Name: In situ TEM of Biological Assemblies in Liquid (Video) | JoVE Journal
Uploaded: 2013-12-30T20:00:00.000+00:00
Duration: 8 min 28 s
Description: Learn in situ TEM imaging of viral complexes in liquid at nanometer resolution using affinity capture microchips. This method reveals dynamic behaviors of rotavirus particles for structural biology research.

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5 minutes

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>

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<li>Parsons, D. F., Matricardi, V. R., Moretz, R. C., Turner, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+microscopy+and+diffraction+of+wet+unstained+and+unfixed+biological+objects%5BTitle%5D)+AND+%22Adv.+Biol.+Med.+Phys%22%5BJournal%5D)">Electron microscopy and diffraction of wet unstained and unfixed biological objects.</a> Adv. Biol. Med. Phys. 15, 161-270 (1974).</li>
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<li>Hui, S. W., Parsons, D. F., Cowden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+diffraction+of+wet+phospholipid+bilayers%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Electron diffraction of wet phospholipid bilayers.</a> Proc. Natl. Acad. Sci. U.S.A. 71, 5068-5072 (1974).</li>
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<li>Dukes, M. J., Ramachandra, R., Baudoin, J. P., Gray Jerome, W., de Jonge, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+locations+of+gold-labeled+proteins+in+a+whole+mount+eukaryotic+cell+obtained+with+3nm+precision+using+aberration-corrected+scanning+transmission+electron+microscopy%5BTitle%5D)+AND+%22J.+Struct.+Biol%22%5BJournal%5D)">Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy.</a> J. Struct. Biol. 174, 552-562 (2011).</li>
<li>Yuk, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+EM+of+colloidal+nanocrystal+growth+using+graphene+liquid+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">High-resolution EM of colloidal nanocrystal growth using graphene liquid cells.</a> Science. 336, 61-64 (2012).</li>
<li>Peckys, D. B., de Jonge, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+gold+nanoparticle+uptake+in+live+cells+with+liquid+scanning+transmission+electron+microscopy%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy.</a> Nano Lett. 11, 1733-1738 (2011).</li>
<li>Klein, K. L., Anderson, I. M., de Jonge, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transmission+electron+microscopy+with+a+liquid+flow+cell%5BTitle%5D)+AND+%22J.+Microsc%22%5BJournal%5D)">Transmission electron microscopy with a liquid flow cell.</a> J. Microsc. 242, 117-123 (2011).</li>
<li>Degen, K., Dukes, M., Tanner, J. R., Kelly, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+affinity+capture+devices-a+nanoscale+purification+platform+for+biological+in+situ+transmission+electron+microscopy%5BTitle%5D)+AND+%22Rsc.+Adv%22%5BJournal%5D)">The development of affinity capture devices-a nanoscale purification platform for biological in situ transmission electron microscopy.</a> Rsc. Adv. 2, 2408-2412 (2012).</li>
