We thank Prof. A. Paul Alivisatos at the University of California, Berkeley and Prof. Taeghwan Hyeon at Seoul National University for the helpful discussion. This work was supported by IBS-R006-D1. W.C.L. gratefully acknowledges support from the research fund of Hanyang University (HY-2015-N).

1. Synthesis of Nanoparticles
<ol>
	<li>Synthesis of platinum nanoparticles
	<ol>
		<li>Combine 17.75 mg of ammonium hexachloroplatinate(IV) ((NH4)2Pt(IV)Cl6), 3.72 mg of ammonium tetrachloroplatinate(II) ((NH4)2Pt(II)Cl4), 115.5 mg of tetramethylammonium bromide, 109 mg of poly(vinylpyrrolidone) (MW: 29,000), and 10 mL of ethylene glycol with a stir bar in a 100 mL 3-neck round bottom flask equipped with a rubber septum.</li>
		<li>Equip the flask with a reflux condenser and purge under vacuum. Stir the reaction mixture with a magnetic stir bar at 1,000 rpm.</li>
		<li>Heat the reaction mixture to 180 &#176;C in a heating mantle at a rate of 10 &#176;C/min under a flow of argon.</li>
		<li>Maintain the temperature (180 &#176;C) for 20 min. The solution becomes a dark brown color.</li>
		<li>Remove the flask from the heating mantle to allow it to cool to room temperature.</li>
		<li>Transfer the product to a 50 mL centrifuge tube. Add 30 mL of acetone to the product to precipitate the nanoparticles and centrifuge the sample at 2,400 x g for 10 min. 
		NOTE: For safety, this process should be performed in a fume hood.</li>
		<li>Discard the supernatant and re-disperse the black precipitate with 10 mL of ethanol.</li>
		<li>Precipitate the product with 30 mL of toluene and centrifuge the suspension at 2,400 x g for 10 min. Discard the supernatant and re-disperse the black precipitate with 10 mL of ethanol. Repeat this process 3 times.</li>
		<li>Add 5 mL of oleylamine to the platinum nanoparticles and transfer the dispersion to a 100 mL round-bottom flask. Reflux the dispersion overnight under magnetic stirring at 1,000 rpm to allow the ligand exchange reaction to occur.</li>
		<li>Centrifuge the ligand-exchanged nanoparticles at 10,000 x g for 30 min to separate them from the solution.</li>
		<li>Discard the supernatant and disperse the platinum nanoparticles in 10 mL of a hydrophobic solvent, such as toluene, hexane, or chloroform.</li>
	</ol>
	</li>
	<li>Synthesis of lead selenide nanoparticles
	<ol>
		<li>Preparation of lead oleate
		<ol>
			<li>Combine 758 mg of lead acetate trihydrate (Pb(Ac)2&#183;3H2O), 2.5 mL of oleic acid, and 10 mL of diphenyl ether with a stir bar in a 100 mL 3-neck round bottom flask equipped with a rubber septum.</li>
			<li>Equip the flask with a reflux condenser.</li>
			<li>Degas the mixture at 70 &#176;C under vacuum condition for 2 h and stir with a magnetic stir bar at 1,000 rpm.</li>
			<li>Purge the flask with argon and then cool it to room temperature.</li>
		</ol>
		</li>
		<li>Preparation of trioctylphosphine selenide (TOPSe)
		<ol>
			<li>In a separate vessel, combine 474 mg of selenium and 6 mL of trioctylphosphine (TOP) under an inert atmosphere, using a glovebox to prevent exposure of the reagents to air.</li>
			<li>Dissolve selenium powder in TOP by ultrasonication at 110 W, 40 kHz, and mix using a vortex mixer until the solution becomes visibly transparent.</li>
		</ol>
		</li>
		<li>In a separate 250 mL 3-neck round bottom flask, degas 15 mL of diphenyl ether at 120 &#176;C under vacuum condition with magnetic stirring at 1,000 rpm for 30 min.</li>
		<li>Purge the flask with argon, and then heat the diphenyl ether to 230 &#176;C.</li>
		<li>Rapidly inject both the lead oleate and the TOPSe solutions into the pre-heated diphenyl ether. The temperature will drop to some extent upon injection. Set the aging temperature to 170 &#176;C.</li>
		<li>Maintain the temperature of the mixture at 170 &#176;C for 10 min with vigorous stirring to allow the growth of lead selenide nanoparticles to occur.</li>
		<li>Remove the flask from the heating mantle to allow it to cool to room temperature.</li>
		<li>Prepare two 50 mL centrifuge tubes and divide the product into equal volumes and transfer to the tubes. Add 30 mL of ethanol to each tube to precipitate the nanoparticles and centrifuge the suspension at 2,400 x g for 10 min.</li>
		<li>Discard the supernatant and re-disperse the black precipitate with 10 mL of toluene.</li>
		<li>Precipitate the product with 30 mL of ethanol and centrifuge the suspension at 2,400 x g for 10 min. Discard the supernatant and re-disperse the black precipitate with 10 mL of toluene. Repeat this process 3 times.</li>
		<li>Discard the supernatant and disperse the lead selenide nanoparticles in 10 mL of a hydrophobic solvent, such as toluene, hexane, or chloroform.</li>
	</ol>
	</li>
</ol>
2. Liquid-cell Fabrication
<ol>
	<li>Silicon nitride liquid-cell (Figure 3a)
	<ol>
		<li>Deposition of silicon nitride layer
		<ol>
			<li>Deposit a low-stress silicon nitride film onto 100 &#181;m-thick silicon wafers (4 inch) by low-pressure chemical vapor deposition (LPCVD) at 835 &#176;C and a pressure of 140 mTorr with a flow of 100 sccm dichlorosilane and 50 sccm ammonia. Control the thickness of silicon nitride layer to be ~25 nm by varying the deposition time. The deposition rate is 2.5 - 3.0 nm/min, but varies slightly for each CVD.</li>
