The authors acknowledge Dr. Michael J. Friedlander, Director of the Virginia Tech Carilion Research Institute for encouraging our research endeavors. This project was supported by development funds to S.M.M and D.F.K. and in part by the Nano-Bio initiative of the Institute for Critical Technology and Applied Science at Virginia Tech.

1. Prepare Affinity Capture Devices16
<ol>
	<li>Clean the silicon nitride E-chips by incubating them in 15&#160;ml of acetone for 2 min followed by 15&#160;ml of methanol&#160;for 2 min (Figure&#160;1A). Allow chips to dry under laminar air-flow.</li>
	<li>Incubate the dried chips on a heated stir plate (without stirring) for 1.5 hr at 150 &#176;C, then allow them to cool to room temperature before use.</li>
	<li>Use Hamilton syringes to compose lipid mixtures in small glass tubes to contain 25% chloroform, 55% DLPC (1,2-dilauroyl-phosphocholine) in chloroform (1 mg/ml) and 20% Ni-NTA lipid (1,2-dioleoyl-iminodiacetic acid-succinyl-nickel salt) in chloroform (1 mg/ml) for a total volume of 40 &#956;l.</li>
	<li>Apply a 1&#160;&#956;l aliquot of the mixture over each 15&#160;&#956;l drop of Milli-Q water on a piece of Parafilm in a humid Petri dish (Figure&#160;1B). Incubate samples on ice for at least 60 min.</li>
</ol>
2. Capture Macromolecules17
<ol>
	<li>Place an E-chip with a 150&#160;nm integrated spacer on top of a monolayer sample and incubate for 1 min (Figure&#160;1C).</li>
	<li>Gently lift the chip off of the sample and add a 3&#160;&#956;l aliquot of His-tagged Protein A. Incubate for 1 min at room temperature (Figure&#160;1D).</li>
	<li>Blot away the excess drop using Whatman #1 filter paper and immediately add a 3&#160;&#956;l aliquot of antibody solution. Incubate for 1 min at room temperature.</li>
	<li>Remove the excess solution using a Hamilton syringe and immediately add a 1&#160;&#956;l aliquot of&#160;rotavirus DLPs (0.1 mg/ml) in buffer solution containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl2 and 10 mM CaCl2. Incubate for at least 2 min at room temperature. The preparation of DLPs has been described previously18.</li>
</ol>
3. Assemble the Microfluidic Chamber and Load the In situ Specimen Holder
<ol>
	<li>Load the wet E-chip containing the viral sample into the tip of the microfluidic specimen holder. Glow-discharge a second flat E-chip for 1 min then loaded on top of the spacer chip (Figure&#160;2, panels 1-5).</li>
	<li>Alternatively, to increase the contrast of biological macromolecules, heavy metal stain (e.g.&#160;0.2% uranyl formate) can be added to wet specimens prior to placing the second E-chip on the wet specimen. However, the E-chip containing the specimen will need to be washed with Milli-Q water prior to adding the contrast reagent.</li>
	<li>Sandwich the entire assembly together to form a sealed enclosure, held in place mechanically within the holder by 3 brass screws (Figure&#160;2, panels 6-8).</li>
	<li>Following assembly, the tip of the holder is pumped to 10-6 Torr using a turbo-pumped dry pumping station before placing the holder inside the TEM.</li>
</ol>
4. Imaging in Liquid Using a Transmission Electron Microscope
<ol>
	<li>Load the&#160;in situ specimen holder into a Transmission Electron Microscope (FEI Company) equipped with a LaB6 filament and operating at 120 kV.</li>
	<li>Turn on the TEM filament and adjust the eucentric height of the microscope stage with respect to the specimen by using the wobbler function to tilt the sample from -15&#176; to +15&#176; back and forth in the column. This procedure adjusts the stage in the z-direction to help account for the proper thickness of the liquid chamber. This step also ensures an accurate magnification is used when recording images.</li>
	<li>Record images along the edges and in the corner regions of the microfluidic chamber first. These areas typically contain the thinnest solution. Record images of specimens under low-dose conditions (1-3 electrons/&#197;2) using a CCD camera. Use nominal magnifications of 6,000X - 30,000X.</li>
	<li>Determine the proper defocus value by focusing at the edge of fluidic chamber. Use a value of -1.5 &#956;m to record images at 30,000X magnification. If thick solution is encountered or if contrast agent is not used in the specimen preparation, use higher defocus values in the range of -2 to -4 &#956;m.</li>
	<li>To ensure solution is contained in the microfluidic chamber throughout the experiments, focus the electron beam until the bubbles are formed in the liquid within the device.</li>
</ol>

