, current and working distance may vary due to different SEMs.</li>
		</ol>
		</li>
		<li>Magnify the feature to 1,000 &#215; and twist the &#34;Contrast&#34;, &#34;Brightness&#34;, &#34;Coarse&#34;, and &#34;Fine&#34; knobs on the MUI to optimize the image of the particle feature.</li>
		<li>Center the first hole in the live image of the electron beam imaging area by twisting the &#34;X&#34; and &#34;Y&#34; shift knobs on the MUI board. Enlarge the images with particles to magnification 200,000&#215; by twisting the &#34;Magnification&#34; knob on the MUI board. Select the screen resolution &#34;1,024 &#215; 884&#34; from the list box on the toolbar.</li>
		<li>Set the scan rate as 30 &#181;s from the list box on the toolbar. Press the F4 key to take the snapshot of the current image shown in the electron beam imaging area.</li>
		<li>Press Ctrl + S keys to save the image as.tif file to the desired location with the defined filename including an incremental number.</li>
		<li>Zoom out by twisting the &#34;Magnification&#34; knob to locate the next adjacent hole. Repeat the operations in steps 5.1.4 - 5.1.6 to image the AlOOH particles in the rest of the holes.</li>
	</ol>
	</li>
	<li>Conduct elemental analysis using EDX
	<ol>
		<li>Insert the Energy Dispersive Spectroscopy (EDS) detectors into the chamber.</li>
		<li>Select the ETD detector on the microscope control monitor and SE mode for viewing the sample on the electron beam imaging area. Set the accelerating voltage to 8 keV, the current to 0.47 nA and the WD to 7 mm as described in step 5.1.2.</li>
		<li>Enlarge the AlOOH particles in each hole with magnification 200,000X by twisting the &#34;Magnification&#34; knob on the MUI board. 
		NOTE: Keep the electron beam focused on the same spot so as to provide more localized elemental information. An image of AlOOH is provided in Figure 1a.</li>
		<li>Open the associated EDAX software. 
		NOTE: The associated software may vary due to different instruments&#39; configurations.</li>
		<li>Click &#34;start recording new spectra&#34; in the user interface (UI) to collect the EDX spectrum. Select &#34;Peak ID&#34; to choose the probable elements of the spectrum. Type in observed elements, e.g., oxygen in this case, into the &#34;Element&#34; field. Click &#34;Add&#34; to apply the element to the spectrum.</li>
		<li>Click on &#34;File&#34; and then click &#34;Save As&#34;. Save the spectral data in.csv format using the desired file name for further plotting using a graphing software.</li>
		<li>Repeat the operations in steps 5.3.3 - 5.3.6 to record the EDX spectrum from each hole.</li>
		<li>After finishing the imaging and spectrum recording for each of the holes, turn off the electron beam by clicking &#34;Beam On&#34; button on the &#34;Beam Control&#34; page when the electron beam imaging area is on. Vent the SEM chamber by clicking &#34;Vent&#34; on the same page. Carefully take the sample off the stage by removing all the tapes after the chamber door is open.</li>
		<li>Repeat the procedure to conduct the control experiments using the DI water and an empty microchannel.</li>
	</ol>
	</li>
</ol>
6. Plot the EDX Spectrum
<ol>
	<li>Import the.csv spectrum file into a graphing software.</li>
	<li>Plot the spectrum using the energy level as the x-axis and the intensity received and processed by the EDX as the y-axis to show the reconstructed spectra, as shown in Figures 2a, 2b and 2c.</li>
</ol>