<li>Gilmore, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+viral+assemblies+in+a+nanoscale+biosphere%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">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/pubmed?term=((Purification+and+characterization+of+bovine+rotavirus+cores%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">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/pubmed?term=((SPIDER+and+WEB%3A+processing+and+visualization+of+images+in+3D+electron+microscopy+and+related+fields%5BTitle%5D)+AND+%22J.+Struct.+Biol%22%5BJournal%5D)">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/pubmed?term=((Near-atomic+resolution+using+electron+cryomicroscopy+and+single-particle+reconstruction%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Bayesian+view+on+cryo-EM+structure+determination%5BTitle%5D)+AND+%22J.+Mol.+Biol%22%5BJournal%5D)">A Bayesian view on cryo-EM structure determination.</a> J. Mol. Biol. 415, 406-418 (2012).</li>
<li>Kelly, D. F., Abeyrathne, P. D., Dukovski, D., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Affinity+Grid%3A+a+pre-fabricated+EM+grid+for+monolayer+purification%5BTitle%5D)+AND+%22J.+Mol.+Biol%22%5BJournal%5D)">The Affinity Grid: a pre-fabricated EM grid for monolayer purification.</a> J. Mol. Biol. 382, 423-433 (2008).</li>
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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><tbody><tr><td>E-chips, spacer chip</td><td>Protochips, Inc.</td><td>EPB-52TBD</td><td>400 &amp;mu;m x 50 &amp;mu;m window</td></tr><tr><td>E-chip, top chip</td><td>Protochips, Inc.</td><td>EPT-45W</td><td>400 &amp;mu;m x 50 &amp;mu;m window</td></tr><tr><td>Ni-NTA lipid</td><td>Avanti Polar Lipids</td><td>790404P</td><td>Powder form</td></tr><tr><td>DLPC (12:0) lipid</td><td>Avanti Polar Lipids</td><td>850335P</td><td>Powder form</td></tr><tr><td>Volumetric flasks&amp;nbsp;</td><td>Fisher Scientific</td><td>20-812A; 20-812C</td><td>1 ml; 5 ml&amp;nbsp;</td></tr><tr><td>Hamilton Syringes</td><td>Hamilton Co.</td><td>80300, 80400</td><td>1-10 &amp;mu;l; 1-25 &amp;mu;l</td></tr><tr><td>Whatman #1 filter paper</td><td>Whatman</td><td>1001 090</td><td>100 pieces, 90 mm</td></tr><tr><td>Glass Petri dishes</td><td>Corning</td><td>70165-101</td><td>100 mm x 15 mm</td></tr><tr><td>Glass Pasteur pipettes</td><td>VWR</td><td>14673-010</td><td>14673-010</td></tr><tr><td>Glass culture tubes</td><td>VWR</td><td>47729-566</td><td>6 mm x 50 mm</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A11-1</td><td>1 L</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412-1</td><td>1 L</td></tr><tr><td>Chloroform&amp;nbsp;</td><td>Electron Microcopy Sciences</td><td>12550</td><td>100 ml&amp;nbsp;</td></tr><tr><td>His-tagged Protein A</td><td>Abcam, Inc.</td><td>ab52953</td><td>10 mg</td></tr><tr><td>Milli-Q water system</td><td>EMD Millipore Corp.</td><td>Z00QSV001</td><td>Ultrapure Water</td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td>500 g</td></tr><tr><td>Equipment&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Poseidon &lt;em&gt;In situ&lt;/em&gt; specimen holder</td><td>Protochips, Inc.</td><td></td><td>&amp;nbsp;FEI compatible</td></tr><tr><td>FEI Spirit BioTwin TEM</td><td>FEI Co.</td><td></td><td>120 kV</td></tr><tr><td>Eagle 2k HS CCD camera</td><td>FEI Co.</td><td></td><td>10&amp;nbsp;&amp;Aring;/pixel sampling at 30,000X</td></tr><tr><td>Gatan 655 Dry pump station</td><td>Gatan, Inc.</td><td></td><td>Pump holder tip to 10&lt;sup&gt;-6&amp;nbsp;&lt;/sup&gt;range</td></tr><tr><td>PELCO easiGlow, glow discharge unit</td><td>Ted Pella, Inc.</td><td></td><td>&amp;nbsp;Negative polarity mode</td></tr><tr><td>Isotemp heated stir plate</td><td>Fisher Scientific</td><td></td><td>Heat to 150 &amp;ordm;C for 1.5 hr</td></tr></tbody></table>

