This work was supported by NSF award number DMR-1610126.

1. Hot exfoliation of 2-D materials
NOTE: This procedure is done inside a glovebox.
<ol>
	<li>Tape Preparation
	<ol>
		<li>Cut a length of tape (see Table of Materials) that is &#8776;5-10 cm long and &#8776;2 cm wide. Place it, sticky side up, on the working area. Fold the ends of the tape for easier handling.</li>
		<li>Using tweezers, deposit the desired material approximately one-quarter of the way down the length of the tape by repeatedly pressing the material into the tape.</li>
		<li>Further distribute the material by folding the tape in half, sticking it to itself, and pulling it apart so that the material covers an area of at least 1 cm2. Depending on the material, repeat this multiple times: 1-2 times for black phosphorus, or several times for graphite or hexagonal boron nitride.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Using the desired method, such as a carbide-tipped scribe, cleave an oxidized silicon wafer or other desired substrate into chips suitable for the experiment, &#8804;1 cm wide. Clean the chips by sonicating for 2 min in acetone, followed by isopropanol (IPA), at relatively low power (we used 12 W). Blow the chips dry with N2.</li>
		<li>Using the prepared tape, firmly press the deposited material onto the substrate. Apply firm pressure with a thumb or gently press with tweezers so the material contacts the chip as much as possible</li>
		<li>Place the tape with substrate (substrate side down) on a hotplate at 120 &#176;C for 2 min.</li>
		<li>Allow the substrate to cool to RT and carefully remove it from the tape. Soak in acetone for 20 min to remove tape residue. Rinse with IPA for 30 s and dry the substrate with nitrogen. Depending on the material, further options for cleaning may be available, such as a forming gas anneal.</li>
	</ol>
	</li>
</ol>
2. Hermetic transfer cell construction, operation, and maintenance
<ol>
	<li>Construction
	<ol>
		<li>Construct the cell (Figure 2) out of the desired material (we used aluminum). It is 30 mm in diameter and 17.6 mm tall when closed. Fabrication drawings are available at&#160;http://churchill-lab.com/useful-things.</li>
		<li>Make the base 16.2 mm tall with a raised sample platform that is threaded with &#190; - 10 threads with a vent cut into the threads. Where the cap meets the base, make an inset for an O-ring (see Table of Materials).</li>
		<li>Make the cap 8.6 mm tall with matching female threads through the center.</li>
		<li>Recess the cap by 0.2 mm to accommodate a 24 mm diameter x 0.1 mm thick coverglass window (here, borosilicate glass).</li>
		<li>Apply a small amount of vacuum grease to the all sides of the O-ring and drop it into the base inset.</li>
		<li>Before affixing the window to the cap of the cell, clean the cap in acetone and IPA to remove any oil or debris left by the machining process.</li>
		<li>Attach the window to the cell cap using epoxy. Thoroughly mix the epoxy according to the manufacturer&#39;s specifications. Here, parts A and B are combined in a 1:1.8 ratio by weight.</li>
		<li>Apply a small amount of epoxy to the recessed area on the cap and spread it around as evenly as possible.</li>
		<li>Set a 0.1 mm thick, 24 mm diameter coverglass (borosilicate glass in this case) into the recess and gently press it into the epoxy. Ensure the window is level with the top of the cap and there that there are no bubbles in the epoxy.</li>
		<li>Wipe up any extra epoxy so that nothing protrudes from the surface of the cap. Allow the epoxy to cure for the manufacturer prescribed time at room temperature.</li>
	</ol>
	</li>
	<li>Operation 
	NOTE: This procedure is done inside a glovebox.
	<ol>
		<li>Using the desired method, affix a prepared sample to the cell base (double sided tape, glue, etc.). The cell is designed to accommodate samples up to 1 cm wide and 0.7 mm thick, including the adhesive.</li>
		<li>Firmly screw the cap onto the base. This makes a seal between the cap and base by compressing the O-ring. Make sure that the pressure inside the transfer cell does not exceed 3 mbar above the ambient pressure.</li>
		<li>Check that the sample sits just below the window. The sample can now be safely removed from the glovebox.</li>
	</ol>
	</li>
	<li>Window repair
	<ol>
		<li>Using tweezers, remove any broken glass that is not firmly affixed to the epoxy. Break up what glass remains (using a carbide-tipped scribe or other method) so that the epoxy beneath is exposed. 
		CAUTION: Wear gloves and eye protection when removing broken glass.</li>
		<li>Soak the cap in a 50:50 mix of acetone and trichloroethylene (TCE) for 1-2 h or until the epoxy softens and begins to separate from the cap. Rinse in IPA for 30 s.</li>
		<li>Peel off any loose epoxy and scrape the remaining epoxy from the surface with a razor blade. Take care not to damage the surface of the cap. Repeat the previous step if necessary.</li>
		<li>Scrub the recessed area with acetone until the surface is clean of any epoxy residue. The cell window can now be replaced following the aforementioned steps.</li>
	</ol>
	</li>
</ol>
3. Example uses of the transfer cell
<ol>
	<li>Optical analysis
	<ol>
		<li>For flake imaging, place the transfer cell under the microscope. The cell can be used with any conventional microscope. When focusing, be careful not to crash the objective into the fragile widow.</li>
		<li>Proceed with desired method for finding material flakes.</li>
	</ol>
	</li>
	<li>Polarized Raman spectroscopy
	<ol>
		<li>For polarization-resolved Raman spectroscopy, align a laser spot to a flake of interest. In this case we use 633 nm wavelength and 50 &#181;W power and a 100x objective lens. For black phosphorus, low laser power is required to prevent damage to the flake.</li>
		<li>Using a half-wave plate, vary the polarization angle.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Hot exfoliation retains the ability of typical mechanical exfoliation to produce pristine thin flakes while also avoiding many downfalls of alternatives. Like typical mechanical exfoliation, this technique is not limited to a small subset of materials. Hot exfoliation can be applied to any material that can be exfoliated using room-temperature mechanical exfoliation as long as the material tolerates heating to 120 &#176;C for 2 min in an inert atmosphere. We also note that it has been shown20 that the heating time and temperature (above 100 &#176;C) do not make any noticeable difference in flake density. Along with increased contact, average flake size can also be improved by increasing the bond strength between the substrate and the flakes. One way to do this would be by treating the substrate with O2 plasma but this would also make the flakes hard or impossible to pick up for use in devices requiring heterostructure fabrication20. 
The transfer cell can be constructed from any suitable metal. We used aluminum because it is easy to machine but it should be noted that TCE (used to remove epoxy) is corrosive to aluminum when unstabilized, heated, or mixed with water. Stainless steel would be more durable and less reactive with TCE. However, we have not seen any corrosive effects using this method at RT. For imaging and analysis with high numerical aperture objectives, construction of the transfer cell is such that, when closed, the bottom of the window is 0.8 mm above the top of the base. With a 0.5 mm thick substrate and 0.1 mm thick adhesive, the sample sits 0.3 mm below the top of the transfer cell. This proximity allows for imaging and analysis with high magnification and relatively short working distance objectives. Exfoliated material can be clearly seen at 5, 20, 50 times magnification allowing for easy identification of thin flakes. At higher magnifications, spherical aberrations caused by the window significantly degrades the image quality. Provided that the sample substrate is less than 0.7 mm thick, there is no risk of over tightening the cell. When the cap is screwed down, excess gas is expelled through the vent in the threads. During construction, the precise location of the vent is not important, but it is important that it is not obstructed by the sample, vacuum grease or anything else. The vent prevents the fragile 0.1 mm thick window from breaking due to overpressure when the cap is screwed down. The window can only withstand pressure changes of a few mbar.
The coverglass window used for the transfer cells is made of borosilicate glass but for optical analysis at wavelengths other than visible to near-infrared, other window materials may be use. For the best imaging, care should be taken when installing the glass window. If not seated properly, the distance between the sample and window could be larger than expected. Especially for small working distance objectives, this could cause the objective to crash into and break the window. Also, some epoxies will cure faster at higher temperatures, but because metals and glass have different thermal expansion coefficients, the widow will deform after cooling back to room temperature. The epoxy should be cured at the same temperature at which it will be used (i.e., if the cell will be used at room temperature), the epoxy should also be cured at room temperature.