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<li>Clark, S. B., Buchanan, M., Wilmarth, B. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aClark%2C+SB+%22Basic+Research+Needs+for+Environmental+Management%22">Basic Research Needs for Environmental Management</a>. , Department of Energy. (2016).</li>
<li>Mills, O. P., Rose, W. I. <a target="_blank" href="http://www.geo.mtu.edu/~raman/papers/MillsRoseGeosphere2010.pdf">Shape and surface area measurements using scanning electron microscope stereo-pair images of volcanic ash particles.</a> Geosphere. 6, 805-811 (2010).</li>
<li>Li, S., Jiang, F., Yin, Q., Jin, Y. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/0038109896001433">Scanning electron acoustic microscopy of semiconductor materials.</a> Solid State Commun. 99 (11), 853-857 (1996).</li>
<li>Dohnalkova, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+Hydrated+Microbial+Extracellular+Polymers%3A+Comparative+Analysis+by+Electron+Microscopy%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Imaging Hydrated Microbial Extracellular Polymers: Comparative Analysis by Electron Microscopy.</a> Appl Environ Microbiol. 77 (4), 1254-1262 (2011).</li>
<li>Yu, X. -Y., Liu, B., Yang, L. <a target="_blank" href="https://link.springer.com/article/10.1007/s10404-013-1199-4">Imaging liquids using microfluidic cells.</a> Microfluid Nanofluid. 15 (6), 725-744 (2013).</li>
<li>Barshack, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel+Method+for+%22Wet%22+SEM%5BTitle%5D)+AND+%22Ultrastruct+Pathol%22%5BJournal%5D)">A Novel Method for "Wet" SEM.</a> Ultrastruct Pathol. 28 (1), 29-31 (2004).</li>
<li>Cameron, R. E., Donald, A. M. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.1994.tb03445.x/full">Minizing sample evaporation in the Environmental Scanning Microscope.</a> J Microsc. (Oxford, U. K.). 173 (3), 227-237 (1994).</li>
<li>Danilatos, G. D. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.1991.tb03149.x/abstract">REVIEW AND OUTLINE OF ENVIRONMENTAL SEM AT PRESENT.</a> J Microsc (Oxford, U.K.). 162 (3), 391-402 (1991).</li>
<li>Stokes, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+electron+imaging%2C+image+interpretation+and+applications%3A+environmental+scanning+electron+microscopy%5BTitle%5D)+AND+%22Philos+Trans+R+Soc%2C+A%22%5BJournal%5D)">Recent advances in electron imaging, image interpretation and applications: environmental scanning electron microscopy.</a> Philos Trans R Soc, A. 361 (1813), 2771-2787 (2003).</li>
</ol>

The authors have nothing to disclose.

SEM is a powerful technique in surface characterization of organic and inorganic materials on a nanoscale (nm) level with high resolution1. For example, it is widely used for analyzing the solid and dry samples such as geological materials26 and semiconductor27. However, it has limitations in characterizing the wet and liquid samples due to the incompatibility of liquid within the highly vacuumed environment required for electron microscopy<sup class...

Scanning Electron Microscope (SEM) has been widely applied to investigate a variety of specimens by producing high resolution imaging1. The energy dispersive x-ray spectroscopy (EDX) associated with the SEM enables the determination of elemental composition1. Traditionally, SEM is applied for imaging only dry and solid samples. In the last 30 years, Environmental SEM (ESEM) was developed for analyzing the partial hydrated samples in a vapor environment2,3,4,5. However, ESEM is unable to image the wet, fully fluid samples with desired high resolution6. Wet SEM cells were also developed to image wet specimens using SEM7,8; nevertheless, these cells were developed mainly for biological specimens and backscattered electron imaging, and are more accessible for applications with those designs9,10.
To address the challenges in analyzing various samples in their native liquid environment using SEM, we invented a vacuum compatible microfluidic device, System for Analysis at the Liquid Vacuum Interface (SALVI), to enable high spatial resolution secondary electron (SE) imaging and elemental analysis of liquid samples using the high vacuum mode in SEM. This novel technique includes the following unique features: 1) liquid is directly probed in a small aperture of 1 - 2 &#181;m in diameter; 2) liquid is held within the hole by surface tension; and 3) SALVI is portable and can be adapted to more than one analytical platform11,12,13,14,15,16,17,18.
SALVI consists of a 100 nm thick silicon nitride (SiN) membrane and a 200 &#181;m wide microchannel made of polydimethylsiloxane (PDMS) block. The SiN membrane window is applied to seal the microchannel. The fabrication details and key design considerations were detailed in previous papers and patents11,19,20. Currently, a leading manufacturer and distributor of consumable supply for microscopy has purchased the license to sell SALVI devices commercially for liquid SEM applications21,22.
The applications of SALVI in vacuum-based analytical instruments have been demonstrated using a variety of aqueous solutions and complex liquid mixtures including biofilms, mammalian cells, nanoparticles, and electrode materials12,14,17,20,23,24. However, most of the aforementioned work utilized time-of-flight secondary ion mass spectrometry (ToF-SIMS) as the key analysis tool, thus the application of liquid SEM with SALVI has not been explored fully. In this work, SALVI has been used to study larger non-spherical colloidal particles in liquid using liquid SEM imaging and EDX elemental analysis. The sample consists of AlOOH particles synthesized at our laboratory. Submicrometer-sized boehmite particles are known to exist in high-level radioactive waste at the Hanford site. They are slow to dissolve and may cause rheological problems in waste treatment. Therefore, it is important to have the capability to characterize boehmite particles in liquid25. This technical approach can be used to study boehmite in various physicochemical conditions for improved understanding of these particles and related rheological properties. These particles were utilized to demonstrate step-by-step how to apply SALVI to high vacuum SEM in order to study particles suspended in liquid. Key technical points for SALVI and SEM integration and SEM data acquisition are highlighted within the paper.
The protocol provides demonstration of the liquid sample analysis using SALVI and liquid SEM imaging, for those who are interested in utilizing this novel technique in diverse applications of liquid SEM in the future.

