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://www.jove.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://www.jove.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://www.jove.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://www.jove.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

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Bioengineering

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.
Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of &gt; 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.
Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.

The use of energy dispersive X-ray tomography in the scanning transmission electron microscope to characterize elemental distributions within single nanoparticles in three dimensions is described.

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial ...

Engineering

Comprehensive Characterization of Extended Defects in Semiconductor Materials by a Scanning Electron Microscope

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications of semiconductor materials. However, it is still under debate how the defect structure determines the band structure, and therefore, the recombination behavior of electron-hole pairs responsible for the optical and electrical properties of the extended defects. The present paper is a survey of procedures for the spatially resolved investigation of structural and of physical properties of extended defects in semiconductor materials with a scanning electron microscope (SEM). Representative examples are given for crystalline silicon. The luminescence behavior of extended defects can be investigated by cathodoluminescence (CL) measurements. They are particularly valuable because spectrally and spatially resolved information can be obtained simultaneously. For silicon, with an indirect electronic band structure, CL measurements should be carried out at low temperatures down to 5 K due to the low fraction of radiative recombination processes in comparison to non-radiative transitions at room temperature. For the study of the electrical properties of extended defects, the electron beam induced current (EBIC) technique can be applied. The EBIC image reflects the local distribution of defects due to the increased charge-carrier recombination in their vicinity. The procedure for EBIC investigations is described for measurements at room temperature and at low temperatures. Internal strain fields arising from extended defects can be determined quantitatively by cross-correlation electron backscatter diffraction (ccEBSD). This method is challenging because of the necessary preparation of the sample surface and because of the quality of the diffraction patterns which are recorded during the mapping of the sample. The spatial resolution of the three experimental techniques is compared.

The optical, electrical, and structural properties of dislocations and of grain boundaries in semiconductor materials can be determined by experiments performed in a scanning electron microscope. Electron microscopy has been used to investigate cathodoluminescence, electron beam induced current, and diffraction of backscattered electrons.

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications ...

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. ...

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 ...

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 ...

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.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Immunology and Infection

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Biology

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...