עבודה זו נתמכה בחלקה על ידי המכונים הלאומיים לבריאות מענקים AI067548 R01, R01 GM081520, GM086200 R01, R01 AI049194 ו P01 GM066521 כדי GJJ כמו גם את הווארד יוז רפואי במכון, במכון Beckman בקלטק, מתנות קלטק מן גורדון ובטי מור קרן Agouron המכון. MSL נתמך על ידי מענק 2R37-NIH A1041239-06A1 על פמלה ס ביורקמן. לייקה מיקרוסיסטמס חביב בתנאי תוכן וידאו של אוסף cryosection. תוצאות נציג Treponema primitia נאספו ועובדו על גאווין א מרפי, ופורסם למיקרוביולוגיה מולקולרית עם הכותרת &quot;ultrastructures רומן של Treponema primitia והשלכותיהם על תנועתיות&quot;. (מרפי, G. et al. מול. Microbiol. 67, 1184-1195, 2008).

<ol>
	<li>Jensen, G. J., Briegel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+electron+cryotomography+is+opening+a+new+window+onto+prokaryotic+ultrastructure.">How electron cryotomography is opening a new window onto prokaryotic ultrastructure.</a> Curr. Opin. Struct. Biol. 17, 260-267 (2007).</li><li>Li, Z., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryotomography:+a+new+view+into+microbial+ultrastructure.">Electron cryotomography: a new view into microbial ultrastructure.</a> Curr. Opin. Microbiol. 12, 333-340 (2009).</li><li>Frank, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+biological+complexity.">Three-dimensional imaging of biological complexity.</a> J. Struct. Biol. 138, 85-91 (2002).</li><li>Iancu, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryotomography+sample+preparation+using+the+Vitrobot.">Electron cryotomography sample preparation using the Vitrobot.</a> Nat. Protocols. 1, 2813-2819 (2007).</li><li>Studer, D., Graber, W., Al-Amoudi, A., Eggli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+for+cryofixation+by+high-pressure+freezing.">A new approach for cryofixation by high-pressure freezing.</a> J. Microsc. 203, 285-294 (2001).</li><li>Ladinsky, M. S., Pierson, J., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitreous+cryosectioning+of+cells,+facilitated+by+a+micromanipulator.">Vitreous cryosectioning of cells, facilitated by a micromanipulator.</a> J. Microsc. 224, 129-134 (2006).</li><li>Pierson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+technique+of+vitreous+cryo-sectioning+for+cryo-electron+tomography:+Electrostatic+charging+for+section+attachment+and+implementation+of+an+anti-contamination+glove+box.">Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box.</a> J. Struct. Biol. 169, (2009).</li><li>Suloway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated,+sequential+tilt-series+acquisition+with+Leginon.">Fully automated, sequential tilt-series acquisition with Leginon.</a> J. Struct. Biol. 167, 11-18 (2009).</li><li>Fernando, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markov+random+field+based+automatic+image+alignment+for+electron+tomography.">Markov random field based automatic image alignment for electron tomography.</a> J. Struct. Biol. 161, 260-275 (2008).</li><li>Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> J. Struct. Biol. 116, 71-76 (1996).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-axis+tomography:+an+approach+with+alignment+methods+that+preserve+resolution.">Dual-axis tomography: an approach with alignment methods that preserve resolution.</a> J. Struct. Biol. 120, 343-352 (1997).</li></ol>

אין ניגודי אינטרסים הכריז.

אנו מציגים במאמר זה וידאו איך לעשות ECT של תאים חיידקיים. מוגבלת על ידי מרחב וזמן, אנחנו רק להראות את פרוטוקול כללי. פרטים נוספים ניתן למצוא מאמרים, מידע מקוון או אחרים הניתנים על ידי מייצרת הציוד ומפתחי תוכנה, מקבוצות מחקר ECT. בהתאם מינים שונים חיידקי למד, הציוד המשמש לבין האינטרס מבניים בתאים, את הפרוטוקול ואת הפרמטרים ניסיוני צריכים להיבדק אופטימיזציה.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Poly-L-Lysine</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P8920</td>
<td>0.1% solution</td>
</tr>
<tr>
<td>MKIII Vitrobot</td>
<td>&nbsp;</td>
<td>FEI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gold colloid solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1527-25ml</td>
<td>10 nm</td>
</tr>
<tr>
<td>Bovine serum Albumin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P2489</td>
<td>10%</td>
</tr>
<tr>
<td>Nr 1 Filter paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1001 055</td>
<td>&nbsp;</td>
<tr>
<td>Dextran</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>31389</td>
<td>15-25%, MW 39000</td>
</tr>
<tr>
<td>Brass-domed planchet</td>
<td>&nbsp;</td>
<td>Swiss Precision Company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bal-Tec HPm 010 high pressure freezer</td>
<td>&nbsp;</td>
<td>Leica Microsystems Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cryo-ultra microtome UC6/FC6</td>
<td>&nbsp;</td>
<td>Leica Microsystems Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Diamond cryo trim 45</td>
<td>&nbsp;</td>
<td>Diatome US Inc</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Static line II ioniser</td>
<td>&nbsp;</td>
<td>Diatome US Inc</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CX100 Dry shipper</td>
<td>&nbsp;</td>
<td>Taylor-Wharton-Cryogenics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MSH loading station</td>
<td>&nbsp;</td>
<td>Gatan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ti E/B inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Custom</td>
</tr>
<tr>
<td>Cryo LM stage</td>
<td>&nbsp;</td>
<td>FEI</td>
<td>&nbsp;</td>
<td>Custom</td>
</tr>
<tr>
<td>TF30-polara</td>
<td>&nbsp;</td>
<td>FEI</td>
<td>&nbsp;</td>
<td>With UltraCam from Gatan</td>
</tr>
<tr>
<td>Amira</td>
<td>&nbsp;</td>
<td>Visage Imaging</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electron cryotomography of bacterial cells