<table><tbody><tr><td>Carbon Coater</td><td>Cressington</td><td>208 Carbon</td><td>It is accompanied with thickness monitor MTM-10.</td></tr><tr><td>SEM</td><td>FEI</td><td>Quanta 3D FEG</td><td>It provides highly resolved scanning electron microscopy and elemental analysis.</td></tr><tr><td>System for Analysis at the Liquid Vacuum Interface (SALVI)</td><td>Pacific Northwest National Laboratory</td><td>N/A</td><td>SALVI is a unique, vacuum compatible microfluidic cell that enables the characterization of the liquid sample using vacuu- based scientific instrument.</td></tr><tr><td>PEEK Union</td><td>Valco</td><td>ZU1TPK</td><td>The polyether ether ketone union is used for connecting the inlet and outlet of SALVI</td></tr><tr><td>Syringe</td><td>BD</td><td>309659</td><td>1 mL</td></tr><tr><td>Pipette</td><td>Thermo Fisher Scientific</td><td>21-377-821</td><td>Range: 100 to 1,000 mL</td></tr><tr><td>Pipette Tip 1</td><td>Neptune</td><td>2112.96.BS</td><td>1,000 &amp;micro;L</td></tr><tr><td>Pipette Tip 2</td><td>Rainin</td><td>17001865</td><td>20 &amp;micro;L</td></tr><tr><td>Syringe Pump</td><td>Harvard Apparatus</td><td>70-2213</td><td>It is used to inject the liquid sample into the SALVI device.</td></tr><tr><td>pH meter</td><td>Fisher Scientific/accumet</td><td>13-636-AP72</td><td>It is used for measuring the pH of AlOOH in DI water.</td></tr><tr><td>Barnstead Ultrapure Water System, UV/UF</td><td>Thermo Scientific Barnstead</td><td>Nanopure diamond D11931</td><td>It is used for producing DI water.</td></tr><tr><td>Centrifuge tubes</td><td>Fisher scientific/Falcon</td><td>15-527-90</td><td>15 mL</td></tr><tr><td>Bransonic ultrasonic cleaner</td><td>Sigma-Aldrich</td><td>2510</td><td>It is used to ultrasonicate the AlOOH liquid sample.</td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td>11106015</td><td>XS64</td></tr><tr><td>AlOOH</td><td>Pacific Northwest National Laboratory</td><td>N/A</td><td>It is synthesized by scientists at Pacific Northwest National Laboratory.</td></tr><tr><td>xT microscope Control</td><td>FEI</td><td>Quanta 3D FEG</td><td>Default microscope control software of SEM Quanta 3D FEG</td></tr><tr><td>EDAX Genesis software</td><td>EDAX</td><td>N/A</td><td>The software is used for collecting the EDX elemental information of the samples.</td></tr><tr><td>Teflon tubing</td><td>SUPELCO</td><td>58697-U</td><td>It is used for introducing the sample into the microchannel and holding adequate volume of liquid.</td></tr></tbody></table>

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.

The representative results are presented to show how the particles are imaged and analyzed using in situ liquid SEM imaging coupled with EDX. The results include SE images and EDX spectra. The SE images were obtained at 100,000X and 200,000X magnification levels in Figure 1. Figure 1a depicts the SE image of the AlOOH, Figure 1b DI water, and Figure 1c the hole ...

Watch this Scientific Journal Video about In Situ Characterization of Boehmite Particles in Water Using Liquid SEM at JoVE.com

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 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 ...

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Research

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Chemistry

9.2K Views.  Pacific Northwest National Laboratory. 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.

