We thank Tilo Schmutzler for the preparation of the HAuCl4 solution. Furthermore, we thank R. Christian Martens for proof reading. Financial support by the German Research Foundation (DFG) via the Research Training Group GRK 1896 &#8220;In situ microscopy with electrons, X-rays and scanning probes&#8221; and through the Cluster of Excellence EXC 315/2 EAM &#8220;Engineering of Advanced Materials&#8221; is gratefully acknowledged.

1. Fabrication of microwell-based liquid cell templates
<ol>
	<li>Remove organic residues and native oxide layers from a 175 &#181;m thick, single crystalline, boron-doped (1 &#8211; 30) &#8486;cm, 100 mm diameter (100) silicon wafer. Apply an oxidation step with H2O2 and TMAH, followed by a HF dip in 1 &#8211; 5% HF solution.</li>
	<li>Thermally oxidize the wafer in a dry oxygen atmosphere at 800 &#176;C to grow an oxide layer with a thickness of 11 nm (Figure 2a). 3% Dichlorethene (DCE) is used to bind metallic contamination.</li>
	<li>Deposit a stoichiometric Si3N4 layer via low-pressure chemical vapor deposition (LPCVD). The Si3N4 layer thickness defines the well depth. Choose a value suited for the planned experiment (e.g., 500 nm) (Figure 2b).</li>
	<li>Define the lateral well geometry by structuring the front side via photolithography and reactive ion etching (RIE) (Figure 2c). Suitable dimensions are for example circular structures with 2.5 &#181;m radius arranged in hexagonal arrays. Choose the well distance carefully (for example, 5 &#181;m), to avoid instabilities in the structure.</li>
	<li>Deposit another 20 nm of stoichiometric Si3N4 by LPCVD, which forms the bottom membrane of the liquid cell (Figure 2d). Follow the procedure described above (see step 1.3).</li>
	<li>Use a second photolithography/RIE step to structure the backside which later defines the geometrical dimensions of the LC frame and its TEM windows (Figure 2e) (Frame diameter: 3 mm).</li>
	<li>Via bulk-micromachining in 20% KOH at 60 &#176;C, remove the Si in the predefined area and create a free-standing Si3N4 membrane (Figure 2f).</li>
	<li>Remove residual metal ions in a final cleaning step with 10% HCl solution and deionized (DI) water.</li>
</ol>
2. Transfer of graphene onto TEM grids
<ol>
	<li>Wet the tissue on which the commercially acquired few-layer (6 &#8211; 8) CVD-graphene on PMMA is placed. Immerse the PMMA-coated graphene in a Petri dish filled with DI water (Figure 3a). 
	NOTE: The number of layers of graphene can be determined using feasible techniques32,33.</li>
	<li>Place the graphene layer on a filter paper and cut it into pieces suitable to cover all fabricated wells (e.g., 4 mm&#178;) (Figure 3b).</li>
	<li>Re-immerse the cut pieces into the Petri dish (Figure 3c).</li>
	<li>Use a TEM grid coated with a support layer of holey carbon to fish the produced pieces out of the DI water. To do so, carefully dive the grid into the water and catch the graphene floating on the surface. Hold the grid with anti-capillary tweezers (Figure 3d,e). 
	NOTE: Take care that the graphene site of the graphene-PMMA stack stays on top during the whole procedure. Otherwise, the subsequent PMMA-removal will lift-off the graphene layer.</li>
	<li>Let the sheets dry for a few hours.</li>
	<li>Remove the PMMA protection layer in an acetone bath for 30 min and consecutively add further cleaning steps by immersing in ethanol and DI water without drying the sample in between. Use a flat vessel (e.g., a Petri dish) to simplify the specimen transfer afterwards.</li>
	<li>Dry the sample afterwards for 30 min at ambient conditions.</li>
</ol>
3. Specimen preparation
<ol>
	<li>Prepare the specimen for incorporation in the GSMLC. To do so, prepare a 1 mM stock solution by solving 196.915 mg of HAuCl4&#183;3H2O crystals in 0.5 L of DI water.</li>
	<li>Take the desired amount of specimen from the stock solution. Here, 0.5&#160;&#181;L is applied. This can be done by using a syringe or an Eppendorf pipette.</li>
</ol>
4. GSMLC loading
<ol>
	<li>Rinse the fabricated liquid cell template with acetone and ethanol.</li>
	<li>Apply an ambient O2/N2 (20%/80%) plasma for 5 min to enhance the wettability of the membrane.</li>
	<li>Dispense 0.5&#160;&#181;L of specimen solution on the template or the graphene layer. Ensure a smooth working procedure to minimize changes in concentration due to evaporation.</li>
	<li>Place the TEM grid onto the micro-patterned Si3N4 layer with the graphene facing the template. Press the graphene-coated TEM grid onto the template. Be careful not to destroy the bottom Si3N4 membrane.</li>
	<li>Remove excess solution with a tissue to accelerate the cell drying and thus mitigate concentration changes (Figure 4a). After approx. 2 &#8211; 3 min, the graphene-Si3N4 van-der-Waals interaction sufficiently seal the liquid cell (Figure 4b). Alternatively, leave the cell to dry out completely without removing the excess solution. The latter offers a higher success rate in the cell processing. However, evaporation-based concentration changes in the specimen solution are expected to be more severe when using this approach. 
	NOTE: The successful drying process can be verified with a contrast change in the periphery (compare Figure 4a,b).</li>
	<li>Carefully remove the TEM grid with a tweezer by pushing a tweezer tip between the grid and the GSMLC frame. 
	NOTE: Rash movements might break the underlying membrane. To reduce shear force damage, start from the grid site parallel to the smaller window edge.</li>
	<li>Check, whether at least one membrane of the GSMLC is still intact via optical microscopy (Figure 4c). If all membranes were broken, LCTEM would be impossible.</li>
</ol>
5. TEM Imaging and video analysis
<ol>
	<li>Load the sample to a (S)TEM directly after preparation using a standard TEM holder. 
	NOTE: As it is reported for GLCs19, GSMLCs can dry out over time. Therefore, the time between loading and imaging should be minimized.</li>
	<li>Image the sample with a suitable imaging technique, depending on both the sample and microscope. Here, a (S)TEM device operated at an acceleration voltage of 300 kV is utilized. Use a low dose to minimize beam-induced artifacts and a short exposure time to avoid movement-related blurring34. In case of long-term experiments, block the beam to reduce radiation damage. 
	NOTE: Due to a better temporal resolution, TEM is to be preferred over STEM for kinetic analyses34&#160;and reduced ion reduction35. STEM, however, is preferred for investigation into thick liquid layers and high-Z elements due to its higher spatial resolution in thick specimens34,35.</li>
	<li>Image segmentation method
	<ol>
		<li>Use a suitable image processing platform to extract features of interest. For particle tracking and analysis, use the open source ImageJ-distribution FIJI36.</li>
		<li>Utilize the Analyze Particles function to gain precise information (projected area, barycenter) of every particle in each frame. 
		NOTE: This function requires binary images.</li>
		<li>Connect the particles between the frames with the help of the plugin TrackMate37. By default, TrackMate is searching for bright particles on a dark background, so invert the images (in the case of BF-TEM) before starting TrackMate.</li>
		<li>Combine the results of TrackMate and Analyze Particles with a suitable script utilizing the Python-based open source ecosystem SciPy38,39.</li>
		<li>Use FIJI to extract the precise contours of more complex structures such as dendrites. Here, Analyze Particles can be applied, as well (see inset of Figure 6a). 
		NOTE: It might be feasible to analyze features of interest manually.</li>
	</ol>
	</li>
</ol>

We have nothing to disclose.

In contrast to commercially available liquid cells, custom-made GSMLCs have the advantage that they can be designed to fit into readily available TEM holders and do not require an expensive, dedicated liquid cell TEM holder.
The GSMLC architecture demonstrated here combines aspects of SiLCs and GLCs that could potentially lead to unique advantages. On the one hand, SiLCs allow for a precise determination of cell position and shape, but require relatively thick Si3N4 membranes to reduce bulging effects while ultimately reducing the achievable resolution. GLCs, on the other hand, exhibit exceptionally thin membrane walls consisting of graphene, yet suffer from random pocket sizes and positions. By combining these two membrane approaches via GSMLCs, the resolution limitation caused by the cell boundaries35&#160;can be bypassed. As the well structure is fabricated directly into the Si3N4 layer, the actual Si3N4 membrane can be constructed even smaller than in SiLCs, simplifying HRTEM analyses which has already been demonstrated in GSMLCs6. Still, it should be noted that HRTEM in general is possible with SiLCs as well48. Moreover, large viewing areas can be realized without severe window bulging due to the small membrane areas of the individual specimen chambers. Thereby, bulging-related thickness increase35&#160;can be ruled out to a large extent, as shown by Dukes et al.49. This is demonstrated in Figure 7, where a representative high-angle annular dark field (HAADF) STEM image of al loaded GSMLC is displayed. This image was acquired using a Dual-beam system. Since the image brightness acquired in this setup is directly related to the specimen thickness, it is clearly visible that the sealed microwells exhibit only small negative bulging. Kelly et al.24 have demonstrated that the negative bulging and partial well drying visible in Figure 7 depends on the well diameter. Reducing the well diameter is therefore a feasible approach to homogenize the liquid thickness even further.
Due to the equilibrium pocket shape of GLCs, the liquid thickness is also strongly site-dependent35. SiLCs follow the design of two membranes stemming from different Si wafers. By replacing the top Si3N4 membrane with graphene, liquid-cell fabrication is simplified. This means that possible delamination of two bonded Si-wafers during the subsequent wet etching steps can be avoided and the alignment of two wafer pieces during the cell loading is omitted. The flat surface on one side of this cell architecture enables complementary in situ analysis methods such as EDXS analysis of the specimen6, which is restricted in conventional SiLC architectures by shadowing effects at steep Si edges50.
Sealing prepatterned microwells with graphene on both the bottom and top well site has been demonstrated before24,25. Applying two graphene membranes may enhance the achievable resolution. A twofold graphene transfer, however, would complicate the preparation process further; especially since this has proven to be the most sensitive preparation step (see below). Furthermore, the above discussed membrane bulging is expected to be even more critical in case of two graphene membranes, because graphene is much more flexible than a Si3N4 layer. In those architectures, the microwells were constructed using sequential focused ion beam (FIB) milling. While this approach has proven to yield high-quality results, FIB milling is complicated and expensive cell production technique. Utilizing massively parallel single-shot patterning techniques that are already standard in today&#8217;s semiconductor industry such as nanoimprint- or photolithography, however, has the major advantage of being fast, cheap and scalable for mass production.
It should be noted that the approach presented here does not allow for liquid flow operation, which is achievable by other designs28. Since the loading and liquid volume are comparable for GSMLCs and GLCs, a contamination of high vacuum due to rupture of the membrane can be avoided19. This eliminates the need for a cumbersome seal check. Though the advantages of SiLCs and GLCs have been combined, the disadvantages of both approaches are still present in GSMLCs. The fabrication of the cells requires a clean room infrastructure for silicon technology, which is not necessarily present in TEM laboratories. In addition, the liquid loading is not trivial. It requires a dedicated training, similar to graphene cells. This, however, is also true for commercially available systems. Here, the most sensitive preparation step is the TEM-grid removal after the graphene transfer because rash movements or jittering is likely to break the Si3N4 layer. The redundant membrane windows, however, enhance the chances of preserving at least one membrane area. As a consequence, the yield (amount of operable GSMLC chips) achieved by a trained experimenter is three out of four6, and thus exceeds the one achieved with graphene-based cells (one to two out of four)19.
As with GLCs, the liquid encapsulation in GSMLCs is based on van-der-Waals interactions18. Consequently, interface contamination could lower the success rate in processing of GSMLCs19. Furthermore, depending on the Hamaker constant of the to-be-encapsulated liquid phase, the wetting characteristics during the loading procedure (and thus the achievable yield) may differ51&#160;and therefore the preparation can be complicated. Our experience shows that this is the case if, for example, amphiphilic species are present.
The GSMLC architecture enables flexible configuration of well-depths, allowing for adaptation to various experimental prerequisites. Moreover, the architecture is suitable for electron tomography investigations over a broad tilt-angle range of &#177;75 &#176;, which would also allow for in situ electron tomography52. Therefore, in situ and post mortem tomography of specimen in liquid could also be established with GSMLCs.

Modern materials science, chemistry and cell biology require a deep understanding of underlying dynamic processes and effects at the sub-micron scale. Despite the power of advanced optical microscopy techniques such as stimulated-emission-depletion fluorescence microscopy1, direct imaging techniques to access detailed morphologies require electron microscopy. In particular, in situ (scanning) transmission electron microscopy (S)TEM has been shown to illuminate valuable insights into process dynamics by encapsulating liquids in dedicated, vacuum-tight cells2. Various experiments such as quantitative investigations of nanostructure formation kinetics and thermodynamics3,4,5,6, imaging of biological specimens7,8,9,10 and studies of energy storage-related mechanisms11,12 along with comprehensive studies of corrosion process dynamics13 or nanobubble physics14,15,16 have unraveled many phenomena using (S)TEM that were not accessible using standard microscopy techniques.
During the last decade, two major approaches to realize in situ liquid cell TEM (LCTEM) have been established. In the first approach, the liquid is encapsulated in a cavity between two Si3N4 membranes produced via Si process technology17, whereas in the second, small liquid pockets are formed between two graphene or graphene oxide sheets10,18. The handling of both silicon-based liquid cells (SiLCs) and graphene-based liquid cells (GLCs) has been demonstrated19,20,21. Although both approaches have undergone significant improvements22,23,24,25, they still lack in the combination of the respective advantages. In general, a tradeoff exists between encapsulating the sample in often undefined graphene pockets with a small liquid volume that enables high-resolution imaging18, and well defined cell volumes resulting in thicker membranes and liquid layers, which provide an environment closer to the natural situation in bulk liquid26 at the expense of resolution2. Furthermore, some experiments depend on a liquid flow26,27 which has only been realized in SiLC architectures and requires a dedicated TEM holder28.
Here, we present the fabrication and handling of a liquid cell approach for high-performance in situ LCTEM via static graphene-supported microwell liquid cells (GSMLCs) for TEM analyses. A sketch of the GSMLC is presented in Figure 1. GSMLCs have proven to be capable of enabling in situ high-resolution transmission electron microscopy (HRTEM) results6 and are also feasible for in situ scanning electron microscopy29. Their Si technology-based frame allows for mass production of reproducibly shaped cells with tailored liquid thickness and extra-thin membranes from a single wafer. The graphene membrane covering these cells also mitigates electron beam-induced perturbations8,30,31 since the electron beam passes through the top graphene membrane first. The flat topography of the cells allow for complementary analysis methods such as energy-dispersive X-ray spectroscopy (EDXS)6 without any shadowing effects arising from the liquid cell itself, enabling a variety of high-quality in situ liquid cell electron microscopy experiments.

<table><tbody><tr><td>Acetone</td><td>VWR Chemicals</td><td>50488858</td><td>VLSI</td></tr><tr><td>Deionized water</td><td></td><td></td><td>own production</td></tr><tr><td>Dumont Anti-Capillary tweezers</td><td>Carl Roth GmbH + Co. KG</td><td>LH72.1</td><td>0203-N5AC-PO Dumoxel alloyed</td></tr><tr><td>Ethanol</td><td>VWR Chemicals</td><td>85651.360</td><td>VLSI</td></tr><tr><td>FIJI Is Just ImageJ</td><td>FIJI.sc</td><td>Version 1.51</td><td></td></tr><tr><td>Gold Quantifoil, Amorphous Carbon TEM Grids</td><td>Plano GmbH</td><td>S173-8</td><td>R 2/2 Au 300 mesh</td></tr><tr><td>HAuCl&lt;sub&gt;4&lt;/sub&gt; &amp;middot; 3 H&lt;sub&gt;2&lt;/sub&gt;O crystal</td><td>Alfa Aesar</td><td>36400.06</td><td>5 g</td></tr><tr><td>Jupyter Notebook</td><td>Project Jupyter</td><td>Version 5.7.2</td><td></td></tr><tr><td>Matplotlib-Package</td><td>John Hunter, Darren Dale, Eric Firing, Michael Droettboom and the Matplotlib development team</td><td>Version 3.0.2</td><td></td></tr><tr><td>NumPy-Package</td><td>NumPy developers</td><td>Version 1.15.4</td><td></td></tr><tr><td>Pandas-Package</td><td>AQR Capital Management, LLC, Lambda Foundry, Inc. and PyData Development Team</td><td>Version 0.23.4</td><td></td></tr><tr><td>Python</td><td>Python Software Foundation</td><td>Version 3.7</td><td></td></tr><tr><td>Scipy-Package</td><td>SciPy developers</td><td>Version 1.1.0</td><td></td></tr><tr><td>Seaborn-Package</td><td>Michael Waskom</td><td>Version 0.9.0</td><td></td></tr><tr><td>Si wafer</td><td>Siegert Wafer GmbH</td><td></td><td>Thin silicon (100) wafer 175 +/-5 &amp;micro;m, 4&quot;, p-type, boron doped (1-30 Ohm cm), double-sided polished</td></tr><tr><td>single tilt TEM holder</td><td>Philips</td><td></td><td>Ensure that cell fits</td></tr><tr><td>Transmission Electron Microscope</td><td>Philips</td><td>CM 30 (S)TEM</td><td>300 kV</td></tr><tr><td>Trivial Transfer Graphene</td><td>ACS Material</td><td>TTG60011</td><td>PMMA-covered, 6 -- 8 MLs</td></tr></tbody></table>

preparation of graphene-supported microwell liquid cells for in situ transmission electron microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

After loading of the cell, a successful graphene transfer is indicated by a differently shaded appearance on the wells under an optical microscope. This is visible, for example, in the right membrane of Figure 3c. As mentioned, it is crucial to carefully remove the TEM-grid in order to not break the thin Si3N4 layer. In case of a broken membrane, lucent and curved residuals are clearly visible in the optical microscope, as shown in the left two membranes of Figure 3c. Due to the multiple viewing areas in the utilized GSMLC design, the cell can be used as long as at least one membrane is intact. Broken membranes can be used for TEM alignment without exposing the specimen to the electron beam.
A successful encapsulation of the specimen solution can be verified during electron microscopy. Figure 5 presents individual micrographs of Supplementary Video 1, where the dissolution of an ensemble of nanoparticles and the growth of a dendritic structure is statistically evaluated in a GSMLC. Besides the drift-induced movement of the image, minor individual orthogonal particle movements are visible, indicating that particles in solution are present. Furthermore, the prevalence of particle dissolution proves that a wet-chemical reaction is present which would not be possible without a successful liquid enclosing. Other typical indications for enclosed liquids are beam-induced bubble formation19 or particle motion. The presence of Au particles in graphene-featured cells alone does not conclusively indicate a liquid environment, since the particles could also stem from the graphene-induced reduction of HAuCl440. A quantification of the oxygen peaks of the enclosed liquid via electron energy loss spectroscopy (EELS) can also be performed to verify a liquid environment41.
In order to gain insights into particle growth and dissolution kinetics, it is important to investigate each particle individually rather than to analyze the development of average parameters42. It is also crucial to exclude particles at the frame edges that are only partially captured by the camera because drift effect-related position changes of such particles might be mistaken as growth or dissolution processes. Etching is believed to be caused by oxidative species generated by electron beam-induced radiolysis43. In order to yield sufficient statistics, computational single particle tracking is required. By estimating the growth exponent &#945; of the equivalent radius variation of individual particles over time, information of the underlying reaction kinetics can be obtained. To do so, it is possible to introduce an equivalent radius based on the projected particle area, even if not all particles are completely spherical6,44. Figure 5b shows the tracking of equivalent radii over time for six representative particles which are highlighted in Figure 5a. Figure 5c shows the distribution of &#945; based on 73 dissolving particles from the present study. Only particles where an allometric model explains the radius decline to at least 50% (adjusted coefficient of determination) are regarded.
Furthermore, a dendrite structure emerges rapidly after about 42 s in the same well depicted in Figure 6a. Dendrite formation is another typical, well documented process in liquid cells45,46. To quantify dendrite growth, the structural outlines (see inset in Figure 6a) are analyzed. The evolution of tip radius and velocity over time (see Figure 6b,c) reveals the expected hyperbolic relationship47&#160;(Figure 6d). Dendrite growth is caused by local supersaturation of Au-ions due to the aforementioned particle etching. In Figure 5a, it is clearly visible that particles are still dissolving whilst the oversaturated system relaxes into dendrite growth. This may be caused by local concentration variations in both the Au-ions and the oxidative species as a result of the high viscosity of the liquid in the GSMLC which has been observed before6. A detailed discussion of this phenomenon, however, is beyond the scope of this work.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59751/59751fig1.jpg" /> 
Figure 1: Sketch of a GSMLC: Schematics of the structure of a graphene-supported microwell liquid cell. Reprinted from https://pubs.acs.org/doi/abs/10.1021/acs.nanolett.8b03388&#160;6. Further permissions are to be directed to the American Chemical Society (ACS). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59751/59751fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59751/59751fig2.jpg" /> 
Figure 2: Fabrication of GSMLC-frames. The fabrication process of GSMLC-frames is schematically sketched. (a) Oxidation of Si wafer after cleaning. (b) LPCVD of Si3N4. (c) Front side Si3N4 patterning by photolithography and RIE to define the cell volume. (d) Deposition of Si3N4 to form the bottom cell window. (e) Back-side lithography and RIE. (f) Bulk micromachining with KOH to create a freestanding Si3N4 membrane containing microwells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59751/59751fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59751/59751fig3.jpg" /> 
Figure 3: Transfer of few-layer CVD-graphene with PMMA protection layer onto a TEM-grid. The transfer of few-layer CVD-graphene on PMMA onto the top of a holey carbon-coated TEM-grid is displayed. (a) Immersion of the few-layer CVD graphene on PMMA in a Petri dish filled with DI water. (b) Transferred graphene/PMMA stack on a filter paper is cut into pieces suitable to cover the GSMLC-frames. (c) Re-immersion of a cut graphene/PMMA piece. (d) Transfer of the graphene/PMMA layer onto a holey carbon-coated TEM grid (e) Graphene/PMMA stack after a successful transfer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59751/59751fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59751/59751fig4.jpg" /> 
Figure 4: Removal of the top TEM grid. The drying process of a loaded GSMLC is documented with the help of an optical microscope. (a) A graphene-coated TEM grid is placed on top of the GSMLC directly after loading. The graphene layer is visible as turquoise rectangle covering all three viewing areas. Its outlines are roughly sketched by the black rectangle. (b) An almost completely adhered membrane is visible by the contrast change between the wet (dark, compare with (a)) and the adhered area (turquoise) after approximately two minutes. (c) A GSMLC after the lift-off of the TEM grid is shown, revealing two broken membranes (left and middle), and one membrane with successfully loaded and sealed microwells (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59751/59751fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59751/59751fig5v2.jpg" /> 
Figure 5: Representative development of nanoparticle radii. The radius development of 183 individual particles has been tracked. (a) Image sequence taken from Supplementary Video 1. Six representative particles are highlighted. The colored circles correspond to the obtained equivalent radius. (b) Logarithmic plot of the particle radii. (c) The histogram of 73 particles where a negative allometric exponent &#945; has been determined using an automated routine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59751/59751fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59751/59751fig6.jpg" /> 
Figure 6: Dendrite dynamics: The tip radius of five dendrite branches is analyzed. Error bars account for the respective standard deviation. (a) Image sequence taken from Supplementary Video 1 showing the emerging dendrite, which is visible after about 42 s. The inset in the right image shows the evolving dendrite contours. Here, the pink outlines correspond to 42.09 s, red to 42.7 s, and purple to 43.3 s. (b) Development of the (averaged) tip radius over time. (c) The mean tip velocity plotted over time. (d) The averaged tip radius logarithmically plotted against the averaged tip velocity, revealing a hyperbolic dependency (orange curve). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59751/59751fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59751/59751fig7.jpg" /> 
Figure 7: SEM Image of a loaded GSMLC: A representative SEM image acquired in HAADF STEM mode in an SEM of a loaded GSMLC at low acceleration voltage (29 kV) is displayed. Besides the prominent 5 &#181;m wide microwells, two partially overlapping circular holey carbon grids (2 &#181;m diameter) stemming from the graphene transfer elucidated above is visible. The first carbon grid stems from an unsuccessful graphene transfer. It is clearly visible that the membrane shading stays mostly constant over the well region, but slightly darkens towards the well center. This accounts for weak, negative bulging. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59751/59751fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1: In situ video showing representative results of a liquid cell bright field TEM study of etching of Au nanoparticles and subsequent growth of a dendrite structure caused by supersaturation of the surrounding specimen solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59751/Supplementary_Video_1.avi">Please click here to download this file.</a>

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Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

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

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in liquid environments. This manuscript describes the process for making graphene liquid cells through the example of graphene liquid cell transmission electron microscopy (TEM) experiments of gold nanocrystal etching. The protocol for making graphene liquid cells involves coating gold, holey-carbon TEM grids with chemical vapor deposition graphene and then using those graphene-coated grids to encapsulate liquid between two graphene surfaces. These pockets of liquid, with the nanomaterial of interest, are imaged in the electron microscope to see the dynamics of the nanoscale process, in this case the oxidative etching of gold nanorods. By controlling the electron beam dose rate, which modulates the etching species in the liquid cell, the underlying mechanisms of how atoms are removed from nanocrystals to form different facets and shapes can be better understood. Graphene liquid cell TEM has the advantages of high spatial resolution, compatibility with traditional TEM holders, and low start-up costs for research groups. Current limitations include delicate sample preparation, lack of flow capability, and reliance on electron beam-generated radiolysis products to induce reactions. With further development and control, graphene liquid cell may become a ubiquitous technique in nanomaterials and biology, and is already being used to study mechanisms governing growth, etching, and self-assembly processes of nanomaterials in liquid on the single particle level.

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching

Graphene liquid cell electron microscopy can be used to observe nanocrystal dynamics in a liquid environment with greater spatial resolution than other liquid cell electron microscopy techniques. Etching premade nanocrystals and following their shape using graphene liquid cell Transmission Electron Microscopy can yield important mechanistic information about nanoparticle transformations.

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in ...

Preparation of Graphene Liquid Cells for the Observation of Lithium-ion Battery Material

In this work, we introduce the preparation of graphene liquid cells (GLCs), encapsulating both electrode materials and organic liquid electrolytes between two graphene sheets, and the facile synthesis of one-dimensional nanostructures using electrospinning. The GLC enables in situ transmission electron microscopy (TEM) for the lithiation dynamics of electrode materials. The in situ GLC-TEM using an electron beam for both imaging and lithiation can utilize not only realistic battery electrolytes, but also the high-resolution imaging of various morphological, phase, and interfacial transitions.

Here, we present a protocol for the fabrication and preparation of a graphene liquid cell for in situ transmission electron microscopy observation, along with a synthesis of electrode materials and electrochemical battery cell tests.

In this work, we introduce the preparation of graphene liquid cells (GLCs), encapsulating both electrode materials and organic liquid electrolytes between ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...

Biochemistry

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 ...

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass optical fiber inserted into the electron column above the objective lens polepiece, and a laser, an adjustable light source, was used to fabricate the device. After the illuminator has been calibrated using an external measuring system, it allows one to adjust the intensity of the lighting to the needs of the observed process. This lighting system was utilized to image antimicrobial photodynamic therapy phenomena, which are currently the subject of intense research. The sample was prepared by spotting a suspension of bacteria on a carbon, graphene, or silicon nitride substrate, blotting the excess solution, spotting the photosensitizer solution, blotting the excess liquid again, and then assembling the liquid cell with a second substrate or graphene film. The process of the imaging experiment itself includes choosing the right place for observation with the use of low magnification and a minimum dose of electrons, and then cyclical activation of the light source to capture subsequent images at specified intervals with the minimum amount of electrons necessary. The electron dose of each exposure and the time and intensity of lighting used need to be carefully recorded due to the complexity of the observed phenomena as, at the same time, the process is both light- and electron-driven. After the actual experiment is performed, additional control observations must be made, in which the same doses of electrons are used but without additional light influence and smaller doses of electrons are used for higher doses of light. This makes it possible to distinguish light-induced microstructural effects from those caused by electrons in both the fields of life and materials science.

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The present protocol describes transmission electron microscope (TEM) modifications with a light illumination system, the fabrication of liquid cells, and in situ TEM observations of light-induced interactions between bacterial cells and a photosensitizer. The sample preparation methods, electron beam damage, and imaging are also discussed.

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...

Biology

Coating TEM Grids with a Graphene Monolayer

Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample preparation for cryoEM requires preserving specimens in a thin layer of vitreous ice, typically suspended within the holes of a fenestrated support film. However, all commonly used sample preparation approaches for cryoEM studies expose the specimen to the air-water interface, introducing a strong hydrophobic effect on the specimen that often results in denaturation, aggregation, and complex dissociation. Further, preferred hydrophobic interactions between regions of the specimen and the air-water interface impact the orientations adopted by the macromolecules, resulting in 3D reconstructions with anisotropic directional resolution. 
Adsorption of cryoEM specimens to a monolayer of graphene has been shown to help mitigate interactions with the air-water interface while minimizing the introduction of background noise. Graphene supports also offer the benefit of substantially lowering the required concentration of proteins required for cryoEM imaging. Despite the advantages of these supports, graphene-coated grids are not widely used by the cryoEM community due to the prohibitive expense of commercial options and the challenges associated with large-scale in-house production. This paper describes an efficient method for preparing batches of cryoEM grids that have nearly full coverage of monolayer graphene.

Author Spotlight: Enhancing CryoEM Resolution Using Graphene-Coated Grids 

Here, we describe a protocol for applying a single monolayer of graphene to electron microscopy grids and how to prepare them for use in cryoEM structure determination.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

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