Die Autoren möchten sich Eugenio Vivas, Kurt Heungens, Eric Martens, Charles Cowles, Darby Sugar, Eric Stabb und Todd Ciche für ihre Beiträge danken für die Entwicklung dieses Protokolls und Werkzeuge verwendet. KEM und JMC wurden von den National Institutes of Health (NIH) National Research Service Award T32 (AI55397 &quot;Mikroben in Health and Disease&quot;) unterstützt. JMC wurde von der National Science Foundation (NSF) Graduate Research Fellowship unterstützt. Diese Arbeit wurde durch Zuschüsse aus dem National Science Foundation (IOS-0.920.631 und IOS-0950873) unterstützt.

<ol>
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Die hier beschriebene Protokoll stellt ein Verfahren zur optischen Detektion von Bakterien in einer Nematode Host (Abbildung 1). Diese Methode nutzt die optische Transparenz von Nematoden und die Fähigkeit zur Fluoreszenz-Label Bakterien, mit der in vivo-Analyse von Bakterien innerhalb des Nematoden Host (Abbildung 3). Insbesondere, identifiziert dieser Ansatz bakteriellen Lokalisation innerhalb seines Wirtes. Durch Zählen einer Nematoden Bevölkerung und Scoring für bakteri...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Lipid Agar (Steril) </td><td></td><td></td><td> 8 Gramm Nährbrühe, 15 Gramm Agar, 5 g Hefeextrakt, 890 ml Wasser, 10 ml 0,2 g / ml MgCl 2. 6H 2 0, 96 ml Maissirup Lösung *, 4 ml Maisöl * Stir Medien während Giesserplatten * In sterile Wirkstoff nach dem Autoklavieren </td></tr><tr><td> Corn Syrup Lösung (Steril) </td><td></td><td></td><td> 7 ml Maissirup, 89 ml Wasser Mix und Autoklaven </td></tr><tr><td> Egg Lösung </td><td></td><td></td><td> 16,6 ml 12% ige Natriumhypochlorit, 5 ml 5M KOH, 80 ml Wasser </td></tr><tr><td> Lysogenie Broth (Steril) </td><td></td><td></td><td> 5 Gramm Hefeextrakt, 10 g Trypton, 5 g Salz, 1 L Wasser Mix und Autoklaven </td></tr><tr><td> Zentrifugieren </td><td> Fischer </td><td> 13-100-675 </td><td> Jede Mikrofuge die Mikrozentrifugenröhrchen hält funktionieren </td></tr><tr><td> Zentrifugieren </td><td> Beckman </td><td> 366802 </td><td> Große Tischzentrifuge, die 15 ml und 50 ml konischen Röhrchen hält </td></tr><tr><td> Sterile 60 mm X 15 mm Petrischale </td><td> Fischer </td><td> 0875713 </td><td></td></tr><tr><td> 50 ml Zentrifugenröhrchen </td><td> Fischer </td><td> 05-539-6 </td><td></td></tr><tr><td> 15 ml Zentrifugenröhrchen </td><td> Fischer </td><td> 05-531-6 </td><td></td></tr><tr><td> Sterile 100 mm X 20 mm Petrischale </td><td> Fischer </td><td> 0875711Z </td><td> Tiefer als Standard-Petrischalen </td></tr><tr><td> 24-Well-Platte </td><td> Greiner Bio-One &lt;/ Td&gt; <td> 662000-06 </td><td></td></tr><tr><td> Mikroskop </td><td></td><td></td><td> Das Mikroskop muss florescent Funktionen kompatibel mit Ihrem Fluorophor </td></tr><tr><td> Paraformaldehyd </td><td> Electron Microscopy Sciences </td><td> 15710 </td><td></td></tr><tr><td> PBS (Steril) </td><td></td><td></td><td> 8 g NaCl 0,2 g KCL 1,44 g Na 2 HPO 4 0,24 g KH 2 PO 4 1 l Wasser Den pH-Wert von 7,4 und Wasser auf 1 l und Autoklav </td></tr><tr><td> Mikrozentrifugenröhrchen </td><td> Fischer </td><td> 05-408-138 </td><td> 2 ml oder 1,5 ml Röhrchen </td></tr><tr><td> Shaker </td><td></td><td></td><td> Jeder Schüttler, der die Flüssigkeit sanft bewegen verursacht wird funktionieren </td></tr><tr><td> Diaminopimelinsäure </td><td> Sigma </td><td&gt; D-1377 </td><td> Wenn nötig, zu ergänzen Medien auf eine Konzentration von 1 mM oder </td></tr></tbody></table>

