This work is funded by the National Key R&amp;D Program under Grant No. 2016YFA0300901 2016YFA0300903 and 2017YFA0205003, the National Natural Science Foundation of China under Grant No. 11634001, 11290162/A040106. Y.J. acknowledges support by National Science Fund for Distinguished Young Scholars and Cheung Kong Young Scholar Program. J. G. acknowledges support from the National Postdoctoral Program for Innovative Talents.

NOTE: The experiments are performed on water molecules adsorbed on the Au-supported NaCl(001) film (Figure 1a) at 5 K with an ultrahigh-vacuum (UHV) cryogenic STM equipped with Nanonis electronic controller.
1. Fabrication of Experimental Sample
<ol>
	<li>Clean the Au(111) single crystal
	<ol>
		<li>Pump the gas line to the pressure of ~10-7 mbar and then flush the gas line with Ar gas. Put through the pump/flush cycle for three times. 
		NOTE: Each pump/flush cycle takes about 30 min.</li>
		<li>Fill the gas line with Ar gas to the pressure of 2 bar, thus prohibiting the atmosphere to permeate through the gas line.</li>
		<li>Put the Au(111) crystal on the heater stage, which is mounted on the manipulator in the UHV chamber (base pressure of 1.4&#215;10-10 mbar).</li>
		<li>Clean the Au(111) single crystal by cycles of Ar+ ion sputtering for 15 min (p(Ar) = 5&#215;10&#8722;5 mbar, 1.0 kV, 6 &#181;A) and subsequent annealing at about 900 K for 5 min. 
		NOTE: The annealing temperature should be decreased slowly, otherwise a high density of step edges will form on the Au surface. 3 - 5 sputtering/annealing cycles are usually used.</li>
		<li>Transfer the Au(111) sample to the STM scanning stage, and check the cleanliness with STM (Inset of Figure 1b).</li>
	</ol>
	</li>
	<li>Deposition of NaCl on the Au(111) substrate
	<ol>
		<li>Degas the NaCl source. Slowly increase the current applied on the Knudsen cell until the temperature of the source reaches 670 K. Degas the NaCl source several times until the pressure of the chamber is below 4&#215;10-9 mbar. 
		NOTE: The increasing rate of the current depends on the outgassing rate of the NaCl source to maintain the pressure of the chamber below 1&#215;10-8 mbar.</li>
		<li>Put the Au(111) sample on the manipulator and adjust the position of the Au sample to make the sample face the shutter of the Knudsen cell. 
		NOTE: The temperature of the Au(111) substrate could be decreased below room temperature (77-300 K) by cooling down the manipulator head with a continuous flow of liquid nitrogen</li>
		<li>Increase the current applied on the Knudsen cell until the temperature of the source reaches 640 K, and let the evaporation flux stabilize for 5 min before opening the shutter.</li>
		<li>Open the shutter and deposit the NaCl onto the Au(111) sample held at 290 K for 2 min.</li>
		<li>Transfer the Au-supported NaCl sample to the STM scanning stage. Check the coverage and size of the bilayer NaCl(001) islands on the Au(111) substrate with STM (Figure 1b).</li>
	</ol>
	</li>
	<li>Purify the water under vacuum by freeze-pump-thaw cycles21 to remove remaining impurities.
	<ol>
		<li>Prepare three sealed-off glass-UHV adapters. Place water H2O, D2O, and D2O:HOD:H2O isotopic mixture solutions (2 mL) into three adapters separately, and mount the adapters on the gas line (Figure 2). 
		NOTE: The D2O:HOD:H2O isotopic mixtures can be obtained by mixing the ultrapure H2O and D2O with equal amounts under ultrasonic oscillation for 10 min.</li>
		<li>Freeze the liquid water with liquid nitrogen. Make sure that the gas line is pumped to the pressure of ~10-7 mbar before freezing.</li>
		<li>Open the diaphragm-sealed valve and pump off the atmosphere for 15 min. Then close the diaphragm-sealed valve and thaw the solution. 
		NOTE: Gas bubbles evolve from the solution when it is thawing. 
		CAUTION: Let the frozen solvent thaw by itself. Thawing the solution with water bath may cause the glass vessel to break. To freeze and thaw the solution quickly, replace glass-UHV adapters with metal-UHV adapters, though the solution in the metal vessel is invisible.</li>
		<li>Repeat steps 1.3.2-1.3.3 until no gas bubbles evolve from the solution as the solution thaws. Put through the freeze-pump-thaw cycle at least three times.</li>
		<li>Close the bellows-sealed valve and leave the gas line in vacuum. Then open the diaphragm-sealed valve, and let the water vapor fill in the gas line.</li>
	</ol>
	</li>
	<li>Dose water molecules in situ onto the sample surface
	<ol>
		<li>Decrease the temperature of the sample to 5 K. Open the leak valve slowly to make the pressure of the STM UHV chamber increase to 2&#215;10-10 mbar. 
		NOTE: Water molecules flow into the UHV chamber through the dosing tube, which points to the shutter of the shield. The distance between the shutter and the sample (in the shield) is about 6 cm. The base pressure of STM chamber are better than 7&#215;10-11 mbar. The deposition rate is about 0.01 bilayer min-1.</li>
