Name: plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy
Uploaded: 2017-05-05T12:30:00
Description: Watch this Scientific Journal Video about Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy at JoVE.com

We express our gratitude to M. Bouchecareilh, E. T&#233;taud, S. Duvezin-Caubet, and A. Devin for their help and comments on the manuscript. We are grateful to the electronic imaging pole of Bordeaux Imaging Centre where the images were taken. This work was supported by Centre National de la Recherche Scientifique.

1. Preparation of Formvar Grid Film
NOTE: Perform chloroform manipulation under a fume hood using personal protective equipment (gloves, lab coat, glasses). Use 400 mesh electron microscopy copper grids. With other grid types (other mesh sizes, gold and nickel grids), the quality of freezing is worse.
<ol>
	<li>Prepare a 100 mL solution of 0.3% formvar in chloroform. Allow it to dissolve overnight without agitation.</li>
	<li>Put 400 mesh (number of holes per square inch)electron microscopyc...

<ol>
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TEM is a powerful method for the ultrastructural observation of organelles, cells, and tissues. Cryofixation/freeze-substitution is currently the best method for the preservation of both ultrastructure and protein antigenicity. Chemical fixatives penetrate and act very slowly thereby allowing structural rearrangements before the complete stabilization of the ultrastructure2. Conversely, cryofixation/freeze-substitution instantly stabilizes cellular structures4. However, cry...

Sample preparation is critical for the success of any electron microscopy study. Conventional EM fixation was the primary method for fixing tissue or cells for transmission electron microscopy (TEM)1. First, aldehydes and osmium tetroxide are used to chemically fix the material at room temperature. Then, the material is dehydrated with organic solvents, infiltrated, and embedded in epoxy resin. This method is dependent upon the penetration rate of fixatives into the cell. Consequently, the artifacts and extraction of cellular contents are usually observed2.
Cryofixation is clearly a better alternative for the preservation of cellular structures3, keeping them intact. The highest quality of TEM images4 in thin resin sections can be obtained using the cryofixation/freeze substitution method. The aim of this technique is to obtain vitrified biological samples without ice crystals formation or containing ice crystals small enough not to damage the ultrastructure of the cells. High-pressure freezing (HPF) and ultra-rapid cryofixation, which is also called plunge freezing (PF), are two methods to cryofix samples. HPF immobilizes molecules in the cell instantaneously and avoids the damage caused by conventional EM fixation. Several types of freezing machines and automatic substitution devices have been developed5. The freezing machines and consumables (liquid nitrogen, specimen carriers, etc.) are expensive, but they allow for producing high-quality electron micrographs6,7. PF is a technique that was used in the early 1950s and is described in the literature as simple and cheap8.During the PF procedure, the prepared sample is frozen at a rapid rate to obtain ice crystals size less than 3 to 5 nm. To this end, the sample is plunged into a liquid cryogen, such as ethane, propane, or an ethane-propane mixture. Since the 1950s, improvements of PF have been done to make this technique available to a greater number of users. High-pressure freezing is currently the only viable way to freeze a large variety of samples thicker than 50 &#181;m (up to a thickness of 200 &#181;m for disc-shaped samples)5, whereas PF is widely used to image small objects (&lt;100 nm), such as macromolecular complexes suspended in a thin film of amorphous ice9. Larger samples, for example eukaryotic cells, can be cryofixed by PF, but requires specimen holders, such as capillary copper tubes or sandwich systems8,10,11. 
Here, a rapid, easy-to-use and low-cost plunge freezing/freeze-substitution technology usable for various suspension cell cultures is presented.