Various layered materials that can be exfoliated down to a single atomic layer have generated interest across a broad range of fields. However, investigation and application of many of these materials is complicated by the fact that they are unstable in air and quickly oxidize or hydrate when exposed. For example; black phosphorus is a semiconductor with a tunable direct band gap, high mobility and anisotropic optical and electrical properties1,2,3,4,5 but is unstable in air and will deteriorate in less than an hour6,7 due to interactions with oxygen8. CrI3 has been recently shown to exhibit two-dimensional ferromagnetism9,10,11 but, when exposed to air, it degrades almost instantly11.
Devices made from these materials can be protected from air by working in a glovebox and encapsulating them in a chemically inert material such as hexagonal boron nitride12,13. However, when developing these devices, it is often necessary to identify and analyze the flakes before encapsulation. This analysis requires either removing the sample from the inert environment of the glovebox or putting the analysis equipment in the glovebox. Removing the sample, even for a short time, risks damage via oxidation or hydration, while placing the necessary equipment inside a glovebox can be costly and cumbersome. To remedy this, we designed a hermetic transfer cell that safely encloses a sample, keeping it in an inert environment, so that it can be removed from the glovebox. While in the transfer cell, a sample sits 0.3 mm below a glass window to allow easy identification of flakes under a microscope as well as the use of optical analysis techniques such as photoluminescence or Raman spectroscopy.
Some two-dimensional materials, in addition to being air sensitive, are also difficult to exfoliate into thin flakes with the typical mechanical exfoliation method because a relatively high cleavage energy, relatively weak in-plane bonds, or both. Other methods, such as CVD growth14,15, liquid exfoliation16, or gold mediated exfoliation17,18 have been developed for producing thin layers but may result in less than pristine flakes and only work for certain materials. Although exfoliation of graphene at elevated temperatures has been known to produce large flakes for at least a decade19, this technique has been quantitatively characterized recently for both graphene and Bi2Sr2CaCu2Ox flakes20. Here, we demonstrate that hot exfoliation improves exfoliation yield also for black phosphorus, a material that is notoriously difficult to exfoliate. This technique, together with a hermetic transfer cell, facilitates the exfoliation and analysis of air sensitive, two-dimensional materials.

