המחברים מבקשים להודות לאוג&#39;ניו Vivas, הקורט Heungens, אריק מרטינס, צ&#39;ארלס קאולס, דארבי סוכר, אריק Stabb, וטוד Ciche על תרומתם לפיתוח של פרוטוקול זה וכלים בשימוש. KEM וJMC נתמכו על ידי המכונים הלאומיים לבריאות (NIH) בפרס לאומי לחקר שרותים T32 (AI55397 &quot;חיידקי מחלה ובריאות&quot;). המרכז למוסיקת ירושלים נתמך על ידי מלגת מחקר למוסמכי קרן לאומית למדע (NSF). עבודה זו נתמכה על ידי מענקים מהקרן הלאומי למדע (IOS-0920631 וIOS-0950873).

<ol>
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אין ניגודי האינטרסים הכריזו.

הפרוטוקול המתואר כאן מספק שיטה לזיהוי האופטי של חיידקים בתוך מארח נמטודות (איור 1). שיטה זו מנצלת את השקיפות האופטית של נמטודות ויכולת fluorescently חיידקי תווית, מה שמאפשר בניתוח vivo של חיידקים בתוך פונדקאי נמטודות (איור 3). באופן ספציפי, גישה זו מזהה ל?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות (אופציונלי) </td></tr><tr><td> אגר שומנים (עקר) </td><td></td><td></td><td> 8 גרם מרק מזין, 15 אגר גרם, שמרי תמצית 5 גרם, מים 890 מ&quot;ל, 10 מ&quot;ל 0.2 גר &#39;/ המ&quot;ל MgCl 2. 6H 2 0, * 96 מ&quot;ל סירופ תירס פתרון, 4 מיליליטר שמן תירס * מערבב תקשורת בעת שמזגה צלחות * להוסיף מרכיב עקר אחרי מעוקר </td></tr><tr><td> פתרון סירופ תירס (עקר) </td><td></td><td></td><td> סירופ תירס 7 מ&quot;ל, 89 מיליליטר מים תמהיל וחיטוי </td></tr><tr><td> פתרון ביצה </td><td></td><td></td><td> 16.6 מ&quot;ל 12% hypochlorite נתרן, 5 המ&quot;ל KOH 5M, מי 80 מ&quot;ל </td></tr><tr><td> Lysogeny מרק (עקר) </td><td></td><td></td><td> 5 גרם תמצית שמרים, 10 גרם tryptone, 5 גרם מלח, 1 ליטר מים תמהיל וחיטוי </td></tr><tr><td> Microfuge </td><td> דיג </td><td> 13-100-675 </td><td> כל microfuge שמחזיק צינורות microfuge יעבוד </td></tr><tr><td> סרכזת </td><td> Beckman </td><td> 366802 </td><td> צנטריפוגה בראש הטבלה הגדולה שמחזיקה 15 מ&quot;ל ו 50 צינורות חרוטי מ&quot;ל </td></tr><tr><td> סטרילי 60 מ&quot;מ X 15 מ&quot;מ צלחת פטרי </td><td> דיג </td><td> 0875713 </td><td></td></tr><tr><td> 50 צינורות צנטריפוגות מ&quot;ל </td><td> דיג </td><td> 05-539-6 </td><td></td></tr><tr><td> 15 צינורות צנטריפוגות מ&quot;ל </td><td> דיג </td><td> 05-531-6 </td><td></td></tr><tr><td> 100 מ&quot;מ X 20 מ&quot;מ צלחת פטרי סטרילית </td><td> דיג </td><td> 0875711Z </td><td> עמוק יותר מצלחות פטרי הרגילות </td></tr><tr><td> צלחת 24 היטב </td><td> גריינר ביו אחד &lt;/ Td&gt; <td> 662000-06 </td><td></td></tr><tr><td> מיקרוסקופ </td><td></td><td></td><td> מיקרוסקופ צריך יכולות ניאון תואמות fluorophore </td></tr><tr><td> Paraformaldehyde </td><td> מדעי מיקרוסקופ אלקטרונים </td><td> 15710 </td><td></td></tr><tr><td> PBS (עקר) </td><td></td><td></td><td> 8 גרם NaCl 0.2 גרם KCL 1.44 גרם Na 2 HPO 4 0.24 גרם KH 2 PO 4 1 ליטר מים התאם לpH של 7.4 ליטר ומים עד 1 וחיטוי </td></tr><tr><td> צינורות Microfuge </td><td> דיג </td><td> 05-408-138 </td><td> 2 מ&quot;ל או 1.5 מ&quot;ל צינורות </td></tr><tr><td> מטרף </td><td></td><td></td><td> כל מביא שגורם לנוזל בעדינות להעביר יעבוד </td></tr><tr><td> חומצת Diaminopimelic </td><td> סיגמא </td><td&gt; D-1377 </td><td> במידת צורך, להוסיף מדיה לריכוז או 1 mM </td></tr></tbody></table>

