Name: preparation and observation of thick biological samples by scanning transmission electron tomography
Uploaded: 2017-03-12T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography at JoVE.com

This work was funded by two ANR grants (ANR-11-BSV8-016 and ANR-10-IDEX-0001-02). We also acknowledge the PICT-IBiSA for providing access to chemical imaging equipment.

<ol>
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We have no conflict of interest to disclose.

במאמר זה, אנו מציגים פרוטוקול הכנת מדגם קונבנציונלי יחד עם מדריך צעד-אחר-צעד לביצוע ניתוח 3D על דגימות ביולוגיות עבות באמצעות STEM באמצעות מוקד טומוגרפיה. הטבעת שרף של דגימות ביולוגיות שמשה במשך עשרות שנתי 26, ופרוטוקולים חלופיים מותאמים מיני מדגמים שונים נ...

מאז ה -1970 המוקדמות, טומוגרפיה גישות במיקרוסקופ אלקטרוני הילוכים (TEM) היה בשימוש נרחב על האפיון המבני של דגימות ביולוגיות 1, 2, 3. הערעור של טומוגרפיה אלקטרונים הילוכים היה שזה יכול לשמש כדי לחקור מגוון רחב של מבנים ביולוגיים בקנה מידה ננומטרי, מן הארכיטקטורה של תאים ואת ultrastructure של אברונים למבנה מתחמי וחלבונים macromolecular. אף על פי כן, טומוגרפיה אלקטרוני הילוכים לא יכולה לשמש כדי לחקור דגימות עבות מאוד (יותר מ -0.5 מיקרומטר). ואכן, דגימות עבות לייצר אלקטרונים מפוזרים מדי, שהניבו נמוך יחס אות לרעש תמונות (SNR). בנוסף, טומוגרפיה כרוך האוסף של תמונות של דגימות מוטות, עם העובי לכאורה של המדגם עולה, עם זווית ההטיה. למרות פיזור קשיח ניתן לסנןמתוך באמצעות מסנני אנרגיה, הכמות הקריטית של אלקטרונים דרושים תמונות SNR גבוהות הוא הגיעה בקושי TEM. לפיכך, דגימות ביולוגיות עבות נחקרו רק באמצעות חתך 4. כמה דוגמאות לא יכול להיות פרוס: מסוימים עשויים להתדרדר במידה ולא נחתכים, ואחרים צריכים להיחקר בשלמותם על מנת להבין את המורכבות שלהם. גישה חלופית היא להשתמש TEM סריקה במצב 5, 6, 7, 8. סריקה מיקרוסקופי אלקטרוני הילוכים (STEM), המסלול האופטי של האלקטרונים שונה מזו TEM הקונבנציונלי. אלקטרונים העובר דרך המדגם ללא פיזור ניתן לאסוף על הציר האופטי עם גלאי שדה בהיר (BF) 9, ואילו אלה מפוזרים בצורה אלסטית ניתן לאסוף בזווית מסוימת המצייר האופטי עם גלאי כהה שדה (DF).