		<li>Open the shutter. Dose the water molecules onto the Au-supported NaCl surface for 1 min. Then close the shutter and the leak valve.</li>
		<li>Check the coverage of water molecules on the Au-supported NaCl(001) surface with STM. Isolated water monomers form on the sample surface (Figure 1c).</li>
	</ol>
	</li>
</ol>
2. Preparation of the Cl-Terminated Tip
<ol>
	<li>Fabricate an electrochemically etched tungsten (W) tip.
	<ol>
		<li>Place 0.3 mm W wire into a 3 Mol/L NaOH etching solution with an immersion length of about 2 mm.</li>
		<li>Apply a 5 V dc potential to the W wire with respect to a platinum ring electrode inserted into the NaOH solution.</li>
		<li>Stop the etching process when the suspended W wire fell off. Clean the etched W tip with distilled water and ethanol. Then transfer the W tip into the scanner. 
		NOTE: The electrochemically etched W tip can be used for one year before exchange.</li>
	</ol>
	</li>
	<li>Apply voltage pulses (2-10 V) and controlled crashing procedures (0.25-0.4 nm) on the STM tip until the atomic Cl atoms of the NaCl surface are resolved. 
	NOTE: The STM tip is poked on a clean region of the Au(111) surface.</li>
	<li>Position the STM tip over the center of one Cl atom (Figure 3a). Bring the bare STM tip close to the NaCl surface in proximity with the set point of V = 5 mV and I = 5 n A (Figure 3b).</li>
	<li>Retract the tip to original set point (Figure 3c) and scan the same area. Check the obtainment of the Cl-tip by improved resolution and a missing Cl atom in the STM image of NaCl (Figure 3d-e). 
	NOTE: Unsuccessful cases may occasionally occur, for example, when the Cl atom doesn&#39;t transfer to the STM or multiple Cl/Na atoms adsorb on the tip. If this occurs, repeat the steps 2.2-2.5.</li>
</ol>
3. Orbital Imaging of Water Monomer
<ol>
	<li>Shape the STM tip with voltage pulse (2-10 V) and controlled crashing (0.25-0.4 nm) procedures.</li>
	<li>Scan the water molecules adsorbed on the NaCl(001) surface with 10 nm by 10 nm frame at 5 K.</li>
	<li>Focus on one individual water monomer and zoom in. Scan the water monomer in a systematic way as functions of the bias (-400-400 mV) and the tunneling current (50-300 pA). 
	NOTE: With a bare STM tip, the highest occupied (HOMO) and lowest unoccupied (LUMO) molecular orbitals of water appear at positive and negative bias, respectively22. Once the tip is Cl-terminated, only HOMO emerges (Figure 4a), and the LUMO feature is not observed throughout the accessible bias range (from -400 mV to 400 mV). Even under larger bias voltage, the water molecules will be unstable due to vibrational excitation.</li>
</ol>
4. Single-Molecule Vibrational Spectroscopy
<ol>
	<li>Setup of the digital lock-in and bias spectroscopy module (Nanonis electronic controller)
	<ol>
		<li>Setup of bias spectroscopy module: Select the current, LIX1 (dI/dV spectra signal), and LIX2 (d2I/dV2 spectra signal) channels. Set the setting time as 50 ms, and integration time as 300 ms. Increase the integration time and sweep times to obtain smooth spectra. Tune the Z offset to take the bias spectroscopy at different tip heights. Make sure the Z-controller set holds and lock-ins runs during measurement 
		NOTE: Setting time is defined as: the time to wait after changing the bias to the next level and before starting to acquire data to avoid transient effect induced by the bias change. Integration time is defined as: the time during which the data are acquired and averaged.</li>
		<li>Setup of lock-in module 
		NOTE: The scanning tunneling spectroscopy, dI/dV and d2I/dV2 spectra, are acquired simultaneously using a lock-in amplifier by demodulating the first and second harmonics of the tunneling current, respectively.
		<ol>
			<li>Modulate the bias and demodulate the current. Set the modulation frequency as a few hundred Hz and modulation amplitude as 5-7 mV. Make sure there is no mechanical and electronic noise at the set point frequency and the corresponding second harmonic frequency.</li>
			<li>Set the first harmonic phase: Switch to the Z-controller module. Set the tip lift to 10 nm and turn off the feedback. Switch to the lock-in module and turn on the lock-in button (green). Click the first harmonic auto phase and record the phase. Repeat the auto phase at least five times and take the average. Then subtract 90 degrees from the averaged phase to get the phase of the junction.</li>
			<li>Set the second harmonic phase: Position the STM tip on the Au(111) substrate and start the bias spectroscopy sweep from -1.5 V to 1.5 V. Select the channel LIX 1 and function dY/dX, which together show the derivative of dI/dV spectrum. Find a prominent peak feature in the spectrum and set the corresponding energy as the bias. Turn on the lock-in and keep the STM system in tunneling. Click the second harmonic auto phase at least five times and take the average. 