<table><tbody><tr><td>Ablestik 286 epoxy</td><td>Loctite</td><td>256 6 OZ TUBE KIT</td><td>air-tight epoxy</td></tr><tr><td>Acetone</td><td>EDM Millipore Corporation</td><td>67-64-1</td><td></td></tr><tr><td>Circular coverglass, 24 mm dia, 0 thickness</td><td>Agar Scientific</td><td>AGL46R22-0</td><td>window glass</td></tr><tr><td>Dicing tape</td><td>Ultron systems</td><td>1009R</td><td>exfoliation tape</td></tr><tr><td>High-Vacuum grease</td><td>Dow Corning</td><td>1597418</td><td>O-ring grease</td></tr><tr><td>Isopropanol</td><td>VWR Chemicals</td><td>BDH20880.400</td><td></td></tr><tr><td>Silicon wafer, 300 nm oxide</td><td>University Wafer</td><td>E0851.01</td><td>flake substrate</td></tr><tr><td>Silicon wafer, 90 nm oxide</td><td>Nova Electronic Materials</td><td>HS39626-OX</td><td>flake substrate</td></tr></tbody></table>

exfoliation and analysis of large-area, air-sensitive two-dimensional materials

We describe methods for producing and analyzing large, thin flakes of air-sensitive two-dimensional materials. Thin flakes of layered or van der Waals crystals are produced using mechanical exfoliation, in which layers are peeled off a bulk crystal using adhesive tape. This method produces high-quality flakes, but they are often small and can be hard to find, particularly for materials with relatively high cleavage energies such as black phosphorus. By heating the substrate and the tape, two-dimensional material adhesion to the substrate is promoted, and the flake yield can be increased by up to a factor of ten. After exfoliation, it is necessary to image or otherwise analyze these flakes but some two-dimensional materials are sensitive to oxygen or water and will degrade when exposed air. We have designed and tested a hermetic transfer cell to temporarily maintain the inert environment of a glovebox so that air-sensitive flakes can be imaged and analyzed with minimal degradation. The compact design of the transfer cell is such that optical analysis of sensitive materials can be performed outside of a glovebox without specialized equipment or modifications to existing equipment.