		</ol>
		</li>
		<li>Top and bottom chips 
		NOTE: Please refer to reference25 for a detailed description of the microfabrication process.
		<ol>
			<li>Spin coat 10 mL of positive photoresist on the silicon nitride/silicon wafer at 3,000 rpm for 30 s, using enough photoresist to wet the wafer completely. 
			NOTE: Ultrathin silicon wafers are easily broken during spinning. We usually attach the ultrathin wafer on another silicon wafer with a thickness of 500 &#181;m using photoresist to avoid breakage. After spin-coating, the thin wafer is separated from the thick wafer by immersion in acetone. This technique can also be used in the following process for spin coating on ultrathin silicon wafers.</li>
			<li>Bake the wafer on a hot plate at 85 &#176;C for 60 s. 
			NOTE: Do not bake the prepared wafer at temperatures of &#62;110 &#176;C. Baking the photoresist at high temperatures will cause the photoresist to change from a positive to a negative photoresist.</li>
			<li>Expose the photoresist-coated wafer to ultraviolet light (365 nm) for 10 s through a chromium mask (Figure 1a).</li>
			<li>Immerse the photoresist-coated wafer into 50 mL of developer solution for 40 s. Soak the developed wafer in 50 mL of deionized water for 1 min to wash the wafer. Repeat the washing process twice.</li>
			<li>Etch the silicon nitride for 1 min using a reactive ion etcher with a flow of 50 sccm of sulfur hexafluoride.</li>
			<li>Immerse the top chip and bottom chip in acetone for 2 min to remove the photoresist. Shake the dish gently by hand to effectively remove the photoresist.</li>
			<li>Etch the silicon with an aqueous solution of potassium hydroxide (30 mg/mL) at 85 &#176;C for 1.5 - 2 h. Use a water bath to ensure that the etching solution has a uniform temperature profile. Uneven temperature will lead to some areas being excessively etched while some are incompletely etched.</li>
			<li>When the window appears to be completely etched by the naked eye, stop the etching and take the cell out of the etching solution. Tilt the cell when removing it from the etching solution to avoid the chance of the window being broken by buoyancy, which can occur if lifted horizontally.</li>
			<li>Repeat these procedures with a mask for the bottom chip (Figure 1b). 
			NOTE: As the windows of the top and bottom chip are very thin, about 25 nm, they are very fragile. Therefore, extreme care should be taken when handling. The window side should always be facing upwards when the chip is rested on a surface.</li>
		</ol>
		</li>
		<li>Bonding top and bottom chips
		<ol>
			<li>Spin coat 10 mL of positive photoresist on the silicon nitride/silicon wafer at 3,000 rpm for 30 s.</li>
			<li>Bake the wafer on a hot plate at 90 &#176;C for 60 s.</li>
			<li>Expose the photoresist-coated wafer to ultraviolet light (365 nm) for 10 s through a chromium mask (Figure 1c).</li>
			<li>Immerse the photoresist-coated wafer into 50 mL of developer solution for 40 s. Soak the developed wafer in 50 mL of deionized water for 1 min to wash the wafer. Repeat the washing process twice.</li>
			<li>Deposit a ~100 nm thick layer of indium onto the bottom chip using a thermal evaporator. The indium layer is used as both a spacer and a sealing material.</li>
			<li>Immerse the chip in acetone for 2 min to remove the photoresist. Shake the dish gently by hand to effectively remove the photoresist.</li>
			<li>Align the bottom and top chips using an aligner and bond them at 100 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Silicon liquid-cell (Figure 3b)
	<ol>
		<li>Preparation of top and bottom chips
		<ol>
			<li>Use p-type silicon-on-insulator (SOI) wafers with layer thicknesses of 100 nm, 400 nm, and 600 &#181;m for the top silicon, buried SiO2, and handling silicon layers, respectively.</li>
			<li>Perform wet oxidation of the SOI wafer in an oxidation furnace at 950 - 1100 &#176;C to grow a layer of silicon oxide with a thickness of 170 nm.</li>
			<li>Etch the silicon oxide by dipping the SOI wafer into a buffered oxide etch (BOE) solution at room temperature for 2 min, which is a wet etchant of silicon oxide. The purpose of steps 2.2.1.2 and 2.2.1.3 is to reduce the thickness of the top silicon layer of the SOI wafer to 25 nm.</li>
			<li>Deposit a layer of low-stress silicon nitride with a thickness of 25 nm onto the SOI wafers (4-inch) by LPCVD using the same process conditions as in step 2.1.1.1.</li>
			<li>Pattern the silicon nitride using the same processes and mask patterns as in steps 2.1.2.1 - 2.1.2.4.</li>
			<li>Immerse the wafers in acetone for 2 min to remove the photoresist. Shake the dish gently by hand to effectively remove the photoresist.</li>
			<li>Etch the silicon oxide with the BOE solution for 30 s.</li>
			<li>Etch the silicon with an aqueous solution of potassium hydroxide (500 mg/mL) at 80 &#176;C for 7 - 12 h. Trimethylamine hydroxide (TMAH) aqueous solution can also be used for the silicon etching.</li>
			<li>Etch the silicon nitride with 85 % phosphoric acid at 160 &#176;C for 10 min.</li>
			<li>Etch the silicon oxide with the BOE solution at room temperature for 3 min.</li>