			NOTE: Since the second harmonic signals are usually very weak, the phase can fluctuate wildly. When decreasing the tip height to increase the intensity of the signal, the fluctuation of the phase will be much smaller (a few degrees) and the second harmonic phase will be more accurate.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Tip-enhanced IETS of a D2O monomer
	<ol>
		<li>Scan a water monomer with Cl-tip at the set point of V=100 mV and I=50 pA.</li>
		<li>Position the Cl-tip on the NaCl surface and take the bias spectroscopy as the background signal. Then position the Cl-tip on the water monomer and start the bias spectroscopy sweep.</li>
		<li>If the dI/dV and d2I/dV2 spectra of water are featureless, simply follow the background NaCl surface (blue curves of Figure 4c-d). Decrease the tip height by tuning the Z offset until the vibrational features emerge in the spectra (red curves of Figure 4c-d). 
		&#8203;NOTE: For the IETS measurement, a long integration time (~1s) and multiple sweeps are needed. For a D2O water monomer, set the bias range from -360 mV to 360 mV. For H2O/HOD water monomers, sweep the bias from -475 mV to 475 mV. Comparing with D2O, H2O, and HOD, water monomers are more easily disturbed and even swept away during IETS measurement.</li>
	</ol>
	</li>
	<li>H-bond strength
	<ol>
		<li>Repeat steps 4.2.2-4.2.3 and tune the sweep bias range to focus on the stretching mode of water monomers. The IETS of water D2O, H2O, and HOD are presented and discussed in 23.</li>
		<li>Obtain the H-bonding energy by converting from the redshift of the H-bonded OH stretching frequency (relative to the free OH stretching energy) using this empirical formula: 
		&#916;H = 1.3 &#215; &#8730;&#916;v&#160; &#160; (1) 
		NOTE: &#916;H is the H-bonding energy, in kJ/mol; &#916;v is the redshift of the OH stretching mode, in cm-1. Convert the unit of H-bonding strength to meV by: 1kJ/mol=10.4 meV/atom. To apply Eq. 1 to the OD stretching mode, the quantity &#916;v should be multiplied by a factor: v(OH) / v(OD) = 1.3612, where v(OH) and v(OD) are the OH and OD stretching frequencies of the free HOD molecule, respectively.</li>
	</ol>
	</li>
</ol>
5. Molecular Manipulation
<ol>
	<li>Construction of a water tetramer (Figure 5a)
	<ol>
		<li>Scan an area containing four water monomers. Position the Cl-tip on top of a monomer at the set point of V=100 mV and I=50 pA. Decrease the height to the set point of V=10 mV and I=150 pA to enhance the tip-water interaction.</li>
		<li>Move the Cl-tip along the predesigned trajectories. Then retract the tip to the initial set point (V=100 mV, I=50 pA), and rescan the same area to check that the water dimer is formed.</li>
		<li>Repeat the steps 5.1.1-5.1.2 to form the water trimer and tetramer. 
		NOTE: The above manipulation process could be realized by the Nanonis controller (Scan control-Follow me module). Setup of the Scan control-Follow me module: 
		Bias: 10 mV 
		Speed: 500 pm/s 
		Z-ctrl Setpoint: 150 pA 
		Switch on/off Z-Ctrl: green 
		Time to wait: 1s 
		Current Gain: LN 10^9 
		Path: Click RECORD button and draw up the designed trajectories on the image, then click the STOP button. 
		Click the EXECUTE button and the STM tip will move along the predesigned trajectories with the setpoint in the Follow me module. If the water monomer doesn&#39;t move, decrease the tip height (smaller bias and bigger current) during manipulation.</li>
	</ol>
	</li>
	<li>Chirality switching of a water tetramer (Figure 6)
	<ol>
		<li>Scan a water tetramer with the Cl-tip. Change the set point to V=5 mV, I=5 pA, and position the tip slightly off the center of the water tetramer.</li>
		<li>In the Z-controller module, define a distance to lift the tip when the Z-controller is switched off (e.g., Tip Lift: -230 pm). Turn off the Z-controller feedback. Bring the tip close to the water tetramer (~230 pm).</li>
		<li>Record the current trace, which shows two different levels, indicating that tetramer has undergone reversible interconversion between two H-bonding chirality.</li>
		<li>Leave the current at the high level and switch on the Z-controller feedback. Retract the tip to the original set point (V=5 mV, I=5 pA). Then scan the water tetramer with the set point of V=10 mV and I=100 pA to check the chiral state of the water tetramer.</li>
		<li>Repeat steps 5.2.1-5.2.4 at least 10 times to confirm the corresponding chiral state of water tetramer at the high current level.</li>
		<li>Repeat steps 5.2.1- 5.2.4 at least 10 times, but leave the current at the low level to check the corresponding chiral state of water tetramer.</li>
		<li>Record the tunneling trace for 20 min, which contains a few hundred switching events</li>
		<li>Plot the distribution of the times the tetramer spent in the low and high level of the current trace, respectively.</li>
		<li>Fit the distribution to an exponential decay (Figure 7). Then get the fitted time constant. Inverse the time constant to yield the switching rate.</li>
	</ol>
	</li>
</ol>

