Nous tenons à remercier Harald Hess et Eric Betzig pour l&#39;accès au microscope PALM pour preuve de principe expériences, Richard Fetter pour les protocoles de partage de fixation, des réactifs et des encouragements. Nous remercions Michael Davidson, Geraldine Seydoux, Stefan Eimer, Rudolf Leube, Keith Nehrke, Christian Frøkjr-Jensen, Aude Ada-Nguema et Marc Hammarlund pour des constructions d&#39;ADN. Nous remercions également Carl Zeiss Inc pour fournir un accès à l&#39;Zeiss PAL-M, une version bêta du microscope Zeiss Elyra P.1 PALM.

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Production et le libre accès à cet article est sponsorisé par Carl Zeiss, Inc

Nous décrivons ici comment préserver protéines fluorescentes en plastique, de localiser les protéines fluorescentes dans les sections, et l&#39;image de l&#39;ultrastructure en microscopie électronique. Les protéines ont été localisées en dessous de la limite de diffraction en microscopie PALM à résolution nanométrique. Pour adapter ce protocole aux spécimens particuliers, les paramètres suivants doivent être considérés: fluorophore, la quantification et l&#39;alignement. Le choix d&#39;une protéine fluorescente ou fluorophore organique dépend de l&#39;application et du système modèle. Nous avons testé une variété de protéines fluorescentes, y compris EGFP, YFP, Citrine, mEosFP, mEos2, tdEos, Morange, PA-mCherry et Dendra 12. La préservation de la fluorescence de chaque fluorophore était similaire, ce qui suggère que toutes les protéines fluorescentes peuvent être conservés à l&#39;aide de la méthode décrite. Nous avons choisi tdEos car il a bien exprimé dans C. elegans, les protéines demeurées fonctionnelles lorsqu&#39;il est fusionné à tdEos, et parce que sonphoto-activation caractéristiques sont optimales pour la microscopie PALM. Cependant, l&#39;expression d&#39;agrégation ou pas de tdEos a parfois été observée 12. Selon l&#39;application, un fluorophore différent peut-être mieux. Dans de nombreux cas, il n&#39;est pas nécessaire d&#39;utiliser une protéine fluorescente photo-activée. Simple microscopie électronique corrélative de fluorescence ne nécessite pas de photo-activé protéine fluorescente. GFP ou des colorants organiques peuvent être utilisés à fluorescence image à partir de protéines marquées dans les sections ci-dessus de la limite de diffraction. Par exemple, une boîte d&#39;image d&#39;un axone dans un neuropile en utilisant la microscopie par fluorescence et de corréler le signal de fluorescence d&#39;un axone particulier dans une micrographie électronique d&#39;imagerie par fluorescence sur l&#39;un microscope à fluorescence. D&#39;autres techniques de super-résolution, telles que la microscopie d&#39;émission stimulée épuisement (STED) 12, la microscopie appauvrissement de l&#39;état fondamental suivi par le retour molécule individuelle (GSDIM) 13, etmicroscopie illumination structurée (SIM) 14, n&#39;ont pas besoin de photo-activés protéines fluorescentes. En outre, de super-résolution des techniques d&#39;imagerie utilisant des colorants organiques 9,15,16 ou la propriété intrinsèque des sondes fluorescentes 17 sont facilement applicables. Dans PALM, le nombre de molécules peuvent être quantifiés en raison de fluorescence de chaque molécule est séparé spatialement et temporellement. Cependant, la quantification peut être trompeuse pour quatre raisons: oxydation, sous-estimation, surdénombrement, et la surexpression. Tout d&#39;abord, une fraction des protéines fluorescentes peuvent être dénaturé ou oxydée pendant le traitement d&#39;échantillons 5,12. Bien que ~ 90% du signal de fluorescence a été préservée grâce à la fixation et l&#39;inclusion dans notre protocole, l&#39;oxydation de la protéine fluorescente peut se produire après que l&#39;échantillon a été sectionné et la surface exposée à l&#39;oxygène. D&#39;autre part, l&#39;activation des protéines photo-activables est stochastique, et ainsi de multiples molécules peuvent êtreactivés dans une tache de diffraction donnée limitée 8. Fluorescence des molécules multiples apparaîtra comme une tache, et donc le nombre total de protéines vont être sous-estimés. Troisièmement, un problème similaire peut conduire à surdénombrement. Dans PALM, chaque protéine fluorescente est localisée puis «effacées» par le blanchiment. Toutefois, les protéines fluorescentes peut revenir de l&#39;état sombre sans être définitivement blanchi 18. Ces molécules seront ensuite comptée plusieurs fois. Quatrièmement, les protéines marquées sont exprimés en transgènes et sont souvent présents en plusieurs exemplaires, ce qui peut conduire à une surexpression. Par conséquent, la quantification de PALM peut être utilisée pour estimer, mais pas de déterminer avec précision le nombre de molécules dans un lieu donné. L&#39;alignement d&#39;une image PALM avec une micrographie électronique peut aussi être difficile à cause de la différence de résolution en microscopie optique et électronique et la distorsion provoquée par le faisceau d&#39;électrons. Les particules d&#39;or servent bien loclisés marqueurs repères dans micrographies électroniques. Cependant, fluoresecence de particules d&#39;or n&#39;est pas photo-activé, et apparaît comme une grande tache limitée par la diffraction. Ainsi, la mise en place d&#39;une image de fluorescence sur une micrographie électronique est une estimation. Distorsions peuvent également survenir des interactions des électrons avec la partie plastique. Résines acryliques tels que les GMA sont moins stables sous le faisceau d&#39;électrons, et les dimensions de la matière plastique peut être modifié. Dans ces circonstances, l&#39;alignement de la fluorescence avec ultrastructure peut exiger une transformation non linéaire des marqueurs fiduciaires.