			<li>Repeat the procedures of steps 2.2.1.1 - 2.2.1.10 with a mask for the bottom chip (Figure 1b).</li>
		</ol>
		</li>
		<li>Bonding top and bottom chips
		<ol>
			<li>Spin coat 10 mL of positive photoresist on the silicon nitride/silicon wafer at 3,000 rpm for 30 s.</li>
			<li>Bake the wafer on a hot plate at 90 &#176;C for 60 s.</li>
			<li>Expose the photoresist-coated wafer to ultraviolet light (365 nm) for 10 s through a chromium mask (Figure 1c).</li>
			<li>Immerse the photoresist-coated wafer into 50 mL of developer solution for 40 s. Soak the developed wafer in 50 mL of deionized water for 1 min to wash the wafer. Repeat the washing process twice.</li>
			<li>Deposit a ~100 nm thick layer of indium onto the bottom chip using a thermal evaporator. The indium layer is used as both a spacer and a sealing material.</li>
			<li>Immerse the chip in acetone for 2 min to remove the photoresist. Shake the dish gently by hand to effectively remove the photoresist.</li>
			<li>Align the bottom and top chips using an aligner and bond them at 100 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Liquid-cell TEM
<ol>
	<li>Loading solution
	<ol>
		<li>Add 20 &#181;L of the nanoparticle dispersion (protocol 1.1 and 1.2) into a 5 mL vial and dry in the air for 10 min. Disperse the nanoparticles in a solvent mixture (1 mL of o-dichlorobenzene, 250 &#181;L of pentadecane, and 10 &#181;L of oleylamine). 
		NOTE: Drying under extreme conditions, such as high temperature, low pressure, and for extended durations may result in poor nanoparticle dispersions. Since the ligand-to-particle interactions are dynamic, there is a high probability of the agglomeration of particles after ligand detachment under extreme conditions. Since o-dichlorobenzene, pentadecane, and oleylamine have different vapor pressures, the drying process should be carried out immediately before the loading process to keep the solvent ratio constant.</li>
		<li>Inspect the liquid-cell using an optical microscope. 
		NOTE: If any of the windows of a liquid-cell are broken, do not use the cell.</li>
		<li>Load ~100 nL of the nanoparticle dispersion into the reservoirs (Figure 2a and 2b) of the liquid-cell. An injector equipped with an ultrathin capillary (Figure 2c) can be used to load the small amount of dispersion into reservoirs of the liquid-cell effectively. 
		NOTE: In general, the injected amount of nanoparticle dispersion exceeds the capacity of the reservoir. If the dispersion overflows from the reservoir, the liquid-cell may not completely seal. Therefore, any overflowing dispersion outside the reservoir must be absorbed by the tip of a filter paper cut into a fan shape. Avoid contact with the window during absorption.</li>
		<li>Expose the liquid-cell to the air for 10 min to dry the o-dichlorobenzene.</li>
		<li>Apply vacuum grease to one side of a copper aperture grid with a size of 2 mm and a hole-size of 600 &#181;m and cover the liquid-cell with the greased side of the aperture grid to create a sealed environment. 
		NOTE: Vacuum grease deposited on the window greatly reduces the TEM resolution. Thus, failure to match the hole of the copper aperture grid with the window of the cell on the first attempt should not be corrected; rather, the cell should be discarded and a new one prepared.</li>
	</ol>
	</li>
	<li>TEM measurement
	<ol>
		<li>Place the liquid-cell in a standard TEM holder. The liquid-cell is designed to fit into a standard holder. 
		NOTE: Use gloves to avoid contamination of the TEM holder.</li>
		<li>Set the TEM in continuous image acquisition mode. Capture TEM images at an acceleration voltage of 200 kV with a current density of ~700 A/m2. 
		NOTE: Frequently check the TEM pressure level. If the pressure level is abnormal, stop imaging immediately and remove the holder from the TEM chamber as quickly as possible. The low contrast of the liquid-cell TEM causes difficulty in focusing the image. Fine alignment and focusing can be performed easily by an initial coarse focus on edge of the window. The drying rate can be controlled by changing the current density. The 2D drying rate can be measured by tracking the size of the evolving drying patches. However, it is difficult to quantify the drying rate in terms of volume.</li>
		<li>Open the original TEM images using the ImageJ software package. Select the multi-point icon of the program and assign the centers of all the individual nanoparticles of the image, and then extract the x and y coordinates of the selected particles.</li>
		<li>Calculate the radial distribution function (RDF): 
		<img alt="Equation 1" src="/files/ftp_upload/56335/56335eq1.jpg" /> 
		where r is the interparticle distance, N is the number of particles, A is the area covered by the particles, &#961; is 2D areal density of particles (N/A), <img alt="Equation 2" src="/files/ftp_upload/56335/56335eq2.jpg" /> is the position vector from particle j to particle k, and &#948;(r) is the Dirac delta function. We use&#160;<img alt="Equation 3" src="/files/ftp_upload/56335/56335eq3.jpg" /> where a = 0.8 nm as the Dirac delta function for the realistic calculation.</li>
	</ol>
	</li>
</ol>