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Authors have nothing to disclose.

To probe the internal structure, dynamics, and vibrational spectroscopy of water molecules adsorbed on the solid surfaces, paying particular attention to the degrees of freedom of hydrogen, some experimental steps are of crucial importance, which will be discussed in the following paragraphs.
The orbital imaging of water molecules is achieved based on two key steps. First, the insulating NaCl films decouple the water electronically from the Au substrate, second the orbital gating effect of the...

The interactions of water with the surfaces of solid materials are involved in various surface reaction processes, such as heterogeneous catalysis, photoconversion, electrochemistry, corrosion and lubrication et al.1,2,3 In general, to investigate interfacial water, spectroscopic and diffraction techniques are commonly used, such as infrared and Raman spectroscopy, sum-frequency generation (SFG), X-ray diffraction (XRD), nuclear magnetic resonance (NMR), neutron scattering4,5,6,7,8. However, these methods suffer from the limitation of spatial resolution, spectral broadening, and averaging effects.
STM is a promising technique to overcome these limitations, which combines the sub-&#197;ngstr&#246;m spatial resolution, atomic manipulation, and single-bond vibrational sensitivity9,10,11,12,13,14. Since the beginning of this century, STM has been extensively applied to investigate the structure and dynamics of water on solid surfaces3,15,16,17,18,19,20. Additionally, vibrational spectroscopy based on STM could be obtained from the second-derivative differential tunneling conductance (d2I/dV2), also known as inelastic electron tunneling spectroscopy (IETS). However, resolving the internal structure, i.e. the H-bond directionality, and acquiring reliable vibrational spectroscopy of water are still challenging. The main difficulty lies in that water is a close shell molecule, whose frontier orbitals are far away from the EF, thus the electrons from the STM tip can hardly tunnel into the molecular resonance states of water, leading to the poor signal-to-noise ratio of molecular imaging and vibrational spectroscopy.
Water adsorbed on the Au-supported NaCl(001) films provides an ideal system for atomic-scale investigation by STM with a Cl-terminated tip (Figure 1a), which is performed at 5 K in the ultrahigh-vacuum (UHV) environment with a base pressure better than 8&#215;10-11 mbar. On one hand, the insulating NaCl films decouple water molecules electronically from the Au substrate so the native frontier orbitals of water are preserved and the lifetime of the electrons residing in the molecular resonant state is prolonged. On the other hand, the STM tip could effectively tune the frontier orbital of water toward the EF via tip-water coupling, especially when the tip is functionalized with a Cl atom. These key steps enable high-resolution orbital imaging and vibrational spectroscopy of water monomers and clusters. In addition, water molecules could be manipulated in a well-controlled manner, due to the strong electrostatic interaction between the negatively charged Cl-tip and water.
In this report, the preparation procedures of the sample and the Cl-terminated tip for STM investigation are outlined in detail in section 1 and 2, respectively. In section 3, we describe the orbital imaging technique, by which the O-H directionality of water monomer and tetramer are resolved. The tip-enhanced IETS is introduced in section 4, which allows the detection of vibrational modes of water molecules at single-bond limit, and determination of the H-bonding strength with high accuracy from the red shift in the oxygen-hydrogen stretching frequency of water. In section 5, we show how the water tetramer can be constructed and switched by controlled tip manipulation. Based on the orbital imaging, spectroscopy, and manipulation techniques, isotopic substitution experiments can be performed to probe the quantum nature of protons in interfacial water, such as quantum tunneling and zero-point motion.