<table border="1"><tbody><tr><td> Le nom du réactif ou de l&#39;équipement </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Haute pression congélateur </td><td> ABRA </td><td> HPM 010 </td><td> EMPACT et HPM 100 de Leica ou hpf-01 de Wohlwend peut également être utilisé. </td></tr><tr><td> Automatisé unité cryosubstitution </td><td> Leica </td><td> AFS 2 </td><td> AFS 1 peut également être utilisé. </td></tr><tr><td> Zeiss PALM </td><td> Zeiss </td><td> ELYRA P.1 </td><td> Nikon et Vutara vendent aussi des microscopes PALM commerciales. </td></tr><tr><td> Microscope électronique à balayage </td><td> FEI </td><td> Nouvelle nano </td><td> D&#39;autres haute résolution des microscopes MEB peuvent être utilisées. </td></tr><tr><td> Acétone </td><td> EMS </td><td> RT10016 </td><td></td></tr><tr><td> L&#39;éthanol </td><td> Sigma-Aldrich </td><td> 459844-1L </td><td></td></tr><tr><td> Osmium tetroxide </td><td> EMS </td><td> RT19134 </td><td></td></tr><tr><td> Le permanganate de potassium </td><td> EMS </td><td> RT20200 </td><td></td></tr><tr><td> L&#39;albumine de sérum bovin </td><td> Sigma-Aldrich </td><td> A3059-50G </td><td></td></tr><tr><td> L&#39;acétate d&#39;uranyle </td><td> Polysciences </td><td> 21447-25 </td><td> pH de l&#39;acétate d&#39;uranyle de cette compagnie est légèrement plus élevé. </td></tr><tr><td> Méthacrylate (GMA) </td><td> SPI </td><td> 02630-AA </td><td> Acide faible, la qualité du TEM. </td></tr><tr><td> N, N-diméthyl-p-toluidine </td><td> Sigma-Aldrich </td><td> D9912 </td><td></td></tr><tr><td> Tubes Cryo </td><td> Nalgene </td><td> 5000-0020 </td><td></td></tr><tr><td> Flacons en verre </td><td> EMS </td><td> 72632 </td><td></td></tr><tr><td> Film de Aclar </td><td> EMS </td><td> 50425-10 </td><td></td></tr><tr><td> BEEM capsule </td><td> EBSciences </td><td> TC </td><td> Polypropylène </td></tr><tr><td> 3/8 &quot;DISC poinçons </td><td> Ted Pella </td><td> 54741 </td><td></td></tr><tr><td> Les nanoparticules d&#39;or </td><td> microsphères-nanospheres.com </td><td> 790122-010 </td><td> Demander 2x solution concentrée </td></tr></tbody></table>

nano-fem: protein localization using photo-activated localization microscopy and electron microscopy

