我々は固定プロトコル、試薬や励ましを共有するための証明の原理実験、リチャード·フェッター用のPalm顕微鏡へのアクセスのためにハラルドヘスとBetzigエリックに感謝します。我々は、マイケルソン、ジェラルディンSeydoux、ステファンアイマー、ルドルフロイベ、キースNehrke、クリスチャンFrøkjr·ジェンセン、DNA構築のためのオードADA-ヌグエマとMarc Hammarlundに感謝します。我々はまた、ツァイスPAL-M、ツァイスELYRA P.1やし顕微鏡のベータ版へのアクセスを提供するためにカールツァイス社に感謝します。

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この記事への生産とフリーアクセスはカールツァイス社が主催している

ここでは、プラスチック製で、電子顕微鏡を用いた超微細構造をセクションで蛍光タンパク質をローカライズし、画像の蛍光タンパク質を保持する方法について説明します。タンパク質は、ナノメートル分解能にPALM顕微鏡を用いた回折限界以下局在していた。特定の標本にこのプロトコルを適応させるために、次のパラメータを考慮する必要があります。フルオロフォア、定量化、および配置。 蛍光タンパク質や有機蛍光色素の選択は、アプリケーションとモデルシステムによって異なります。我々は、EGFP、YFP、シトリン、mEosFP、mEos2、tdEos、mOrange、PA-mCherry、とdendronの複数形12を含む蛍光タンパク質の様々なテストをしている。各蛍光色素からの蛍光の保全は、すべての蛍光タンパク質が記載された方法を用いて保存することができることを示唆して、類似していた。それはCでうまく表現ので、我々はtdEosを選んだelegansは 、タンパク質はtdEosに融合したときに機能的に推移し、そのため光活性化特性は、ヤシの顕微鏡検査に最も適していた。しかし、tdEosの凝集したか失敗した式が、時折12観察されている。 アプリケーションに応じて、異なる蛍光団はより適しているかもしれません。多くの場合、それは光活性化蛍光性タンパク質を使用する必要はありません。単純な相関蛍光電子顕微鏡では、光活性化蛍光タンパク質を必要としません。 GFPまたは有機色素は、回折限界上記のセクションでタグ付けされたタンパク質からの蛍光画像に使用することができます。例えば、人はイメージ、蛍光顕微鏡を用いて神経網の軸索とイメージングによる電子顕微鏡写真で特定の軸索蛍光顕微鏡で蛍光を蛍光シグナルを関連付けることができます。このような誘導放出の枯渇顕微鏡（STED）12、個々の分子リターン（GSDIM）13続いて基底状態の枯渇顕微鏡のような他の超解像技術や、構造化照明顕微鏡（SIM）14は 、光活性化蛍光タンパク質を必要としません。また、有機色素9,15,16または蛍光プローブ17の固有の特性を使用した超解像イメージング技術は、容易に適用可能である。 各分子の蛍光が空間的にも時間的に分離されているので、手のひらに、分子数を定量化することができる。酸化、過少、過大、および過剰発現：しかし、定量化は、4つの理由のために誤解を招く可能性があります。まず、蛍光タンパク質の画分を変性またはサンプル処理5,12間に酸化することができる。蛍光シグナルの約90％が我々のプロトコルでの固定と包埋を通して維持されたが、試料が切削されており、表面が酸素にさらされた後に、蛍光タンパク質の酸化が発生することがあります。第二に、光活性タンパク質の活性化は、確率的であるため、複数の分子ができる所定の回折限界のスポット8で活性化。複数の分子からの蛍光は1スポットとして現れるでしょう、そして、その結果、タンパク質の総数はundercountedされます。第三に、同様の問題は過大につながることができます。手のひらに漂白することにより、それぞれの蛍光タンパク質は、ローカライズされた後、 &quot;消去&quot;。しかし、蛍光タンパク質は18永久に漂白されることなく、暗い状態から戻ることができます。このような分子は、複数回カウントされます。第四に、タグ付けされたタンパク質は、トランスジーンとして表現さ​​れ、しばしば過剰発現につながることができます複数のコピーに存在しています。したがって、Palmから定量化は、与えられた場所での分子の数を推定するが、正確には判断できないために使用することができます。 電子顕微鏡写真とヤシの画像の位置合わせもあるため、電子ビームによる光と電子顕微鏡と歪みで解像度の違いから挑戦することができます。金粒子は、しっかりとlocのサーブ電子顕微鏡写真で基準マーカーを未実現。しかし、金粒子からfluoresecenceは、光活性化ではなく、大規模な回折限界スポットとして表示されます。このように、電子顕微鏡写真上の蛍光画像の配置は推定値です。歪みもプラスチックセクションと電子の相互作用から生じる可能性があります。そのようなGMA等のアクリル系樹脂は、電子ビームの下にあまり安定しており、プラスチック製の寸法を変更することができます。このような状況下では、超微細構造と蛍光を整列する基準マーカーの非線形変換が必要な場合があります。