The goal of exfoliating two-dimensional materials is to isolate atomically thin layers. During the exfoliation process, flakes separate from the bulk crystal, leaving behind flakes of varying thicknesses, with a small probability for some flakes to be monolayers. By increasing the density and size of all exfoliated flakes, hot exfoliation increases the density and lateral size of thin flakes. This is accomplished by increasing the material area that makes close contact with the substrate. While in contact, gasses trapped between the material and the substrate expand during heating and are pushed out from under flakes. The removal of trapped gas allows more of the material to come into close contact with the substrate, thus increasing the amount of exfoliated flakes (Figure 1A,B) as clearly explained in Ref 20. Exfoliations of black phosphorus were performed using the typical mechanical exfoliation and hot exfoliation technique on silicon chips with 90 nm thick SiO2. By measuring the total area of deposited material on a 1 cm x 1 cm silicon chip, it can be seen (Figure 1C) that hot exfoliation deposits 6-10 times more material. We note that in our experience other materials can be picked up from HF-cleaned substrates using polycarbonate following hot exfoliation, including graphene, hexagonal boron nitride, black phosphorus, MnPSe3, and WSe2. We used a 10:1 HF:water solution to clean the SiO2 substrates over a 15 s period. Note, 10% HF etches SiO2 at a rate of 23 nm/min21 , so this process etches our substrates by 6 nm.
We now consider the effectiveness of the hermetic transfer cell (Figure 3A) in maintaining an inert atmosphere when removed from a glovebox. CrI3 is particularly sensitive to hydration and degrades within seconds when exposed to air (Figure 3D). Inside a transfer cell, however, an exfoliated CrI3 sample remained unchanged for 15 hours (Figure 3B) and only began to show signs of degradation (blisters) after 24 hours (Figure 3C). While damage on a scale too small observe optically likely occurs on a shorter timescale, these results demonstrate that the hermetic transfer cell described here slows the sample degradation rate by at least three orders of magnitude (hours inside the cell compared with seconds outside).
To demonstrate use of the transfer cell for optical analysis of air-sensitive materials, we performed polarization-resolved Raman spectroscopy on a relatively thick (&#62; 50 nm) flake of black phosphorus (Figure 4A). The spectra were acquired using 50 &#181;W laser excitation at 632.8 nm with a 100x objective lens. A half-wave plate was used to rotate the polarization of the excitation beam. In Figure 4B, three Raman peaks can be observed in BP at around 466, 438 and 361&#8197;cm&#8722;1, corresponding to Ag2, B2g and Ag1 vibration modes respectively, regardless of the polarization, which agrees well with previous observations in bulk BP crystals for excitation and collection along the z-axis.5,22 The peak positions do not vary with polarization angle. However, the relative intensities of these three modes change significantly with incident light polarization. The vibration mode Ag2, which has the strongest intensity variation with the excitation laser polarization, as shown in Figure 4B,C, is associated with the atomic motion along the armchair direction. Therefore, as previously reported5, this vibration mode provides an effective method to determine the armchair direction of the BP crystal and hence the crystal orientation. In Figure 4C, the Raman intensity shows two maxima within one full rotation, located at 26.5&#176; and 206.5&#176; with respect to the X and Y axes defined in the microscope images, and we conclude that the armchair direction of the BP is oriented at 26.5&#176; for this flake. Similar optical spectroscopy methods can be used to determine crystal orientation and other properties, such as layer number or optical band gap, for other air-sensitive 2-D materials.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58693/58693fig1.jpg" /> 
Figure 1: Distribution of material on an&#160;oxidized silicon chip. (A) Typical sample of black phosphorus exfoliated at room temperature. (B) Typical sample of black phosphorus exfoliated at 120 &#176;C. (C) Histogram of exfoliated black phosphorus area using room temperature(cold) and hot exfoliation. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58693/58693fig1large.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/58693/58693fig2.jpg" /> 
Figure 2: Transfer cell. (A) Picture of a hermetic transfer cell showing separate cap and base. (B) Schematic drawing of transfer. A vent (green) is cut into the threads. Note that the bottom of the base is tapped and threaded for mounting. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58693/58693fig2large.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/58693/58693fig3.jpg" /> 
Figure 3: Transfer cell suppression of flake degradation. (A) Fresh CrI3 in a transfer cell (B) CrI3 in a cell after 15 h. (C) CrI3 in a cell after 24 h Hydration blisters can be seen at this point. (D) CrI3 in air after 24 h in transfer cell and 30 s in air. Large areas of hydrated CrI3 have collected at the edges of the flake. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58693/58693fig3large.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/58693/58693fig4.jpg" /> 
Figure 4: Crystalline orientation identification. (A) Optical micrograph of thick flakes of exfoliated BP. (B) Polarization-resolved Raman spectroscopy of a thick BP flake. (C) Polar plot of Raman intensity averaged over the spectral range in (B) as a function of linear excitation polarization angle (plot origin is zero intensity). The fit is a sine function plus a constant. The dashed line indicates the armchair direction.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58693/58693fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Exfoliation and Analysis of Large-area, Air-Sensitive Two-Dimensional Materials

A method for exfoliating large thin flakes of air sensitive two-dimensional materials and safely transporting them for analysis outside of a glovebox is presented.

We describe methods for producing and analyzing large, thin flakes of air-sensitive two-dimensional materials. Thin flakes of layered or van der Waals ...