Cartographie de la répartition des protéines est essentielle pour comprendre la fonction des protéines dans une cellule. La microscopie à fluorescence est largement utilisé pour la localisation des protéines, mais le contexte subcellulaire est souvent absent dans les images de fluorescence. Immuno-microscopie électronique, d&#39;autre part, peuvent localiser les protéines, mais la technique est limitée par un manque d&#39;anticorps compatibles, la préservation de la morphologie des pauvres et parce que la plupart des antigènes sont pas exposés à la surface de l&#39;échantillon. Approches corrélatives peuvent acquérir l&#39;image de fluorescence d&#39;une cellule toute la première, soit à partir de l&#39;immuno-fluorescence ou de protéines génétiquement marqués. L&#39;échantillon est ensuite fixé et intégré pour la microscopie électronique, et les images sont corrélées 1-3. Cependant, l&#39;image basse résolution fluorescence et le manque de marqueurs fiduciaires obstacle à la localisation précise des protéines. Alternativement, l&#39;imagerie de fluorescence peut être fait après préservant l&#39;échantillon dans du plastique. Encette approche, le bloc est sectionné, et les images de fluorescence et micrographies électroniques de la même section sont corrélées 4-7. Cependant, la limite de diffraction de la lumière dans l&#39;image corrélé obscurcit les emplacements des molécules individuelles, et la fluorescence s&#39;étend souvent au-delà de la limite de la cellule. Nano-résolution de fluorescence par microscopie électronique (nano-FEM) est conçu pour localiser les protéines à l&#39;échelle nanométrique par imagerie les mêmes sections en utilisant la microscopie localisation photo-activé (PALM) et la microscopie électronique. PALM surmonte la limite de diffraction individuels d&#39;imagerie par fluorescence des protéines et ensuite la cartographie du centroïde de chaque tache fluorescente 8-10. Nous décrivons la technique de nano-FEM en cinq étapes. Tout d&#39;abord, l&#39;échantillon est fixe et intégrée en utilisant des conditions qui préservent la fluorescence des protéines marquées. D&#39;autre part, les blocs de résine sont coupés en segments ultraminces (70-80 nm) qui sont montéssur un verre de recouvrement. Troisièmement, la fluorescence est représenté dans ces sections en utilisant le microscope Zeiss PALM. Quatrièmement, électrons structures denses sont imagés dans ces mêmes articles à l&#39;aide d&#39;un microscope électronique à balayage. Cinquièmement, les micrographies de fluorescence et électronique sont alignées en utilisant des particules d&#39;or comme marqueurs fiduciaires. En résumé, la localisation subcellulaire des protéines marquées par fluorescence peut être déterminée à une résolution nanométrique dans environ une semaine.

Watch this Scientific Journal Video about Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy at JoVE.com

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Nous décrivons une méthode pour localiser les protéines marquées par fluorescence en microscopie électronique. Fluorescence est d&#39;abord localisée en utilisant la microscopie localisation photo-activé sur coupes ultrafines. Ces images sont ensuite alignées à micrographies électroniques de la même section.

Nano-FEM: la localisation des protéines en utilisant la microscopie Localisation Photo-actif et microscopie électronique

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

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15.9K vues.  University of Utah. Nous décrivons une méthode pour localiser les protéines marquées par fluorescence en microscopie électronique. Fluorescence est d'abord localisée en utilisant la microscopie localisation photo-activé sur coupes ultrafines. Ces images sont ensuite alignées à micrographies électroniques de la même section.

Vidéo: Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

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Biologie

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Quantification de l&#39;expression protéique et co-localisation utilisant multiplexé Immuno-histochimie Coloration et multispectrale Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Trois dimensions Super Resolution Microscopie de Filaments F-actine par interférométrique photoactive Localisation Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Microscopie multicolore localisation des protéines de la Membrane unique dans les organites des cellules mammifères vivants

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

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.

Préparation de l’échantillon biologique par congélation à haute pression, amélioration du contraste assistée par micro-ondes et minime résine enrobage pour l’imagerie de Volume

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

Visualisation de la structure des ganglions lymphatiques et de la localisation cellulaire à l'aide de la microscopie confocale ex-Vivo

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

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.

Méthode CryoAPEX pour l’analyse de la microscopie électronique de la localisation des protéines membranaires dans les cellules ultrastructuralement conservées

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

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Analyse du réseau α-Actinine dans les cardiomyocytes dérivés de l’iPSC à l’aide d’une microscopie de localisation à molécule unique

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

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.

Préparation de grille cryo-microscopique électronique pour des études résolues dans le temps à l’aide d’un nouveau système robotique, Spotiton

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

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.