<table><tbody><tr><td>Au(111) single crystal</td><td>MaTeck</td><td>NA</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>450006</td><td></td></tr><tr><td>Water, deuterium-depleted&amp;nbsp;</td><td>Sigma Aldrich</td><td>195294</td><td></td></tr><tr><td>Deuterium oxide&amp;nbsp;</td><td>Sigma Aldrich</td><td>364312</td><td></td></tr><tr><td>Sealed-off glass-UHV adapters</td><td>MDC vacuum products</td><td>46300</td><td></td></tr><tr><td>Diaphragm-sealed valve</td><td>any</td><td>NA</td><td></td></tr><tr><td>Bellows-sealed valve</td><td>any</td><td>NA</td><td></td></tr><tr><td>Leak valve</td><td>Kurt J. Lesker&amp;nbsp;</td><td>NA</td><td></td></tr><tr><td>Scanning tunneling microscopy</td><td>CreaTec</td><td>NA</td><td></td></tr><tr><td>Electronic controller.</td><td>Nanonis&amp;nbsp;</td><td>NA</td><td></td></tr><tr><td>Tungsten wire</td><td>any</td><td></td><td>diameter:0.3 mm; purity: 99.95%</td></tr></tbody></table>

probing the structure and dynamics of interfacial water with scanning tunneling microscopy and spectroscopy

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal structure and probing the hydrogen-bond (H-bond) dynamics of the water molecules adsorbed on solid surfaces are fundamental issues of water science, which remains a great challenge owing to the light mass and small size of hydrogen. Scanning tunneling microscopy (STM) is a promising tool for attacking these problems, thanks to its capabilities of sub-&#197;ngstr&#246;m spatial resolution, single-bond vibrational sensitivity, and atomic/molecular manipulation. The designed experimental system consists of a Cl-terminated tip and a sample fabricated by dosing water molecules in situ onto the Au(111)-supported NaCl(001) surfaces. The insulating NaCl films electronically decouple the water from the metal substrates, so the intrinsic frontier orbitals of water molecules are preserved. The Cl-tip facilitates the manipulation of the single water molecules, as well as gating the orbitals of water to the proximity of Fermi level (EF) via tip-water coupling. This paper outlines the detailed methods of submolecular resolution imaging, molecular/atomic manipulation, and single-bond vibrational spectroscopy of interfacial water. These studies open up a new route for investigating the H-bonded systems at the atomic scale.

Figure 1a illustrates the schematic of the STM experimental setup. First, Au(111) substrate is cleaned by sputtering and annealing cycles in the UHV chamber. The clean Au(111) sample shows 22&#215;&#8730;3 reconstructed surface, where the atoms of the surface layer occupy both the hcp and the fcc sites forming herringbone structures (Inset of Figure 1b). The NaCl is evaporated on the Au(111) subs...

Watch this Scientific Journal Video about Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy at JoVE.com

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

Here, we present a protocol to investigate the structure and dynamics of interfacial water at the atomic scale, in terms of submolecular resolution imaging, molecular manipulation, and single-bond vibrational spectroscopy.

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal ...

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Chemical Bonding: Molecular Geometry and Bonding Theories

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8.8K Views.  Peking University. Here, we present a protocol to investigate the structure and dynamics of interfacial water at the atomic scale, in terms of submolecular resolution imaging, molecular manipulation, and single-bond vibrational spectroscopy.