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G., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+Dynamic+Processes+of+Materials+in+Liquid+Using+Transmission+Electron+Microscopy.">Revealing Dynamic Processes of Materials in Liquid Using Transmission Electron Microscopy.</a> J. Vis. Exp. (70), e50122 (2012).</li><li>Hermannsd&#246;rfer, J., de Jonge, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+Dynamic+Processes+of+Nano-sized+Objects+in+Liquid+using+Scanning+Transmission+Electron+Microscopy.">Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy.</a> J. Vis. Exp. (120), e54943 (2017).</li><li>Tsung, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-10+nm+Platinum+Nanocrystals+with+Size+and+Shape+Control:+Catalytic+Study+for+Ethylene+and+Pyrrole+Hydrogenation.">Sub-10 nm Platinum Nanocrystals with Size and Shape Control: Catalytic Study for Ethylene and Pyrrole Hydrogenation.</a> J. Am. Chem. Soc. 131, 5816-5822 (2009).</li><li>Cho, K. S., Talapin, D. V., Gaschler, W., Murray, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+PbSe+Nanowires+and+Nanorings+through+Oriented+Attachment+of+Nanoparticles.">Designing PbSe Nanowires and Nanorings through Oriented Attachment of Nanoparticles.</a> J. Am. Chem. Soc. 127, 7140-7147 (2005).</li><li>Manthiram, K., Beberwyck, B. J., Talapin, D. V., Alivisatos, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeded+Synthesis+of+CdSe/CdS+Rod+and+Tetrapod+Nanocrystals.">Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals.</a> J. Vis. Exp. (82), e50731 (2013).</li><li>Woehl, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Procedures+to+Mitigate+Electron+Beam+Induced+Artifacts+During+in+situ+Fluid+Imaging+of+Nanomaterials.">Experimental Procedures to Mitigate Electron Beam Induced Artifacts During in situ Fluid Imaging of Nanomaterials.</a> Ultramicroscopy. 127, 53-63 (2013).</li><li>Shin, D., 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=Growth+Dynamics+and+Gas+Transport+Mechanism+of+Nanobubbles+in+Graphene+Liquid+Cells.">Growth Dynamics and Gas Transport Mechanism of Nanobubbles in Graphene Liquid Cells.</a> Nat. Commun. 6, 6068 (2015).</li></ol>