היתרון השני של גזע הוא כי אלומת האלקטרונים הממוקדת נסרקה על פני השטח של המדגם, המאפשרת פיקסל אחר פיקסל האוסף של התמונות. למרות אלומת אלקטרונים מרחיב תוך שהם מעבירים דרך המדגם 10, ערכת איסוף המסוים הזה רגיש פחות אלקטרונים מפוזרים inelastically מ TEM קונבנציונאלי. יתר על כן, אין עדשה שלאחר הדגימה ב- STEM, ובכך הימנעות עיוותים כרומטיים שיכול להתרחש TEM. אורכו המצלמה יכול להיות מותאם כך גלאי BF בעיקר מזהה אלקטרוני unscattered. באמצעות גלאי DF ללמוד דגימות עבות אינו מומלץ בגלל פיזור מרובה, אשר מפיק תמונות מדויקות. במקום זאת, גלאי BF יכול לשמש 11. בעוד גזע יכול להפיק תמונות SNR גבוהות, יש לו עומק השדה נמוך יחסית בגלל התכנסות קרן גבוהה יחסית TEM, להקטין את כמות מידע מעמיק כי ניתן להשבה מתוך עבהדגימות. במקרה של מיקרוסקופי STEM תקן סטייה, שבו זווית ההתכנסות יכולה להיות גבוהה ככל 30 mrad, העומק של השדה יכול להיות נמוך מספיק כך שהמידע כי היא בפוקוס מקורו במישור מוקד של רק כמה ננומטרים . הגדרת קרן אלקטרונים במצב מקביל משפר את עומק השדה של אלומת אלקטרונים על חשבון הרזולוציה 12. עם זאת, התקנה זו אינה תמיד אפשרית. בכל פעם יש צורך להשתמש אלומת אלקטרונים התכנס, יש להשתמש בטכניקות אשר משפרות את עומק השדה של אלומת האלקטרונים כאשר לומדים דגימות עבות. מחקרים שנעשה לאחרונה דיווחו על רכישת מספר תמונות בבת מטוסים שונים מוקדי ברחבי המדגם לשחזר את הסכום המקסימאלי של מידע פוקוס 13, 14. שני המחקרים מתארים דרכים שונות לטפל במידע מן המטוסים מוקד שונים. Hovden et al.בשילוב בחלל פורה התמונות שנגבו לעבר מטוסים שונים מוקדים, ואת השיקום הסופי הושג ישירות ההופכי 3D התמר 13. לעומת זאת, Dahmen et al. פתח מנוע שחזור הקרן מתכנס לשחזר בחלל אמיתי נפח 3D מן המטוסים השונים מוקדי 14. המעבדה שלנו גם פתחה שיטת הדמית דגימות ביולוגיות עבות. האסטרטגיה שלנו הייתה שונה משתי השיטות שתוארו לעיל בכך שאנו התמזגנו המידע הוא בפוקוס המטוסים השונים מוקדי ושחזרנו את נפח 3D הסופי בחלל אמיתי באמצעות מקרן קרן מקביל 15. המטרה שלנו היתה לפתח שיטה שיכולה להתבצע בקלות בכל מעבדה במיקרוסקופ אלקטרונים. לשם כך, אנו מכוונים כדי לאסוף את התמונות מוקד כמות מוגבלת של זמן, להשוות את מסגרת הזמן של ניסויים טומוגרפיה קונבנציונליים. בנוסף, ג השיטה המוצעת שלנוולד להיות מותאם לשימוש עם סוגים שונים של יישור ותוכנות שחזור. בהקשר של הפרסום שלנו משנת 2015 15, רצינו לדמיין ולאפיין את ההתאוששות של עומק השדה, כך השתמשנו זווית למחצה התכנסות גדולה של 25 mrad. כאן, אנו מציגים צעד-אחר-צעד פרוטוקול לביצוע באמצעות מוקד ההדמיה ב- STEM פי השיטה שפותחה במעבדה שלנו בשנת 2015 15, ואנחנו מציגים כיצד הנתונים משנת 2015 היה מעובד. שיטה זו משחזרת מידע פוקוס ממטוסים כמה מוקדי ברחבי דגימה ביולוגית עבה (750 ננומטר) ומאפשרת שחזורי 3D באיכות גבוהה. במידת הצורך, את ההבדלים מתודולוגיה זו לעומת השיטות שנוקטים קבוצות אחרות גם מוצגים.