<ol>
	<li>Zhou, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+atomic+resolution+structural+determination+by+single-particle+cryo-electron+microscopy.">Towards atomic resolution structural determination by single-particle cryo-electron microscopy.</a> Curr. Opin. Struct. Biol. 18, 218-228 (2008).</li><li>Wolf, M., Garcea, R. L., Grigorieff, N., Harrison, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+interactions+in+bovine+papillomavirus.">Subunit interactions in bovine papillomavirus.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 6298-6303 (2010).</li><li>Dubochet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+of+vitrified+specimens.">Cryo-electron microscopy of vitrified specimens.</a> Q. Rev. Biophys. 21, 129-228 (1988).</li><li>Unwin, P. N., Henderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+structure+determination+by+electron+microscopy+of+unstained+crystalline+specimens.">Molecular structure determination by electron microscopy of unstained crystalline specimens.</a> J. Mol. Biol. 94, 425-440 (1975).</li><li>Ohi, M., Li, Y., Cheng, Y., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Negative Staining and Image Classification - Powerful Tools in Modern Electron Microscopy. Biol. Proced. Online. 6, 23-34 (2004).</li><li>Adrian, M., Dubochet, J., Fuller, S. D., Harris, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-negative+staining.">Cryo-negative staining.</a> Micron. 29, 145-160 (1998).</li><li>Parsons, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+wet+specimens+in+electron+microscopy.+Improved+environmental+chambers+make+it+possible+to+examine+wet+specimens+easily.">Structure of wet specimens in electron microscopy. Improved environmental chambers make it possible to examine wet specimens easily.</a> Science. 186, 407-414 (1974).</li><li>Parsons, D. F., Matricardi, V. R., Moretz, R. C., Turner, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+and+diffraction+of+wet+unstained+and+unfixed+biological+objects.">Electron microscopy and diffraction of wet unstained and unfixed biological objects.</a> Adv. Biol. Med. Phys. 15, 161-270 (1974).</li><li>Hui, S. W., Parsons, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+diffraction+of+wet+biological+membranes.">Electron diffraction of wet biological membranes.</a> Science. 184, 77-78 (1974).</li><li>Hui, S. W., Parsons, D. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+viral+assemblies+in+a+nanoscale+biosphere.">Visualizing viral assemblies in a nanoscale biosphere.</a> Lab Chip. 13, 216-219 (2013).</li><li>Bican, P., Cohen, J., Charpilienne, A., Scherrer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+bovine+rotavirus+cores.">Purification and characterization of bovine rotavirus cores.</a> J. Virol. 43, 1113-1117 (1982).</li><li>Frank, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPIDER+and+WEB:+processing+and+visualization+of+images+in+3D+electron+microscopy+and+related+fields.">SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields.</a> J. Struct. Biol. 116, 190-199 (1996).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-atomic+resolution+using+electron+cryomicroscopy+and+single-particle+reconstruction.">Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 1867-1872 (2008).</li><li>Scheres, S. 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The author, Madeline J. Dukes, is an employee of Protochips, Inc.

In our presented work, we employed the affinity capture approach to tether rotavirus DLPs to a microfluidic platform. This allowed for in situ imaging of macromolecular complexes in a liquid microenvironment. The capture approach is significant with respect to other microfluidic imaging techniques because it localizes biological specimens to the imaging window to negate large diffusive issues that arise while recording images in liquid. However, one of the most critical steps in our protoc...

<a href="https://app.jove.com/t/6605">This corrects</a> the article <a href="https://dx.doi.org/10.3791/50936">10.3791/50936</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/50936">In situ TEM of Biological Assemblies in Liquid</a>. The Representative Results and References sections were&#160;updated.