Video: Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

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 ...

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 ...

Engineering

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 ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

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

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

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

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

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various surfaces and interfaces. This non-invasive optical technique can provide the local molecular-level information with monolayer or submonolayer sensitivity. We here are providing experimental methodology on how to selectively detect the buried interface for both macromolecules and biomacromolecules. With this in mind, interfacial secondary structures of silk fibroin and water structures around model short-chain oligonucleotide duplex are discussed. The former shows a chain-chain overlap or spatial confinement effect and the latter shows a protection function against the Ca2+ ions resulting from the chiral spine superstructure of water.

Being comprehensively utilized, sum frequency generation (SFG) vibrational spectroscopy can help to reveal chain conformational order and secondary structural change happening at polymer and biomacromolecule interfaces.

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various ...

Biochemistry

All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics

The miniaturization of semiconductor devices to scales where small numbers of dopants can control device properties requires the development of new techniques capable of characterizing their dynamics. Investigating single dopants requires sub-nanometer spatial resolution, which motivates the use of scanning tunneling microscopy (STM). However, conventional STM is limited to millisecond temporal resolution. Several methods have been developed to overcome this shortcoming, including all-electronic time-resolved STM, which is used in this study to examine dopant dynamics in silicon with nanosecond resolution. The methods presented here are widely accessible and allow for local measurement of a wide variety of dynamics at the atomic scale. A novel time-resolved scanning tunneling spectroscopy technique is presented and used to efficiently search for dynamics.

We demonstrate an all-electronic method to observe nanosecond-resolved charge dynamics of dopant atoms in silicon with a scanning tunneling microscope.

The miniaturization of semiconductor devices to scales where small numbers of dopants can control device properties requires the development of new ...

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 ...

Visualizing Solution Structure at Solid-Liquid Interfaces using Three-Dimensional Fast Force Mapping

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the solution conditions such as ion concentrations, pH, ligands, and trace additives, as well as the underlying crystallography and chemistry. In this context, three-dimensional fast force mapping (3D FFM) has emerged as a promising tool for investigating solution structure at interfaces. This capability is based on atomic force microscopy (AFM) and allows the direct visualization of interfacial regions in three spatial dimensions with sub-nanometer resolution. Here we provide a detailed description of the experimental protocol for acquiring 3D FFM data. The main considerations for optimizing the operating parameters depending on the sample and application are discussed. Moreover, the basic methods for data processing and analysis are discussed, including the transformation of the measured instrument observables into tip-sample force maps that can be linked to the local solution structure. Finally, we shed light on some of the outstanding questions related to 3D FFM data interpretation and how this technique can become a central tool in the repertoire of surface science.

Here, we present a protocol for using three-dimensional fast force mapping - an atomic force microscopy technique - for visualizing solution structure at solid-liquid interfaces with the subnanometer resolution by mapping the tip-sample interactions within the interfacial region.

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the ...

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant adsorption rates, evaluating equilibrium surface tensions as a function of bulk surfactant concentration, and relating how surface tension changes with changes in the interfacial area following equilibration. Simultaneous visualization of the interface using fluorescence imaging with a high-speed confocal microscope allows the direct evaluation of structure-function relationships. In the capillary pressure microtensiometer (CPM), a hemispherical air bubble is pinned at the end of the capillary in a 1 mL volume liquid reservoir. The capillary pressure across the bubble interface is controlled via a commercial microfluidic flow controller that allows for model-based pressure, bubble curvature, or bubble area control based on the Laplace equation. Compared to previous techniques such as the Langmuir trough and pendant drop, the measurement and control precision and response time are greatly enhanced; capillary pressure variations can be applied and controlled in milliseconds. The dynamic response of the bubble interface is visualized via a second optical lens as the bubble expands and contracts. The bubble contour is fit to a circular profile to determine the bubble curvature radius, R, as well as any deviations from circularity that would invalidate the results. The Laplace equation is used to determine the dynamic surface tension of the interface. Following equilibration, small pressure oscillations can be imposed by the computer-controlled microfluidic pump to oscillate the bubble radius (frequencies of 0.001-100 cycles/min) to determine the dilatational modulus The overall dimensions of the system are sufficiently small that the microtensiometer fits under the lens of a high-speed confocal microscope allowing fluorescently tagged chemical species to be quantitatively tracked with submicron lateral resolution.

This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology.

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

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All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics

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