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plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

In this article, an easy-to-use and low-cost plunge freezing method for ultrastructural (Figures 1 and Figure 2) and immunolabeling (Figure 3) studies is presented. We demonstrate that it is not necessary to have special equipment for the freeze-substitution procedure and that the warming procedure takes less than 6 h instead of the 24 h using a dedicated system.
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Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

The goal of the following experiments is to propose a tool for ultrastructural and immunolocalization studies of suspension cells for visualization in transmission electron microscopy. This technique is easy to use, low cost, and gives excellent preservation of cellular structure. This is a culture of suspension cells.First, the cells are harvested by different centrifugations to obtain a pellet in a microcentrifuge tube. In a second step, the sample is rapidly frozen by plunge freezing which immobilizes the cells and limits the formation of ice crystals. Next, freeze substitution is performed in order to replace water by solvent containing osmium tetroxide as fixative to stabilize cell structures.Results show that plunge freezing, and freeze substitution, preserve perfectly the infrastructure of the cells. Chemical fixation was a primary method for fixing tissue or cells for transmission electron microscopy, but with this method, artifacts and extraction of cellular contents are usually observed. An alternative to chemical fixation is cryofixation.This method represents the best choice for producing the highest quality of morphology in thin resin sections. Plunge freezing is a cryofixation technique that immobilizes molecules in the cell instantaneously to avoid damage caused by conventional chemical fixation. Before plunge freezing the sample, grid cutted with Formvar film must be prepared to be used as support for freezing.Polish glass slide with lint-free cloth until shiny and wipe clean. Dip the glass slide in a solution of Formvar 0.3%in chloroform a few seconds and leave to dry upright in a beaker. Manipulation must be realized under a fume hood using personal protective equipment due to the presence of chloroform.After filling a crystallizer with distillated water, pass over the water surface a glass bar to remove dust. When the film is dry, score the edges with a sharp razor blade so that the film may lift off. Frost the slide by exhaling heavily onto the slide to get a layer of water vapor on the film.Immediately float the film onto the water surface by touching the bottom of the slide with an angle about 30 degrees to the water surface. Slowly lower the slide into the bath, teasing the film off as necessary. The Formvar film settles slowly on the surface of the water.Attach the film on the edge of the crystallizer using tweezers. Gently lay the 400 mesh copper grids face-down on the film in the regions that are of the proper thickness and without wrinkles. The film with grids is picked up by covering them with Joseph paper while they float on the water surface.Pick up the edge of the Joseph paper and the film with grids should come with it. Don't let your fingers touch the water surface or you run the risk of leaving a film in the water. Let the grids dry several hours before using them.For ultrastructural studies, put osmium tetroxide ampoule in a glass flask. Shake well the flask to break the ampoule. Add appropriate volume of acetone 100%to have a final concentration of 4%Stir the flask containing the freeze substitution medium, and dispense 1.5 milliliter aliquots into cryovials with plastic transfer pipet.Close all caps and keep in freezer at minus 84 degrees before using. For protein immunolabeling studies, prepare 0.1%of uranyl acetate in acetone. When the solution becomes clear, filtrate using 0.2 micrometer filter and dispense 1.5 milliliter aliquots into cryovials.Keep in freezer at minus 84 degrees before using. To be sure to have the appropriate consistency of pellets from different cell types, raw yeast cells in minimum of complete liquid medium to obtain one 10 to the nine cells. Escherichia coli in DTY medium to obtain five 10 to the 10 bacterial cells.Leishmania flagellum in AM medium and Trypanosoma brucei in SDM79 medium to obtain five 10 to the eight parasite cells. Transfer the culture medium containing suspension cells to a 50 milliliter propylene tube and centrifuge for three minutes at 1, 500 g. Remove the supernatant and resuspend the pellet in one milliliter of respective cell culture medium.Centrifuge in a microcentrifuge tube for one minute at 3, 900 g. Remove the supernatant completely. For plunge freezing method, propane bottle with a tube connected to the propane cylinder must be used.Propane being potentially explosive, realize the liquefaction in a well-ventilated room or in a fume hood. No open flames are allowed. Personal