Erratum: In situ TEM of Biological Assemblies in Liquid

A common goal of biologists and engineers is to understand the inner-workings of molecular machines. Transmission Electron Microscopes (TEMs) are ideal instruments to visualize these intricate details at near-atomic resolution1-2. In order to sustain the high vacuum system of a TEM, biological samples are typically embedded in thin films of vitreous ice3, sugars4, heavy metal salts5, or some combination thereof6. As a result, images of embedded specimens may reveal only limited snapshots of dynamic processes.
Early attempts to maintain biological specimens hydrated in environmental liquid chambers were undertaken by Parsons and colleagues using differential pumping stages. Electron diffraction patterns of unstained catalase crystals were successfully recorded to a resolution of 3&#160;&#197; in a hydrated state7-8. In addition, phase-separated lipid domains could be examined in hydrated membranes of human erythrocytes9-10. However, motion caused by diffusing liquid and its interference with the electron beam, resulted in severe resolution loss and further experiments using biological specimens were not attempted until recently.
Newly developed microfluidic specimen holders have been introduced that utilize semiconductor microchips to form a micro-scaled environmental chamber. These devices can maintain samples in liquid while they are positioned in a TEM column11-12. This technical breakthrough in TEM imaging has allowed researchers to view, for the first time, progressive events at the molecular level13. We refer to this new modality as &#8220;in situ molecular microscopy&#8221; as experiments can now be performed &#8220;inside&#8221; the EM column14-15. The overall goal of this method is to image biological assemblies in liquid in order to observe their dynamic behaviors at nanometer resolution. The rationale behind the developed technique is to record real-time observations and examine new properties of biological machinery in solution. This methodology expands the use of TEMs for broader purposes in cellular and molecular biology12-16.
In the current video article, we present a comprehensive protocol to assemble and use a commercially available microfluidic specimen holder. These specialized holders utilize silicon nitride microchips produced with integrated spacers to form a liquid chamber that encloses minute volumes of solution. Thin, transparent windows are etched into the microchips for imaging purposes12. We demonstrate the proper use of a microfluidic holder to examine simian rotavirus double-layered particles (DLPs) in liquid using a TEM. To ensure that biological assemblies, such as DLPs, do not rapidly diffuse over great distances while imaging, we employ the Affinity Capture approach to tether them to the surface of the microfluidic chamber16. This molecular capture step has a major advantage over alternative techniques for imaging biological specimens in liquid because it allows for the acquisition of images that will be used for downstream processing routines. This capture step used in conjunction with microfluidic imaging is unique to our procedures17. Readers employing structural biology applications using TEM or microfluidic imaging chambers may consider the use of affinity capture techniques when dynamic observations at the molecular level are the end goal.

<table border="0" cellpadding="0" cellspacing="0" width="832">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">E-chips, spacer chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPB-52TBD</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">E-chip, top chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPT-45W</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ni-NTA lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">790404P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DLPC (12:0) lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">850335P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Volumetric flasks&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">20-812A; 20-812C</td>
			<td style="width:167px;">1 ml; 5 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Hamilton Syringes</td>
			<td style="width:183px;">Hamilton Co.</td>
			<td style="width:245px;">80300, 80400</td>
			<td style="width:167px;">1-10 &#956;l; 1-25 &#956;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Whatman #1 filter paper</td>
			<td style="width:183px;">Whatman</td>
			<td style="width:245px;">1001 090</td>
			<td style="width:167px;">100 pieces, 90 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Petri dishes</td>
			<td style="width:183px;">Corning&#160;</td>
			<td style="width:245px;">70165-101</td>
			<td style="width:167px;">100 mm x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Pasteur pipettes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">14673-010</td>
			<td style="width:167px;">14673-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass culture tubes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">47729-566</td>
			<td style="width:167px;">6 mm x 50 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Acetone</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A11-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Methanol</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A412-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Chloroform&#160;</td>
			<td style="width:183px;">Electron Microcopy Sciences</td>
			<td style="width:245px;">12550</td>
			<td style="width:167px;">100 ml&#160;</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">His-tagged Protein A</td>
			<td style="width:183px;">Abcam, Inc.</td>
			<td style="width:245px;">ab52953</td>
			<td style="width:167px;">10 mg</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Milli-Q water system</td>
			<td style="width:183px;">EMD Millipore Corp.</td>
			<td style="width:245px;">Z00QSV001</td>
			<td style="width:167px;">Ultrapure Water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">HEPES</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">BP310-500</td>
			<td style="width:167px;">500 g</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Equipment&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:245px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Poseidon In situ specimen holder</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;FEI compatible</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">FEI Spirit BioTwin TEM</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>120 kV</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Eagle 2k HS CCD camera</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>10&#160;&#197;/pixel sampling at 30,000X</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">Gatan 655 Dry pump station</td>
			<td style="width:183px;">Gatan, Inc.</td>
			<td style="width:245px;"></td>
			<td>Pump holder tip to 10-6&#160;range</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">PELCO easiGlow, glow discharge unit</td>
			<td style="width:183px;">Ted Pella, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Isotemp heated stir plate</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;"></td>
			<td>Heat to 150 &#186;C for 1.5 hr</td>
		</tr>
	</tbody>
</table>

in situ tem of biological assemblies in liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

Representative images of DLPs in liquid using E-chips that were glow-discharged (Figure&#160;3A) show fewer DLPs in a given viewing area, presumably due to diffusion, in comparison to DLPs that are enriched on Affinity Capture devices (Figure&#160;3B). The addition of uranyl formate in the imaging chamber enhances the contrast of the specimen and hence the visibility of individual DLPs in solution (Figure&#160;3C, top panel). Better contrast allows for downstream image p...

Watch this Scientific Journal Video about In situ TEM of Biological Assemblies in Liquid at JoVE.com

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

in-situ-tem-of-biological-assemblies-in-liquid

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Bioengineering

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Video: In situ TEM of Biological Assemblies in Liquid

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

Engineering

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes.

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a ...

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 ...

Chemistry

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

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.

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.

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

Biology

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

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

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

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

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

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

Biochemistry

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Immunology and Infection

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...