<table border="1"><tbody><tr><td>試薬や機器の名称</td><td>会社</td><td>カタログ番号</td><td>注釈</td></tr><tr><td>高圧冷凍庫</td><td>アブラ州</td><td> HPM 010 </td><td> WohlwendからライカやHPF-01からEMPactとHPM 100を使用することもできます。 </td></tr><tr><td>自動凍結置換ユニット</td><td>ライカ</td><td> AFSの2 </td><td> AFSの1を使用することもできます。 </td></tr><tr><td>ツァイスPALM </td><td>ツァイス</td><td> ELYRA P.1 </td><td>ニコンとVutaraはまた、商業やし顕微鏡を販売しています。 </td></tr><tr><td>走査型電子顕微鏡</td><td> FEI </td><td>ノバナノ</td><td>他の高分解能SEM顕微鏡を使用することができます。 </td></tr><tr><td>アセトン</td><td> EMSの</td><td> RT10016 </td><td></td></tr><tr><td>エタノール</td><td>シグマアルドリッチ</td><td> 459844-1L </td><td></td></tr><tr><td>オスミウムトンetroxide </td><td> EMSの</td><td> RT19134 </td><td></td></tr><tr><td>過マンガン酸カリウム</td><td> EMSの</td><td> RT20200 </td><td></td></tr><tr><td>ウシ血清アルブミンから</td><td>シグマアルドリッチ</td><td> A3059-50G </td><td></td></tr><tr><td>酢酸ウラニル</td><td>ポリサイエンス</td><td> 21447から25 </td><td>この会社の酢酸ウラニルのpHがわずかに高くなっています。 </td></tr><tr><td>グリコールメタクリレート（GMA） </td><td> SPI </td><td> 02630-AA </td><td>低酸、TEM用グレード。 </td></tr><tr><td> N、N-ジメチル-p-トルイジン</td><td>シグマアルドリッチ</td><td> D9912 </td><td></td></tr><tr><td>クライオバイアル</td><td>ナルゲン</td><td> 5000-0020 </td><td></td></tr><tr><td>ガラスバイアル</td><td> EMSの</td><td> 72632 </td><td></td></tr><tr><td> ACLARフィルム</td><td> EMSの</td><td> 50425から10 </td><td></td></tr><tr><td> BEEMカプセル</td><td> EBSciences </td><td> TC </td><td>ポリプロピレン</td></tr><tr><td> 3月8日 &quot;DISCパンチ</td><td>テッドペラ</td><td> 54741 </td><td></td></tr><tr><td>金ナノ粒子</td><td>ミクロス-nanospheres.com </td><td> 790122-010 </td><td> 2倍濃縮液を要求</td></tr></tbody></table>