protective equipment including cryogloves must always be worn when using liquid nitrogen.Put a little quantity of liquid nitrogen in polystyrene tray. Place the brass cup in liquid nitrogen. Bubbles appear.Wait until the bubbles subside. The brass cup is then frozen. Put the tube connected to the propane cylinder in contact with the brass cap wall.Gently open the propane cylinder valve. A lot of smoke forms as propane does not begin to liquefy. When the propane begins to liquefy, the smoke disappears.Stop the liquefaction by closing the propane-cylinder valve and removing rapidly the tube connected to the propane cylinder. Fill the polystyrene tray with liquid nitrogen up to five millimeters from the top of the brass cup. Freeze little brass cups where we will replaced the sample after freezing by putting them in liquid nitrogen.Put one cup for one sample. Be careful not to mix samples. To obtain the appropriate consistency of the pellet, it just necessary to respect culture conditions and centrifugation parameters indicated previously.Freeze the tweezers before using them. Take a 400 mesh copper microscopy grid cutted with Formvar. Plunge a double-edge dissecting needle in the pellet.Put, using the tool, a drop of pellet on the grid. Very rapidly plunge the grid into liquid propane and stir a few seconds. Rapidly transfer the frozen grid in the little brass cup.For each sample, make at least three grids. Don't forget to freeze the tweezers before taking up a grid. Freeze the tweezers before using them.Rapidly remove the cup and transfer the frozen grids into the cryovial using the tweezers. Put the cap back on the cryovial. Repeat the operation for each grid of each sample.Close caps and stir the cryovial to be sure the samples are in freeze substitution medium. Let the cryovials for three days in the minus 84 degrees freezer. Carry the cryovials in a polystyrene tray to avoid a sudden rise in temperature to the minus 30 degree freezer.Let one hour at minus 30 degrees. Carry the cryovials at minus 15 degrees for two hours, then put the samples at four degrees for two hours, and 30 minutes at room temperature. Remove the freeze substitution medium using plastic transfer pipettes and put it in an appropriate waste bottle clearly identified.Put about 500 microliters of 100%acetone. Open the glass vial and stir the cryovial and rapidly put the freeze substitution medium in a glass vial. Rinse one cryovial with 100%acetone.Rinse three times 10 minutes with 100%acetone. Carry the cryovials in a polystyrene tray to avoid a sudden rise in temperature to the minus 30 degree freezer. Let two hours at minus 30 degrees.After putting samples in a polypropylene vial, replace the freeze substitution medium by fresh freeze substitution medium and let two hours at minus 30 degrees. Rinse one hour with 100%acetone and three times one hour with 100%ethanol. Plunge freezing/freeze substitution method resulted in excellent preservation of the intracellular structures of suspension cells.In Saccharomyces cerevisiae, the structures are not shrunken and the cytoplasm appears dense. Moreover, membranes are smooth, which is a proof of a good preservation. All organelles, such as nucleus, mitochondria, and vacuoles can be perfectly identified.Excellent preservation is also observed in Leishmania flagellum, Trypanosoma brucei, and Escherichia coli. In nucleus, double membrane forming nuclear envelope, nuclear pore, spindle pole body, and ribosomes attached on the outer membrane of the nuclear envelope are distinctly visible. In mitochondria, the internal membrane invaginations, called cristae, are clearly visible.Critical organelles are the vacuoles, because large ice crystals can develop easily. In the image, vacuoles are perfectly preserved without ice crystal damage. Plunge freezing/freeze substitution method can be also used to perform protein immunolocalization.The procedure preserves perfectly protein antigenicity, and as mentioned previously, organelles are clearly identified. Different antibodies have been already tested to target different proteins in yeast, such as anti-ATP synthase and anti-porine. Such experiment is also possible for different experimental systems, as in Leishmania, where the flabarin protein was localized.After watching this video, you should have good understanding how to prepare samples with plunge freezing/freeze substitution method for visualizing them in transmission electron microscopy. This technique is rapid, easy to use, low cost, and give excellent preservation of cell infrastructures. Be careful with liquid nitrogen, but especially with osmium tetroxide and uranyl acetate.They should be handled in the fume hood while wearing appropriate personal protective equipment, including lab coat and gloves.

plunge-freezing-tool-for-ultrastructural-immunolocalization-studies

Research

JoVE Journal

Biology

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. 
These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...