<table><tbody><tr><td>Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>2860</td><td></td></tr><tr><td>Epoxy resin</td><td>EMS</td><td>14120</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Add paraformaldehyde powder to PBS heated at approximately 60 &amp;deg;C.&lt;br/&gt; Increase pH by adding 1 N NaOH until no PFA powder is visible.</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G5882&amp;nbsp;</td><td></td></tr><tr><td>Osmium Tetroxyde</td><td>EMS</td><td>19150</td><td></td></tr><tr><td>Uranyl Acetate</td><td>Sigma-Aldrich</td><td>73943</td><td></td></tr><tr><td>Gelatin Capsule</td><td>EMS</td><td>70110</td><td></td></tr><tr><td>Triming and Histo knives</td><td>LFG Distribution</td><td>Diatome diamond knives</td><td></td></tr><tr><td>Electron Microscopy copper grid</td><td>LFG Distribution</td><td>G200-Cu</td><td></td></tr><tr><td>Grid Coating Pen</td><td>LFG Distribution</td><td>70624</td><td></td></tr><tr><td>Specimen Holder</td><td>JEOL</td><td>EM-21311 HTR</td><td></td></tr><tr><td>Electron Microscope</td><td>JEOL</td><td>JEM-2200FS</td><td></td></tr></tbody></table>

preparation and observation of thick biological samples by scanning transmission electron tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

במחקר שלנו, אלקטרונים הואצו עד 200 קילו וולט במיקרוסקופ אלקטרוני הילוכי אקדח פליטת שדה. תמונות נאספו במצב STEM BF באמצעות להתעכב זמן של 20 מיקרו-שניות. לגבי העיצוב של ההטייה סדרה באמצעות מוקד, מצאנו כי מרווחי מוקד 150 ננומטר נתן תוצאות משביעות רצון לחקר ultras...

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Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

הכנה תצפית של דגימות ביולוגיות העבה ידי סריקת הילוכים אלקטרונים טומוגרפיה

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

preparation-observation-thick-biological-samples-scanning

Research

JoVE Journal

Biology

9.2K Views.  Campus Universitaire d'Orsay. This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

Video: Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of the woodboring beetle, Chlorophorus caragana Xie &#38; Wang (2012), for both types of electron microscopy. The scanning electron microscopy (SEM) sample preparation protocol was based on sample chemical fixation, dehydration in a series of ethanol baths, drying, and sputter-coating. By adding Tween 20 (Polyoxyethylene sorbitan laurate) to the fixative and the wash solution, the insect body surface of woodboring beetle was washed more cleanly in SEM. This study&#39;s transmission electron microscopy (TEM) sample preparation involved a series of steps including fixation, ethanol dehydration, embedding in resin, positioning using fluorescence microscopy, sectioning, and staining. Fixative with Tween 20 enabled penetrate the insect body wall of woodboring beetle more easily than it would had been without Tween 20, and subsequently better fixed tissues and organs in the body, thus yielded clear transmission electron microscope observations of insect sensilla ultrastructures. The next step of this preparation was determining the positions of insect sensilla in the sample embedded in the resin block by using fluorescence microscopy to increase the precision of target sensilla positioning. This improved slicing accuracy.

In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

הכנה לדוגמה שיטת סריקה ומיקרוסקופ אלקטרון שידור לתוספות של החיפושית המשעממת

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of ...

Environment

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

שיטה להשגת טורי זגוגית מקטעים של מיקרואורגניזמים במשחק במיקרוסקופ אלקטרונים הילוכים

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

Engineering

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

עיבוד העובר, קליפת ביצה, ופטרייתי תרבות לסריקת מיקרוסקופ אלקטרוני

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

באמצעות Tomoauto: פרוטוקול לתפוקה גבוהה אוטומטית קריו אלקטרון טומוגרפיה

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Bioengineering

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.

הכנת הדוגמא ולמחקר במיקרוסקופ אלקטרונים הילוכים

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

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

זרימת עבודה טומוגרפיה של מערך לרכישה ממוקדת של מידע אודות אמצעי אחסון באמצעות סריקת מיקרוסקופיית אלקטרונים

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

תיאור קרטוגרפי תלת-ממדי של התא על ידי טומוגרפיה של קרני רנטגן רכות של Cryo

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

אסטרטגיות לאופטימיזציה של רכישת נתונים טומוגרפיה אלקטרוגנית

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

טומוגרפיה של קריו-אלקטרונים איסוף נתונים מרחוק וממוצע תת-טומוגרמה

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 &#181;m.