The authors have nothing to disclose.

Platinum nanoparticles with a size of 7 nm were synthesized via the reduction of ammonium hexachloroplatinate (IV) and ammonium tetrachloroplatinate (II) using poly (vinylpyrrolidone) (PVP) as a ligand and ethylene glycol as a solvent and a reducing agent27. A ligand-exchange reaction with oleylamine was performed to disperse the particles in a hydrophobic solvent. Lead selenide nanoparticles were synthesized via the thermal decomposition of lead-oleate complexes using TOP-Se as a selenium source<...

The self-assembly of colloidal nanoparticles is of interest because it provides an opportunity to access collective physical properties of individual nanoparticles11. One of the most effective methods of self-assembly used in practical device-scale applications is self-organization of nanoparticles on a substrate through evaporation of a volatile solvent6,7,8,9,10,11. This solvent evaporation method is a nonequilibrium process, which is largely influenced by kinetic factors such as evaporation rate and changes in nanoparticle-substrate interactions. However, since it is difficult to estimate and control the kinetic factors, the mechanistic understanding of nanoparticle self-assembly by solvent evaporation is not fully mature. Although in situ X-ray scattering studies have provided ensemble-averaged information of the nonequilibrium nanoparticle self-assembly process12,13,14, this technique cannot determine the motion of individual nanoparticles, and their association with the overall trajectory cannot be easily accessed.
Liquid-cell TEM is an emerging tool for tracking the trajectory of individual nanoparticles, enabling us to understand the inhomogeneity of nanoparticle motions and their contribution to ensemble behaviors15,16,17,18,19,20,21,22,23,24,25,26. We have previously used liquid-cell TEM to track the motion of individual nanoparticles during solvent evaporation, showing that the movement of the solvent boundary is a major driving force for inducing nanoparticle self-assembly on a substrate18,19. Herein, we introduce experiments where we can observe the process of nanoparticle self-assembly using liquid-cell TEM. First, we provide protocols for the synthesis of platinum and lead selenide nanoparticles, before introducing the fabrication procedures of liquid-cells for TEM and how to load nanoparticles into the liquid-cell. As representative results, we show snapshot images from TEM movies of nanoparticle self-assembly driven by solvent drying. By tracking individual particles in these movies, we can understand the detailed mechanisms of solvent-drying-mediated self-assembly at a single nanoparticle level. During self-assembly, the platinum nanoparticles on the silicon nitride window mainly follow the movement of the evaporating solvent front because of the strong capillary forces acting on the thin solvent layer. Similar phenomena were also observed for other nanoparticles (lead selenide) and substrates (silicon), indicating that the capillary force of the solvent front is an important factor in particle migration near a substrate.