in situ tem of biological assemblies in liquid

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 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 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 3C, top panel). Better contrast allows for downstream image processing such ...

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

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

The overall goal of this procedure is to image biological assemblies and liquid in order to observe their dynamic behaviors at nanometer resolution. This is accomplished by first preparing the silicon nitride microchips with an affinity capture surface to enable macromolecules to be tethered to the microchip surface, preventing diffusion in liquid. The second step is to load biological samples onto the microchip in order to recruit rotavirus particles.Next, the microchip containing the virus particles is washed with a contrast agent and then loaded onto the microfluidic specimen holder. The final step is to load the top chip and face plate to form a closed vessel encasing the liquid specimen. Ultimately, images are recorded in the TEM to reveal features of the virus sample within the liquid micro chamber.The main advantage of this technique over other existing methods to examine entities in liquid is that our specimen do not rapidly diffuse over great distances while imaging due, do the affinity capture coating applied to the microchips? The implications of this technique extend towards real-time assessments of nanoparticle based drug delivery systems or vaccine development against viral pathogens because we can see for the first time how dynamic nano machines change in solution. Though this method can provide insight into visualizing biological macromolecules, it can also be applied to other systems such as electrochemistry applications.A visual demonstration of this method is critical. Since preparing affinity capture devices and loading the microfluid specimen holder may present a problem to the novice user. To begin clean the silicon nitride e chips by incubating them in 15 milliliters of acetone for two minutes, followed by 15 milliliters of methanol for two minutes.To remove residual photoresist used in the manufacturing process, allow the chips to quickly dry under laminar airflow. Once dry, incubate the chips on a plate heated to 150 degrees Celsius for 1.5 hours. Then allow them to cool to room temperature.Next, use Hamilton syringes to make up a lipid mixture in a small glass tube. By first adding 25%chloroform, then add 55%DLPC in chloroform at one milligram per milliliter. And finally, using a separate syringe, add 20%nickel NTA lipid in chloroform at one milligram per milliliter for a total volume of 40 microliters.Then cut a piece of param to fit inside a 100 by 15 millimeter glass Petri dish, and add nine 15 microliter drops of Milli Q water on the surface of the param. Apply a one microliter aliquot of the lipid mixture over each 15 microliter drop of Milli Q water and place the plate on ice in a humid environment for at least 60 minutes while a lipid monolayer forms on the surface of the drops. Place an EIP with a 150 nanometer integrated spacer on top of the monolayer droplet and incubate them together for one minute.Next, gently lift the chip off of the droplet and add three microliters of hist tagged protein A at 0.01 milligrams per milliliter directly to the surface of the chip. Incubate the chip and protein A for one minute at room temperature. Then blot away the excess liquid using watman number one filter paper and immediately add three microliters of IgG antibody solution at 0.01 milligrams per milliliter.Incubate the sample for one minute at room temperature. Next, remove the excess solution using a Hamilton syringe and immediately add a one microliter aliquot of rotavirus, double layered particles at 0.1 milligrams per milliliter in a heaps buffer solution, and incubate the chip for at least two minutes at room temperature. A second flat IP is prepared by glow discharging for one minute, assemble the microfluidic chamber using a ST.Stereoscope by first loading the tip of the specimen chamber with O-rings.Then load the wet E IP containing the viral sample into the tip of the microfluidic specimen holder. Next, add the glow discharge flat EIP over the top of the specimen sandwich the entire assembly together to form a sealed enclosure held in place mechanically within the holder by three brass screws. Following assembly, pump the tip of the holder down to 10 to the minus six tor.Using a turbo pump dry pumping station before placing the holder inside the TEM, load the I two specimen holder into a transmission electron microscope equipped with a lab six filament operating at 120 kilovolts. Next, turn on the TEM filament and adjust the centric height of the microscope stage with respect to the specimen by using the wobbler function to tilt the sample for minus 15 degrees to plus 15 degrees back and forth. In the column record 6, 000 to 30, 000 times magnification images along the edges and in the corner regions of the microfluidic chamber.First image using a CCD camera at low dose conditions of one to three electrons per angstrom squared. Determine the proper defocus value by focusing at the edge of the fluidic chamber. In general, use a defocus value of minus 1.5 micrometers to record images at 30, 000 times magnification to ensure the 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. The image shown here was taken after two minutes at five electrons per angstrom squared. After five minutes, even more bubbles formed glow discharge, microchips bind.Very few double layered particles in solution presumably due to diffusion. In comparison, the double layered particles are enriched on affinity decorated microchips shown here are representative images and a 3D reconstruction of double layered particles in liquid containing a contrast reagent. The diameter of the reconstruction is 80 nanometers.Class averages of the double layered particles and liquid revealed nicely defined features along their outer surface. Individual panels are 110 nanometers. When performing this procedure, it's important to remember to center the E IPS in the tip of the specimen holder and PrepU the holder prior to inserting it into the electron microscope.Following this procedure, other methods like assessing the transcriptional activity can be performed in order to answer additional questions like, to what extent did the electron beam affect the functional capacity of the viral complexes? After watching this video, you should have a good understanding I how to produce affinity capture devices, decorate them with molecular adapters, load virus specimens into the microfluidic TEM holder and image the specimens using cche microscopy procedures. Don't forget that working with viral pathogens can be extremely hazardous, and precautions such as wearing gloves and protective eyewear should always be taken when performing procedures with BSL two pathogens.

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

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This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

We present a procedure for real-time imaging and elemental composition analysis of boehmite particles in deionized water by in situ liquid Scanning Electron Microscopy.

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

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

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

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

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.

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

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

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

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 µm.

Visualization of Organelles In Situ by Cryo-STEM Tomography

Learn in situ TEM imaging of viral complexes in liquid at nanometer resolution using affinity capture microchips. This method reveals dynamic behaviors of rotavirus particles for structural biology research.

In situ TEM of Biological Assemblies in Liquid

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