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

タンパク質の分布をマッピングすると、細胞内のタンパク質の機能を理解するために不可欠です。蛍光顕微鏡は、広範囲のタンパク質局在するために使われますが、細胞内の文脈はしばしば蛍光画像では欠如しているされています。免疫電子顕微鏡法は、他の一方で、タンパク質をローカライズすることができますが、その方法は互換性のある抗体の不足によって制限され、形態学の貧しい保全とほとんどの抗原は、試料表面に露出されていないため。相関アプローチはどちら免疫蛍光または遺伝的にタグ付けされたタンパク質から、最初の全細胞からの蛍光画像を取得することができます。次に、試料を固定し、埋め込 ​​まれた電子顕微鏡観察のため、画像は1から3まで相関しているされています。しかし、低解像度の蛍光画像と基準マーカの欠如は、タンパク質の正確な局在を排除。 あるいは、蛍光イメージングは​​、プラスチック中の検体を保存した後に行うことができます。でこのアプローチは、ブロックが区分され、蛍光画像と同じ断面の電子顕微鏡写真を4-7相関している。しかし、相関画像の光の回折限界は個々の分子の位置を曖昧にし、蛍光はしばしばセルの境界を越えて延びている。 ナノ分解能蛍光電子顕微鏡（ナノFEM）を撮像することにより、ナノスケールでの光活性化ローカライゼーション顕微鏡（PALM）と電子顕微鏡を使用して同じ部分をタンパク質をローカライズするために設計されています。 Palmはイメージング個々の蛍光タンパク質による回折限界を克服し、その後、各蛍光スポット8-10の重心をマッピングする。 我々は5つのステップでナノFEM手法を概説しています。まず、試料を固定し、タグ付けされたタンパク質の蛍光を維持する条件を使用して、埋め込まれています。第二に、樹脂ブロックが搭載されている極薄のセグメント（70-80 nm）とに区画されているカバーガラス上に。第三に、蛍光はツァイスPALM顕微鏡を用いて、これらのセクションに結像される。第四に、電子密度の高い構造は、走査型電子顕微鏡を用いて、これらの同じセクションに結像される。第五に、蛍光および電子顕微鏡写真は、基準マーカーとしての金粒子を用いて整列されます。要約すると、蛍光標識タンパク質の細胞内局在は、約1週間でナノメートルの分解能で決定することができる。

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

我々は、電子顕微鏡写真で蛍光標識タンパク質をローカライズする方法について説明します。蛍光は、第一超薄切片に光活性化ローカライゼーション顕微鏡を使ってローカライズされます。これらの画像は、同じ断面の電子顕微鏡写真に整列されます。

ナノFEM：写真活性化ローカライゼーション顕微鏡と電子顕微鏡を用いてタンパク質の局在

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

nano-fem-protein-localization-using-photo-activated-localization

Research

JoVE Journal

Biology

15.9K ビュー.  University of Utah. 我々は、電子顕微鏡写真で蛍光標識タンパク質をローカライズする方法について説明します。蛍光は、第一超薄切片に光活性化ローカライゼーション顕微鏡を使ってローカライズされます。これらの画像は、同じ断面の電子顕微鏡写真に整列されます。

ビデオ: 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 ...

生物学

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.

多重化された免疫組織化学染色およびマルチスペクトルイメージングを用いたタンパク質発現と共局在の定量

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.

干渉光活性化ローカライゼーション顕微鏡（iPALM）によってF-アクチンフィラメントの三次元超解像顕微鏡

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.

単一の膜タンパク質生きた哺乳類細胞の細胞内小器官のマルチカラー ローカリゼーション顕微鏡

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.

高圧急速凍結、マイクロ波を用いたコントラスト強調、およびボリューム イメージングのために埋め込む最小限の樹脂による生体試料作製

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.

生体共焦点顕微鏡を用いてリンパ節構造と細胞局在化を可視化

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.

超構造保存細胞における膜蛋白質局在化の電子顕微鏡分析のためのクライオAPEX法

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.

一分子局在顕微鏡を用いたヒトiPSC由来心筋細胞におけるαアクチニンネットワークの解析

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.

新しいロボットシステム、スポティトンを用いた時間分解研究のためのクライオ電子微視的グリッドの準備

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.