<table><tbody><tr><td>ammonium hexachloroplatinate (IV)</td><td>Sigma-Aldrich</td><td>204021</td><td></td></tr><tr><td>ammonium tetrachloroplatinate (II)</td><td>Sigma-Aldrich</td><td>206105</td><td></td></tr><tr><td>tetramethylammonium bromide, 98%</td><td>Sigma-Aldrich</td><td>195758</td><td></td></tr><tr><td>poly(vinylpyrrolidone) powder</td><td>Sigma-Aldrich</td><td>234257</td><td>Mw ~29,000</td></tr><tr><td>ethylene glycol, anhydrous, 99.8%</td><td>Sigma-Aldrich</td><td>324558</td><td></td></tr><tr><td>n-hexane, anhydrous, 95%</td><td>Samchun Chem.</td><td>H0114</td><td></td></tr><tr><td>ethanol, anhydrous, 99.5%</td><td>Sigma-Aldrich</td><td>459836</td><td></td></tr><tr><td>oleylamine, 70%</td><td>Sigma-Aldrich</td><td>O7805</td><td>Technical grade</td></tr><tr><td>lead(II) acetate trihydrate, 99.99%</td><td>Sigma-Aldrich</td><td>467863</td><td></td></tr><tr><td>oleic acid, 90%</td><td>Sigma-Aldrich</td><td>364525</td><td>Technical grade</td></tr><tr><td>diphenyl ether, 99%</td><td>Sigma-Aldrich</td><td>P24101</td><td>ReagentPlus</td></tr><tr><td>selenium powder, 99.99%</td><td>Sigma-Aldrich</td><td>229865</td><td></td></tr><tr><td>tri-n-octylphosphine, 97%</td><td>Strem</td><td>15-6655</td><td>Air sensistive</td></tr><tr><td>Toluene, anhydrous, 99.9%</td><td>Samchun Chem.</td><td>T2419</td><td></td></tr><tr><td>acetone 99.8%</td><td>Daejung Chem.</td><td>1009-2304</td><td></td></tr><tr><td>potassium hydroxide, 95%</td><td>Samchun Chem.</td><td>P0925</td><td></td></tr><tr><td>p-type silicon-on-insulator wafers</td><td>Soitec</td><td>Power-SOI</td><td>for liquid cells with silicon windows</td></tr><tr><td>tetramethylammonium hydroxide, 25% in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>J.T.Baker</td><td>02-002-109</td><td></td></tr><tr><td>AZ 5214 E</td><td>AZ Electronic Materials</td><td>AZ 5214 E</td><td>Positive photorest</td></tr><tr><td>AZ-327</td><td>AZ Electronic Materials</td><td>AZ-327</td><td>AZ 5214 develper</td></tr><tr><td>indium pellets 99.98-99.99%</td><td>Kurt J. Lesker Company</td><td>EVMIN40EXEB</td><td>thermal evaporator target</td></tr><tr><td>1,2-dichlorobenzene, &gt;99%</td><td>TCI</td><td>D1116</td><td></td></tr><tr><td>pentadecane, &gt;99%</td><td>Sigma-Aldrich</td><td>P3406</td><td></td></tr><tr><td>buffered oxide etch 7:1</td><td>microchemicals</td><td>BOE 7-1 VLSI</td><td></td></tr><tr><td>phosphoric acid, 85%</td><td>Samchun Chem.</td><td>P0449</td><td></td></tr></tbody></table>

liquid-cell transmission electron microscopy for tracking self-assembly of nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

The liquid-cell is composed of a top chip and a bottom chip, which are equipped with silicon nitride windows that are transparent to an electron beam with a thickness of 25 nm. The top chip has a reservoir for storing the sample solution and evaporated solvent. The chips are made via conventional microfabrication processing25. The masks used for the top and bottom chips are shown in Figure 1a and 1b, r...