בעוד הרבה ידוע כבר על חילוף החומרים הבסיסי של תאים חיידקיים, שאלות יסוד רבים עדיין ללא מענה מפתיע, כולל למשל איך הם יוצרים ולשמור צורות תאים מסוימים, להקים קוטביות, לבודד הגנום שלהם, ומחלקים. על מנת להבין את התופעות האלה, טכנולוגיות הדמיה כי יש צורך לגשר על הפער בין ההחלטה מיקרוסקופ אור פלואורסצנטי רזולוציה גבוהה יותר שיטות כגון קריסטלוגרפיה רנטגן ספקטרוסקופית NMR. אלקטרונים cryotomography (ECT) היא טכנולוגיה המתעוררים שעושה בדיוק את זה, המאפשר ultrastructure של תאים להיות דמיינו במצב כמעט טבעיות, בשלושה מימדים (3D), עם רזולוציה &quot;macromolecular&quot; (~ 4nm). 1, 2 ב ECT, התאים הם צילמו ב זגוגי, &quot;קפוא hydrated&quot; המדינה מיקרוסקופ אלקטרונים הילוכים cryo (cryoTEM) בטמפרטורה נמוכה (&lt;-180 ° C). עבור תאים דקים (עד ~ 500 ננומטר עובי 3), תאים שלמים הם לצלול קפוא בתוך רשתות התקשורת ברחבי EM ב cryogens כגון אתאן או אתאן / פרופאן תערובות. תאים עבה biofilms יכול להיות גם צילמו במצב זגוגי על ידי &quot;הקפאת בלחץ גבוה&quot; הראשון ולאחר מכן, &quot;cryo-חתך&quot; אותם. סדרה של דו מימדי תמונות הקרנת נאספים אז דרך המדגם כפי שהוא מוטה באופן הדרגתי לאורך אחד או שני צירים. שחזור תלת ממדי, או &quot;tomogram&quot; אז יכול להיות מחושב מן התמונות. בעוד ECT דורש מכשור יקר, בשנים האחרונות, זה נעשה שימוש במעבדות ספורות כדי לחשוף מבנים של איברים חיצוניים שונים, מבנים של מעטפות תאים שונים, עמדות ומבנים של סיבים cytoskeletal, ואת המיקומים של ארכיטקטורות macromolecular גדול מכלולים כגון מנועים flagellar, תאים פנימיים מערכים chemoreceptor. 1, 2 במאמר זה אנו וידאו להמחיש כיצד תאים התמונה עם ECT, כולל תהליכים של הכנת המדגם, איסוף נתונים, שחזור tomogram, ופרשנות של התוצאות באמצעות פילוח וגם מתאם במקרים מסוימים עם מיקרוסקופ אור.

Watch this Scientific Journal Video about Electron Cryotomography of Bacterial Cells at JoVE.com

Electron Cryotomography of Bacterial Cells

אנחנו כאן להמחיש כיצד להשתמש אלקטרונים cryotomography (ECT) כדי ללמוד את ultrastructure של תאים חיידקיים של כמעט ילידי מדינות, כדי &quot;macromolecular&quot; (~ 4 ננומטר) החלטה.

אלקטרונים Cryotomography של תאים חיידקיים

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

electron-cryotomography-of-bacterial-cells

Research

JoVE Journal

Biology

25.2K Views.  California Institute of Technology - Caltech. אנחנו כאן להמחיש כיצד להשתמש אלקטרונים cryotomography (ECT) כדי ללמוד את ultrastructure של תאים חיידקיים של כמעט ילידי מדינות, כדי "macromolecular" (~ 4 ננומטר) החלטה.

Video: Electron Cryotomography of Bacterial Cells

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

מחקרים Chemotaxis חיידקית שימוש מיקרופלואידיקה - ראיון

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

מדידת קשיחות כיפוף של תאים חיידקיים באמצעות מלכודת אופטית

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

משלוח חיידקית של RNAi effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

אינדוקציה וניתוח של אפיתל לmesenchymal מעבר

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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.

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

מקפיא פלאנג: כלי עבור מחקרי ultrastructural ו Immunolocalization של תאי השעית הילוכים מיקרוסקופים אלקטרונים

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

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

ממשק משתמש גרפי למעקב בסיוע תוכנה של ריכוז חלבון בהפרעות ניידות דינמיות

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

ניתוח מדויק, תפוקה גבוהה של התפתחות חיידקים

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

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.

הכנת הדוגמא ביולוגית על ידי הקפאת בלחץ גבוה, שיפור ניגודיות בסיוע מיקרוגל, ושרף מינימלי הטמעה של אמצעי הדמיה

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

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

שיטת ה-קריואיפקס לניתוח מיקרוסקופ אלקטרוני של לוקליזציה של חלבון ממברנה בתוך תאים שהשתמרו באופן מבנית

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

מארז גרפן של תאי יונקים קבועים כימית עבור מיקרוסקופאלקטרונים בשלב נוזלי

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...