Video: In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Environment

Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers

Magnetic beads with ~1.9 &#181;m average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for the purpose of investigating phase change of those liquid segments. The magnetic beads were externally controlled using a magnet, allowing for the beads to bridge the air valve between the adjacent liquid segments. A hydrophobic coating was applied to the inner surface of the tube to enhance the separation between two liquid segments. The applied magnetic field formed an aggregate cluster of magnetic beads, capturing a certain liquid amount within the cluster that is referred to as carry-over volume. A fluorescent dye was added to one liquid segment, followed by a series of liquid transfers, which then changed the fluorescence intensity in the neighboring liquid segment. Based on the numerical analysis of the measured fluorescence intensity change, the carry-over volume per mass of magnetic beads has been found to be ~2 to 3 &#181;l/mg. This small amount of liquid allowed for the use of comparatively small liquid segments of a couple hundred microliters, enhancing the feasibility of the device for a lab-in-tube approach. This technique of applying small compositional variation in a liquid volume was applied to analyzing the binary phase diagram between water and the surfactant C12E5 (pentaethylene glycol monododecyl ether), leading to quicker analysis with smaller sample volumes than conventional methods.

Here, we present a protocol to investigate multi-component phase diagrams using externally controlled magnetic beads as liquid carriers in a lab-in-tube approach. This approach can aid in applications that seek to gather further information on phase change in complex liquid systems.

Magnetic beads with ~1.9 &#181;m average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for ...

Engineering

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 ...

Nanostructured Ag-zeolite Composites as Luminescence-based Humidity Sensors

Small silver clusters confined inside zeolite matrices have recently emerged as a novel type of highly luminescent materials. Their emission has high external quantum efficiencies (EQE) and spans the whole visible spectrum. It has been recently reported that the UV excited luminescence of partially Li-exchanged sodium Linde type A zeolites [LTA(Na)] containing luminescent silver clusters can be controlled by adjusting the water content of the zeolite. These samples showed a dynamic change in their emission color from blue to green and yellow upon an increase of the hydration level of the zeolite, showing the great potential that these materials can have as luminescence-based humidity sensors at the macro and micro scale. Here, we describe the detailed procedure to fabricate a humidity sensor prototype using silver-exchanged zeolite composites. The sensor is produced by suspending the luminescent Ag-zeolites in an aqueous solution of polyethylenimine (PEI) to subsequently deposit a film of the material onto a quartz plate. The coated plate is subjected to several hydration/dehydration cycles to show the functionality of the sensing film.

A protocol for the synthesis of moisture-responsive luminescent Ag-zeolite composites is described in this report.

Small silver clusters confined inside zeolite matrices have recently emerged as a novel type of highly luminescent materials. Their emission has high ...

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

We present a method for achieving sub-nanometer resolution images with amplitude-modulation (tapping mode) atomic force microscopy in liquid. The method is demonstrated on commercial atomic force microscopes. We explain the rationale behind our choices of parameters and suggest strategies for resolution optimization.

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown ...

A Facile Synthetic Method to Obtain Bismuth Oxyiodide Microspheres Highly Functional for the Photocatalytic Processes of Water Depuration

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of materials is highly related to its photocatalytic performance, it is necessary to standardize the synthetic methods in order to obtain the most functional architectures and, thus, the highest photocatalytic efficiency. Here, we report a reliable route to obtain BiOI microspheres via the solvothermal process, using Bi(NO3)3 and potassium iodide (KI) as precursors, and ethylene glycol as a template. The synthesis is standardized in a 150 mL autoclave, at 126 &#176;C for 18 h. This results in 2-3 &#181;m-sized mesoporous microspheres, with a relevant specific surface area (61.3 m2/g). Shortening the reaction times in the synthesis results in amorphous structures, while higher temperatures lead to a slight increase in the porosity of the microspheres, with no effect in the photocatalytic performance. The materials are photo-active under UV-A/visible light irradiation for the degradation of the antibiotic ciprofloxacin in water. This method has demonstrated to be effective in interlaboratory tests, obtaining similar BiOI microspheres in Mexican and Chilean research groups.

This article describes a synthetic method to obtain bismuth oxyiodide microspheres, which are highly functional to perform the photocatalytic removal of organic pollutants, such as ciprofloxacin, in water under UV-A/visible light irradiation.

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of ...