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11.4K Views.  University of Arkansas. A method for exfoliating large thin flakes of air sensitive two-dimensional materials and safely transporting them for analysis outside of a glovebox is presented. 

Video: Exfoliation and Analysis of Large-area, Air-Sensitive Two-Dimensional Materials

The Evolution of Silica Nanoparticle-polyester Coatings on Surfaces Exposed to Sunlight

Corrosion of metallic surfaces is prevalent in the environment and is of great concern in many areas, including the military, transport, aviation, building and food industries, amongst others. Polyester and coatings containing both polyester and silica nanoparticles (SiO2NPs) have been widely used to protect steel substrata from corrosion. In this study, we utilized X-ray photoelectron spectroscopy, attenuated total reflection infrared micro-spectroscopy, water contact angle measurements, optical profiling and atomic force microscopy to provide an insight into how exposure to sunlight can cause changes in the micro- and nanoscale integrity of the coatings. No significant change in surface micro-topography was detected using optical profilometry, however, statistically significant nanoscale changes to the surface were detected using atomic force microscopy. Analysis of the X-ray photoelectron spectroscopy and attenuated total reflection infrared micro-spectroscopy data revealed that degradation of the ester groups had occurred through exposure to ultraviolet light to form COO&#903;, -H2C&#903;, -O&#903;, -CO&#903; radicals. During the degradation process, CO and CO2 were also produced.

Two types of surfaces, polyester-coated steel and polyester coated with a layer of silica nanoparticles, were studied. Both surfaces were exposed to sunlight, which was found to cause substantial changes in the chemistry and nanoscale topography of the surface.

Corrosion of metallic surfaces is prevalent in the environment and is of great concern in many areas, including the military, transport, aviation, ...

Engineering

Preparation of Liquid-exfoliated Transition Metal Dichalcogenide Nanosheets with Controlled Size and Thickness: A State of the Art Protocol

We summarize recent advances in the production of liquid-exfoliated transition metal dichalcogenide (TMD) nanosheets with controlled size and thickness. Layered crystals of molybdenum disulphide (MoS2) and tungsten disulphide (WS2) are exfoliated in aqueous surfactant solution by sonication. This yields highly polydisperse mixtures containing nanosheets with broad size and thickness distributions. However, for most purposes, specific sizes (in terms of both lateral dimension and thickness) are required. For example, large and thin nanosheets are desired for (opto) electronic applications, while laterally small nanosheets are interesting for catalytic applications. Therefore, post-exfoliation size selection is an important step that we address here. We provide a detailed protocol on the efficient size selection in large quantities by liquid cascade centrifugation and the size and thickness quantification by statistical microscopic analysis (atomic force microscopy and transmission electron microscopy). The comparison of MoS2 and WS2 shows that both materials are size-selected in a similar way by the same procedure. Importantly, the dispersions of size-selected nanosheets show systematic changes in their optical extinction spectra with size due to edge and confinement effects. We show how these optical changes are related quantitatively to the nanosheets dimensions and describe how mean nanosheets length and layer number can be extracted reliably from the extinction spectra. The exfoliation and size selection protocol can be applied to a broad range of layered crystals as we have previously demonstrated for graphene, gallium sulphide (GaS) and black phosphorus.

A protocol for the liquid exfoliation of layered materials to nanosheets, their size selection and size measurement by microscopic and spectroscopic techniques is presented.

We summarize recent advances in the production of liquid-exfoliated transition metal dichalcogenide (TMD) nanosheets with controlled size and thickness. ...

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 ...

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

We describe a beamline setup meant to carry out rapid two-dimensional x-ray fluorescence and x-ray microdiffraction mapping of single crystal or powder samples using either Laue (polychromatic radiation) or powder (monochromatic radiation) diffraction. The resulting maps give information about strain, orientation, phase distribution, and plastic deformation.

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Bioengineering

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.

The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

Biochemistry

Characterizing Spherulites with Polarization-Sensitive Two-Photon Microscopy

Polarization-Sensitive Two-Photon Microscopy for a Label-Free Amyloid Structural Characterization

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

Author Spotlight: Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy 

This paper describes how polarization-sensitive two-photon microscopy could be applied to characterize the local organization within label-free amyloid superstructures-spherulites. It also describes how to prepare and measure the sample, assemble the required setup, and analyze the data to obtain information about the local organization of amyloid fibrils.

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Biology

Using a VSFG Microscope for Multimodal, Rapid, Hyperspectral Chemical Analysis

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 &#181;m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 &#181;m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

Author Spotlight: Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures 

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.