Watch this Scientific Journal Video about Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles at JoVE.com

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

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Research

JoVE Journal

Chemistry

12.6K Views.  Institute for Basic Science (IBS). Hier introduceren we een experimentele protocollen voor het real-time opvolgen van een zelf-assemblage proces met behulp van vloeistof-cel transmissie-elektronenmicroscopie.

Video: Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

Engineering

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Biochemistry

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Bioengineering

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes.

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Studying the Effects of Temperature on the Nucleation and Growth of Nanoparticles by Liquid-Cell Transmission Electron Microscopy

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron microscopy. In this paper, we describe how to prepare an in situ heating experiment for studying the effect of temperature on the formation of gold nanoparticles driven by radiolysis in water. The protocol of the experiment is fairly simple involving a special liquid cell with uniform heating capabilities up to 100 &#176;C, a liquid-cell TEM holder with flow capabilities and an integrated interface for controlling the temperature. We show that the nucleation and growth mechanisms of gold nanoparticles are drastically impacted by the temperature in liquid cell. Using STEM imaging and nanodiffraction, the evolution of the density, size, shape and atomic structure of the growing nanoparticles are revealed in real time. Automated image processing algorithms are exploited to extract useful quantitative data from video sequences, such as the nucleation and growth rates of nanoparticles. This approach provides new inputs for understanding the complex physico-chemical processes at play during the liquid-phase synthesis of nanomaterials.

Temperature control during liquid-phase electron microscopy experiments opens up new perspectives of studying the dynamic of nanoparticles in liquid environments mimicking their formation or application media. Using recently developed heating liquid cells, we directly observed the influence of temperature on the nucleation and growth processes of gold nanoparticles in water.

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Direct Synthesis of Gold Nanoparticles for Protein Localization Analysis

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).
Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.
A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

Author Spotlight: Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure  

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various ...

Biology

TEM Workflows, Results Analysis, and Data Management Using a Machine-Vision Approach

A Machine-Vision Approach to Transmission Electron Microscopy Workflows, Results Analysis and Data Management

Transmission electron microscopy (TEM) enables users to study materials at their fundamental, atomic scale. Complex experiments routinely generate thousands of images with numerous parameters that require time-consuming and complicated analysis. AXON synchronicity is a machine-vision synchronization (MVS) software solution designed to address the pain points inherent to TEM studies. Once installed on the microscope, it enables the continuous synchronization of images and metadata generated by the microscope, detector, and in situ systems during an experiment. This connectivity enables the application of machine-vision algorithms that apply a combination of spatial, beam, and digital corrections to center and track a region of interest within the field of view and provide immediate image stabilization. In addition to the substantial improvement in resolution afforded by such stabilization, metadata synchronization enables the application of computational and image analysis algorithms that calculate&#8239;variables between images. This calculated metadata can be used to analyze trends or identify key areas of interest within a dataset, leading to new insights and the development of more sophisticated machine-vision capabilities in the future. One such module that builds on this calculated metadata is dose calibration and management. The dose module provides state-of-the-art calibration, tracking, and management of both the electron fluence (e-/&#197;2&#183;s-1) and cumulative dose (e-/&#197;2) that is delivered to specific areas of the sample on a pixel-by-pixel basis. This enables a comprehensive overview of the interaction between the electron beam and the sample. Experiment analysis is streamlined through a dedicated analysis software in which datasets consisting of images and corresponding metadata are easily visualized, sorted, filtered, and exported. Combined, these tools facilitate efficient collaborations and experimental analysis, encourage data mining and enhance the microscopy experience.

Author Spotlight: A Machine-Vision Approach to Transmission Electron Microscopy Workflows, Results Analysis and Data Management  

Here, we present a protocol to utilize machine-vision software to stabilize dynamic processes during TEM imaging, while concurrently indexing multiple streams of metadata to each image into a navigable timeline. We demonstrate how this platform enables automated calibration and mapping of the electron dose over the course of an experiment.

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

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