visualizing bacteria in nematodes using fluorescent microscopy

Symbiosen, das Zusammenleben von zwei oder mehr Organismen sind in allen Reichen des Lebens verbreitet. Als zwei der am weitesten verbreiteten Organismen auf der Erde bilden Nematoden und Bakterien eine breite Palette von symbiotische Assoziationen, die von Vorteil für pathogene 1-3. Eine solche Vereinigung ist die gegenseitig vorteilhafte Beziehung zwischen Xenorhabdus Bakterien und Steinernema Nematoden, die als Modellsystem Symbiose 4 entstanden ist. Steinernema Nematoden sind entomopathogenen, indem sie ihre bakteriellen Symbionten zu töten Insekten 5. Für die Übertragung von Insekten Gastgeber, besiedeln die Bakterien im Darm der Nematoden der infektiösen jugendlichen Stadium 6-8. In jüngster Zeit haben mehrere andere Nematoden wurde gezeigt, dass Bakterien nutzen zu töten Insekten 9-13, und Untersuchungen haben begonnen Untersuchung der Wechselwirkungen zwischen den Nematoden und Bakterien in diesen Systemen &lt;sup&gt; 9. Wir beschreiben ein Verfahren zur Visualisierung eines bakteriellen Symbionten innerhalb oder auf einer Nematode Host, unter Ausnutzung der optischen Transparenz der Nematoden, wenn durch Mikroskopie angesehen. Die Bakterien werden konstruiert, um ein fluoreszierendes Protein exprimieren, wodurch deren Visualisierung durch Fluoreszenzmikroskopie. Viele Plasmide zur Verfügung, die für Proteine ​​kodierenden Genen tragen, die bei verschiedenen Wellenlängen (dh grün oder rot), und die Konjugation von Plasmiden aus einer Escherichia coli Donor-Stamm in einen Empfänger bakteriellen Symbionten fluoreszieren erfolgreich ist für ein breites Spektrum von Bakterien. Die beschriebenen Methoden wurden entwickelt, um den Zusammenhang zwischen Steinernema carpocapsae und Xenorhabdus nematophila 14 zu untersuchen. Ähnliche Verfahren sind verwendet worden, um andere nematodeninduzierbares Bakterium Assoziationen 9, 15-18 untersuchen und der Ansatz ist deshalb allgemein anwendbar. Der method ermöglicht die Charakterisierung von bakteriellen Präsenz und Lokalisierung innerhalb Nematoden in verschiedenen Stadien der Entwicklung, Einblicke in die Natur des Vereins und der Prozess der Kolonisierung 14, 16, 19. Mikroskopischen Analyse ergibt sowohl Kolonisierung Frequenz innerhalb einer Population von Bakterien und Lokalisierung von Geweben 14, 16, 19-21 hosten. Dies ist ein Vorteil gegenüber anderen Methoden zur Überwachung Bakterien innerhalb Nematodenpopulationen, wie Ultraschallbehandlung 22 oder Schleifen 23, die Durchschnittswerte der Besiedlung bestimmt, die aber nicht zum Beispiel diskriminieren Populationen mit einer hohen Frequenz von niedrigen Lasten aus symbiont Populationen mit eine niedrige Frequenz von hoher Symbionten Lasten. Unterscheiden der Frequenz und der Last besiedelnden Bakterien kann besonders wichtig bei der Durchmusterung oder Charakterisierung bakteriellen Mutanten zur Besiedelung Phänotypen 21, 24. Tatsächlich hat Fluoreszenzmikroskopie in Hochdurchsatzscreening von bakteriellen Mutanten für Defekte in Kolonisierung 17, 18 eingesetzt, und ist weniger aufwendig als andere Verfahren, einschließlich Ultraschallbehandlung 22, 25-27 und Nematoden einzelnen Dissektion 28, 29.

Watch this Scientific Journal Video about Visualizing Bacteria in Nematodes using Fluorescent Microscopy at JoVE.com

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Um die Mutualismus zwischen studieren<em&gt; Xenorhabdus</em&gt; Bakterien und<em&gt; Steinernema</em&gt; Nematoden, Methoden wurden entwickelt, um bakterielle Präsenz und Position innerhalb Nematoden überwachen. Der experimentelle Ansatz, der auf andere Systeme angewendet werden kann, bringt Engineering Bakterien das grün fluoreszierende Protein exprimieren und zu visualisieren, mittels Fluoreszenzmikroskopie Bakterien innerhalb des transparenten Nematoden.

Visualisierung Bakterien in Nematoden durch Fluoreszenzmikroskopie

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

visualizing-bacteria-in-nematodes-using-fluorescent-microscopy

Research

JoVE Journal

Biology

18.7K Aufrufe.  University of Wisconsin-Madison. Um die Mutualismus zwischen studieren Xenorhabdus Bakterien und Steinernema Nematoden, Methoden wurden entwickelt, um bakterielle Präsenz und Position innerhalb Nematoden überwachen. Der experimentelle Ansatz, der auf andere Systeme angewendet werden kann, bringt Engineering Bakterien das grün fluoreszierende Protein exprimieren und zu visualisieren, mittels Fluoreszenzmikroskopie Bakterien innerhalb des transparenten Nematoden.

Serie: Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualisierung der Live- Drosophila Glial-neuromuskulären Synapse mit Fluoreszenzfarbstoffen

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Forschung

Biologie

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualizing Einzelne molekulare Komplexe In Vivo Verwenden der erweiterten Fluoreszenzmikroskopie

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizing Einzel-Molekül-DNA-Replikation mit Fluoreszenz-Mikroskopie

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Intravitalmikroskopie zur Imaging subzellulärer Strukturen in lebenden Mäusen Ausdruck Fluoreszierende Proteine

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Super-Resolution Imaging der zytokinetischen Z-Ring im Live-Bakterien mit Schnelle 3D-Structured Illumination Microscopy (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo Klonale Verfolgung von hämatopoetischen Stamm-und Vorläuferzellen durch Fünf Fluoreszierende Proteine ​​mit Hilfe der konfokalen und Multiphotonen Mikroskopie Markierte

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizing Stromulus Frequenz mit Fluoreszenzmikroskopie

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Split-grün fluoreszierendes Protein System Effektoren geliefert von Bakterien während der Infektion zu visualisieren

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Visualisierung der Lymphknotenstruktur und Zelllokalisierung mittels Ex-Vivo Konfokalmikroskopie

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

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.