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids with adsorbates. Vibrational spectra provide information on the chemical nature of adsorbed species and their structure. Thus, they are very useful for obtaining molecular level understanding of surface species. The IR spectrum of the sample itself gives some direct information about the material. General conclusions can be drawn concerning hydroxyl groups, some stable surface species and impurities. However, the spectrum of the sample is &#34;blind&#34; with respect to the presence of coordinatively unsaturated ions and gives rather poor information about the acidity of surface hydroxyls, species decisive for the adsorption and catalytic properties of the materials. Furthermore, no discrimination between bulk and surface species can be made. These problems are solved by the use of probe molecules, substances that interact specifically with the surface; the alteration of some spectral features of these molecules as a result of adsorption provides valuable information about the nature, properties, location, concentration, etc., of the surface sites.
The experimental protocol for in-situ IR studies of gas/sample interaction includes preparation of a sample pellet, activation of the material, initial spectral characterization through the analysis of the background spectra, characterization by probe molecules, and study of the interaction with a particular set of gas mixtures. In this paper we investigate a zirconium terephthalate metal organic framework, Zr6O4(OH)4(BDC)6 (BDC = benzene-1,4-dicarboxylate), namely UiO-66 (UiO refers to University of Oslo). The acid sites of the UiO-66 sample are determined by using CO and CD3CN as molecular probes. Furthermore, we have demonstrated that CO2 is adsorbed on basic sites exposed on dehydroxylated UiO-66. Introduction of water to the system produces hydroxyl groups acting as additional CO2 adsorption sites. As a result, CO2 adsorption capacity of the sample is strongly enhanced.

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

The use of FTIR spectroscopy for investigation of the surface properties of polycrystalline solids is described. Preparation of sample pellets, activation procedures, characterization with probe molecules and model studies of CO2 adsorption are discussed.

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids ...

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar energy and fuel generation, and electrochemical energy storage. Nanoscale characterization of such interfaces has recently been achieved using cryogenic electron microscopy, thereby providing a new path to advancing our fundamental understanding of interface processes.
This contribution provides a practical guide to mapping the structure and chemistry of solid-liquid interfaces in materials and devices using an integrated cryogenic electron microscopy approach. In this approach, we pair cryogenic sample preparation which allows stabilization of solid-liquid interfaces with cryogenic focused ion beam (cryo-FIB) milling to create cross-sections through these complex buried structures. Cryogenic scanning electron microscopy (cryo-SEM) techniques performed in a dual-beam FIB/SEM enable direct imaging as well as chemical mapping at the nanoscale. We discuss practical challenges, strategies to overcome them, as well as protocols for obtaining optimal results. While we focus in our discussion on interfaces in energy storage devices, the methods outlined are broadly applicable to a range of fields where solid-liquid interface play a key role.

Cryogenic Focused Ion Beam (FIB) and Scanning Electron Microscopy (SEM) techniques can provide key insights into the chemistry and morphology of intact solid-liquid interfaces. Methods for preparing high quality Energy Dispersive X-ray (EDX) spectroscopic maps of such interfaces are detailed, with a focus on energy storage devices.

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar ...

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at length scales down to the atomic level. In situ closed-cell gas reaction (CCGR) studies performed using (scanning) transmission electron microscopy (STEM) can separate and identify localized dynamic reactions, which are extremely challenging to capture using other characterization techniques. For these experiments, we used a CCGR holder that utilizes microelectromechanical systems (MEMS)-based heating microchips (hereafter referred to as &#34;E-chips&#34;). The experimental protocol described here details the method for performing in situ gas reactions in dry and wet gases in an aberration-corrected STEM. This method finds relevance in many different materials systems, such as catalysis and high-temperature oxidation of structural materials at atmospheric pressure and in the presence of various gases with or without water vapor. Here, several sample preparation methods are described for various material form factors. During the reaction, mass spectra obtained with a residual gas analyzer (RGA) system with and without water vapor further validates gas exposure conditions during reactions. Integrating an RGA with an in situ CCGR-STEM system can, therefore, provide critical insight to correlate gas composition with the dynamic surface evolution of materials during reactions. In situ/operando studies using this approach allow&#160;for detailed investigation of the fundamental reaction mechanisms and kinetics that occur at specific environmental conditions (time, temperature, gas, pressure), in real-time, and at high spatial resolution.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

Summary

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VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers

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

Nanostructured Ag-zeolite Composites as Luminescence-based Humidity Sensors

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

A Facile Synthetic Method to Obtain Bismuth Oxyiodide Microspheres Highly Functional for the Photocatalytic Processes of Water Depuration

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO₂ Adsorption in UiO-66 Metal-Organic Framework

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm