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>HAuCl4 &#183; 3 H2O 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 &#181;m, 4&#34;, 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/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/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/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/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/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/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/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/files/ftp_upload/59751/Supplementary_Video_1.avi">Please click here to download this file.</a>

Watch this Scientific Journal Video about Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy at JoVE.com

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

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

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. ...

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9.9K Views.  Friedrich-Alexander University Erlangen-Nürnberg. A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

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

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 ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

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 ...

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.

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 ...

Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a nanometric scale. EFTEM tomography can separate chemical elements that are very difficult to distinguish using other imaging techniques. The experimental protocol described here shows how to create 3D chemical maps to understand the chemical distribution and morphology of a material. Sample preparation steps for data segmentation are presented. This protocol permits the 3D distribution analysis of chemical elements in a nanometric sample. However, it should be noted that currently, the 3D chemical maps can only be generated for samples that are not beam sensitive, since the recording of filtered images requires long exposure times to an intense electron beam. The protocol was applied to quantify the chemical distribution of the components of two different heterogeneous catalyst supports. In the first study, the chemical distribution of aluminum and titanium in titania-alumina supports was analyzed. The samples were prepared using the swing-pH method. In the second, the chemical distribution of aluminum and silicon in silica-alumina supports that were prepared using the sol-powder and mechanical mixture methods was examined.

This paper describes a protocol to achieve 3D chemical maps combining energy filtered imaging and electron tomography. The chemical distribution of two catalyst supports formed by elements that are difficult to distinguish by other imaging techniques was studied. Each application consists of mapping overlapped chemical elements - respectively spaced-ionization edges.

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

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 ...