visualizing bacteria in nematodes using fluorescent microscopy

Symbioses, החיים משותפים של שניים או יותר אורגניזמים, הם נפוצים לאורך כל ממלכות חיים. כשתיים מכל מקום אורגניזמים ביותר על פני אדמה, נמטודות וחיידקים יוצרים מגוון רחב של עמותות סימביוטיים שנע בין מועיל לפתוגניים 1-3. עמותה אחת כזו היא מערכת היחסים הדדיים המועילה בין חיידקי Xenorhabdus ונמטודות Steinernema, שהתפתחה כמערכת מודל של סימביוזה 4. נמטודות Steinernema היא entomopathogenic, באמצעות חיידקי סימביונט להרוג חרקים 5. להעברה בין מארחי חרקים, החיידקים להתנחל המעי של שלב נעורי התולעת של נמטודות 6-8. לאחרונה, כמה מינים נמטודות אחרים הוכחו לנצל חיידקים להדברת חרקים 9-13, וחקירות שהחלו לבחון את יחסי הגומלין בין נמטודות והחיידקים במערכות אלה &lt;sup&gt; 9. אנו מתארים שיטה להדמיה של סימביונט חיידקים בתוך או על מארח נמטודות, מנצלים את השקיפות האופטית של נמטודות כאשר נצפו על ידי מיקרוסקופ. את החיידקים שהונדסו חלבון פלואורסצנטי, המאפשר ההדמיה שלהם על ידי מיקרוסקופ פלואורסצנטי. פלסמידים רבים זמינים שנושאים גני מקודדי חלבונים כי לזרוח באורכי גל שונים (כלומר ירוק או אדום), וצמיד של פלסמידים מזן חיידקי Escherichia תורם לסימביונט חיידקי נמען היא מוצלחת למגוון רחב של חיידקים. בשיטות המתוארות פותחו כדי לחקור את הקשר בין Steinernema carpocapsae וXenorhabdus nematophila 14. שיטות דומות שמשו כדי לחקור עמותות אחרות נמטודות-חיידק 9, 15-18 והגישה לכן היא באופן כללי. נפגשהוד מאפשר אפיון של נוכחות ולוקליזציה חיידקים בתוך נמטודות בשלבים שונים של פיתוח, לספק תובנה על טבעה של העמותה והתהליך של קולוניזציה 14, 16, 19. ניתוח מיקרוסקופי מגלה שכיחות הקולוניזציה בתוך אוכלוסייה ולוקליזציה של חיידקים לארח רקמות 14, 16, 19-21. זה יתרון על פני שיטות אחרות לניטור חיידקים בתוך אוכלוסיות נמטודות, כגון sonication 22 או טחינת 23, אשר יכול לספק רמות ממוצעות של התישבות, אבל לא יכול, למשל, להפלות אוכלוסיות עם שכיחות גבוהה של עומסי סימביונט נמוכים מאוכלוסיות עם תדירות נמוכה של עומסי סימביונט גבוהים. להפלות את התדירות והעומס של חיידקים מיישבים יכול להיות חשוב במיוחד כאשר הקרנה או אפיון מוטציות חיידקים לפנוטיפים קולוניזציה 21, 24. אכן, מיקרוסקופ פלואורסצנטי נעשה שימוש בהקרנת תפוקה גבוהה של מוטציות חיידקים לפגמים ב17 קולוניזציה, 18, ​​והוא פחות מייגע מאשר שיטות אחרות, כולל sonication 22, 25-27 ונתיחת פרט נמטודות 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

כדי ללמוד את ההדדיות בין<em&gt; Xenorhabdus</em&gt; חיידקים<em&gt; Steinernema</em&gt; נמטודות, שיטות פותחו כדי לפקח על נוכחות ומיקום בתוך נמטודות חיידקים. הגישה הניסויית, אשר יכול להיות מיושמת על מערכות אחרות, כרוכה בחיידקי הנדסה לבטא חלבון פלואורסצנטי הירוק והדמיה, באמצעות מיקרוסקופ פלואורסצנטי חיידקים בתוך נמטודות השקופה.

Visualizing חיידקים בנמטודות באמצעות מיקרוסקופיה פלורסנט

 
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

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Biology

18.6K Views.  University of Wisconsin-Madison. כדי ללמוד את ההדדיות בין Xenorhabdus חיידקים Steinernema נמטודות, שיטות פותחו כדי לפקח על נוכחות ומיקום בתוך נמטודות חיידקים. הגישה הניסויית, אשר יכול להיות מיושמת על מערכות אחרות, כרוכה בחיידקי הנדסה לבטא חלבון פלואורסצנטי הירוק והדמיה, באמצעות מיקרוסקופ...

Video: 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.

לדמיין את חיים תסיסנית גליה-neuromuscular צומת עם צבעי ניאון

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

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.

הדמיה מולקולרית מכלולים יחיד In vivo באמצעות מיקרוסקופ פלואורסצנטי מתקדם

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.

חזותי יחיד מולקולת הדנ&quot;א שכפול עם מיקרוסקופ פלואורסצנטי

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.

מיקרוסקופית Intravital למבני ההדמיה subcellular בעכברים חיים לבטא חלבוני פלורסנט

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.

הדמיה ברזולוציה סופר של Cytokinetic Z הטבעת בחיידקים חיים באמצעות-Structured 3D מהיר תאורה מיקרוסקופית (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.

במשובט מעקב vivo של גזע hematopoietic ובתאים המסומנים על ידי חמישה חלבונים ניאון באמצעות Confocal וmultiphoton מיקרוסקופית

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.

חזותי תדר Stromule עם מיקרוסקופ פלואורסצנטי

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.

מערכת חלבון פלואורסצנטי ירוק לדמיין Effectors מועברת בין חיידקים במהלך זיהום

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.

חזותי מבנה צומת לימפה ולוקליזציה לסלולר באמצעות Ex-Vivo Confocal מיקרוסקופ

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.