فإن الكتاب أود أن أشكر أعضاء نوغاليس مختبر في جامعة كاليفورنيا في بيركلي في المساعدة على إنشاء بروتوكولات الأولية وأعضاء وانغ مختبر في جامعة ييل في مساعدتهم لوضع بروتوكولات كاملة. نعترف أيضا من العاملين في البرد EM المرفق وعالية الأداء مركز الحاسوب في كلية ييل للطب لدعمهم. الأب هو رست سميث الأسرة.

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الإعلان عن أي تضارب في المصالح.

في هذا المقال نقدم بروتوكول مفصل لإعداد نموذج إعادة الإعمار وثلاثة الأبعاد للمجمع exosome السلبية باستخدام المجهر الإلكتروني تلطيخ. باستخدام هذه الطريقة ، حصلنا على اعادة اعمار 3D باستخدام طريقة عشوائية الميل المخروطية دون أي علم مسبق للهيكل. عشوائية طريقة الميل المخر?...

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single particle electron microscopy reconstruction of the exosome complex using the random conical tilt method

واحد المجهر الإلكتروني الجسيمات (EM) إعادة الإعمار وأصبح أداة شعبية في الآونة الأخيرة للحصول على ثلاثي الأبعاد (3D) هيكل المجمعات الجزيئات الكبيرة. بالمقارنة مع البلورات بالأشعة السينية ، فإنه لديه بعض مزايا فريدة. أول واحد الجسيمات EM إعادة الإعمار ليست في حاجة لبلورة نموذج البروتين ، والذي هو عنق الزجاجة في البلورات بالأشعة السينية ، وخاصة بالنسبة للمجمعات الجزيئات الكبيرة. وثانيا ، أنها لا تحتاج إلى كميات كبيرة من عينات البروتين. بالمقارنة مع ملليغرام من البروتينات اللازمة لبلورة واحدة التعمير EM يحتاج فقط الجسيمات الدقيقة عدة لترات من محلول البروتين في تركيزات نانو المولي ، وذلك باستخدام أسلوب تلطيخ السلبية EM. ومع ذلك ، على الرغم من الجمعيات القليلة التي تماثل الجزيئات عالية ، جسيم واحد هو EM محدودة في دقة منخفضة نسبيا (أقل من 1 نانومتر القرار) لعينات كثيرة لا سيما تلك دون التماثل. وتقتصر هذه التقنية أيضا حسب حجم الجزيئات قيد الدراسة ، أي 100 كيلو دالتون لعينات الملون سلبيا و300 كيلو دالتون لعينات مجمدة رطب بشكل عام. لعينة جديدة من الهيكل غير معروف ، ونحن عموما استخدام المعادن الثقيلة حل لتضمين الجزيئات التي تلطيخ السلبية. ثم يتم فحص العينة في انتقال مجهرا الكترونيا لاتخاذ ثنائي الأبعاد (2D) micrographs من الجزيئات. من الناحية المثالية ، وجزيئات البروتين وبنية متجانسة 3D كنه يحمل توجهات مختلفة في micrographs. يتم ترقيم هذه micrographs ومعالجتها في أجهزة الكمبيوتر باسم &quot;الجزيئات واحدة&quot;. ثنائي الأبعاد باستخدام المحاذاة وتقنيات التصنيف ، تتجمع جزيئات متجانسة في نفس وجهات النظر إلى طبقات. المتوسطات على تعزيز إشارة من الأشكال الجزيء 2D. بعد أن تعيين جسيمات ذات التوجه النسبي الصحيح (زوايا أويلر) ، سنكون قادرين على إعادة بناء الصور في حجم الجسيمات 2D 3D ظاهري. في واحد الجسيمات 3D إعادة الإعمار ، هو خطوة ضرورية لتعيين بشكل صحيح التوجه السليم لكل جسيم واحد. هناك عدة طرق لتعيين عرض لكل جسيم ، بما في ذلك إعادة الزاوي (1) والميل عشوائي المخروطية (RCT) الأسلوب 2. في هذا البروتوكول ، وصفنا ممارستنا في الحصول على إعادة الإعمار 3D المعقدة باستخدام الخميرة exosome السلبية EM تلطيخ وRCT. وتجدر الإشارة إلى أن لدينا بروتوكول المجهر الإلكتروني ومعالجة الصور يتبع المبدأ الأساسي لRCT ولكن ليست الطريقة الوحيدة لتنفيذ هذه الطريقة. علينا أولا كيف تصف لتضمين عينة البروتين إلى طبقة من ثاني اكسيد اليورانيوم فورمات - بسماكة مماثلة لحجم البروتين ، وذلك باستخدام الشبكة الكربون holey مغطاة بطبقة من الكربون رقيقة فيلم المستمر. ثم يتم إدخال العينة إلى انتقال مجهرا الكترونيا لجمع untilted (0 درجة) ويميل (55 درجة) زوجا من micrographs التي سيتم استخدامها لاحقا لتجهيز والحصول على النموذج الأولي لل3D exosome الخميرة. ولهذه الغاية ، نقوم RCT ثم صقل النموذج الأولي 3D باستخدام طريقة الإسقاط مطابقة التنقيح 3.

Watch this Scientific Journal Video about Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method at JoVE.com

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

توضح هذه المقالة أسلوب قياسي للحصول على ثلاثي الأبعاد (3D) إعادة بناء الجزيئات البيولوجية باستخدام المجهر الإلكتروني تلطيخ السلبية (EM). في هذا البروتوكول ، ونشرح كيفية الحصول على هيكل 3D من exosome السكيراء مجمع البيرة في قرار عشوائي المتوسطة باستخدام أسلوب إعادة الإعمار الميل المخروطية (RCT).

واحد المجهر الإلكتروني التعمير الجسيمات مجمع Exosome باستخدام أسلوب عشوائي الخيمة المخروطية

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

single-particle-electron-microscopy-reconstruction-exosome-complex

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Biology

23.4K Views.  Yale University. توضح هذه المقالة أسلوب قياسي للحصول على ثلاثي الأبعاد (3D) إعادة بناء الجزيئات البيولوجية باستخدام المجهر الإلكتروني تلطيخ السلبية (EM). في هذا البروتوكول ، ونشرح كيفية الحصول على هيكل 3D من exosome السكيراء مجمع البيرة في قرار عشوائي المتوسطة باستخدام أسلوب إعادة الإ...

Video: Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

طريقة لقياس حركية الانصهار الفيروسي على مستوى الجسيمات واحدة

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

المصدر المفتوح تحليل واحد الجسيمات لسوبر قرار المجهري مع VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

التحقيق "تضر الآثار من منخفضة الضغط البلازما التعقيم" على البقاء لجراثيم Bacillus نجحت "باستخدام خلية يعيش مجهرية"

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

والعزلة وتصوير الأسفار لتعمير واحد-الجسيمات Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

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.

إعداد العينات البيولوجية بالتجميد ذات الضغط العالي، وتعزيز النقيض ساعدت الموجات الدقيقة، والحد الأدنى راتنج التضمين لتصوير حجم

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.

طريقة CryoAPEX لتحليل المجهر الإلكتروني لتوطين بروتين الغشاء داخل الخلايا المحفوظة فائقة الهيكلية

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

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing the innate cellular fluorescence signature from each individual live cell in a population distributed in a three-dimensional (3D) space. The innate fluorescence signature of a cell is a collection of fluorescence signals emitted by various biomolecules within the cell. Previous studies established that innate fluorescence signatures reflect various cellular properties and differences in physiological status and are a rich source of information for cell characterization and identification. Innate fluorescence signatures have been traditionally analyzed at the population level, necessitating a clonal culture, but not at the single-cell level. CRIF is particularly suitable for studies that require 3D resolution and/or selective extraction of fluorescence signals from individual cells. Because the fluorescence signature is an innate property of a cell, CRIF is also suitable for tag-free prediction of the type and/or physiological status of intact and single cells. This method may be a powerful tool for streamlined cell analysis, where the phenotype of each single cell in a heterogenous population can be directly assessed by its autofluorescence signature under a microscope without cell tagging.

Here, a protocol is presented for optically extracting and cataloging innate cellular fluorescence signatures (i.e., cellular autofluorescence) from every individual live cell distributed in a three-dimensional space. This method is suitable for studying the innate fluorescence signature of diverse biological systems at a single-cell resolution, including cells from bacteria, fungi, yeasts, plants, and animals.

إعادة بناء خلية واحدة توقيعات الفلورية الفطرية من قبل المجهر Confocal

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing ...

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.

سير عمل التصوير المقطعي للصفيف للحصول المستهدف على معلومات الحجم باستخدام المجهر الإلكتروني المسح الضوئي

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

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.

إعداد الشبكة المجهرية كريو إلكترون للدراسات التي تم حلها عبر الزمن باستخدام نظام روبوتي جديد، Spotiton

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

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

المجهر المباشر لإعادة البناء البصري العشوائي للحوالق خارج الخلية في ثلاثة أبعاد

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...