私たちは、元の単一分子調製プロトコルのためにDr.ティル・ベッキングに感謝します。
この作業は、NSF 許可 1121972 (SS) によってサポートされました。

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著者らは、この論文に競合する金銭的利益を持っていません。

AFMおよび高解像蛍光イメージングによる単一のビリオンイメージングは、CryoEM断層撮影の代替方法として使用することができます。これらの方法論のそれぞれは、それぞれの特定の強みを持っています。例えばAFMは、サンプルまたは内部ウイルスタンパク質のタグ付けに必要のないWTビリオンで行うことができます。ウイルス構造の位置は、ビリオンの弾性特性への寄与から減少する。
新たに開発されたウイルス逆遺伝的アプローチと組み合わせて単一のビリオン超分解能イメージングは、低コピー数ウイルスタンパク質20を局在化するための強力な技術となる。タンパク質を蛍光タンパク質と融合させたタンパク質との置換を伴う組換えウイルスは、これらの逆遺伝的アプローチを用いて作製および精製することができる。超解像イメージングは遺伝的タグ付けによる特異性を有するが、変異型ウイルスの使用が必要である。
AFM と超解像度イメージングは、非常に類似したサンプル準備を利用する無料の方法です。この論文で概説した方法は、初期の単一分子イメージングアッセイの形態であり、ビリオンのトポロジーにほとんど影響を与えずに単一のビリオンを固定することを可能にする。

電荷ベースの非特異的相互作用は、原子間力顕微鏡1,2におけるビリオンの接着に日常的に使用される。これらの技術は、非常に硬いキャプシド3-6で非エンベロープビリオンに使用すると特にうまく機能します。これらの技術はサンプルを固定化するのに非常に有効であるが、それらは表面にタンパク質の非特異的結合を防ぐものではない。非特異的結合は、AFMと様々な抗体を用いてサンプルのインキュベーションを必要とする超解像蛍光技術を用いてビリオンを画像化しようとすると問題を引き起こす可能性がある。ここでは、ビリオンの特定の固定化のためのサンプル調製方法を概説する。
ポリ(エチレングリコール)(PEG)は、ポリ(L-リジン)(PLL)に移植され、ガラス表面に吸着し、ガラス7とのタンパク質の静電相互作用のための重要なブロックを提供する。ガラス表面上の単一分子の固定化を必要とする単分子アッセイは、この性質を利用して、これを用いて、PEGベースの固定化技術8~10を作成した。この調製物はまた、クラトリンケージをガラス表面11に固定化するとともに、コントロールセル接着のための均質なフィブロネクチンコーティングを作成するためにも使用された。
単一分子イメージング方法からガラス表面へのPLL-g-PEG吸着に基づくサンプル調製方法を採用し、静脈性口内炎ウイルス(VSV)の単一ウイルスのイメージングに適用しました。これらの単一のビリオンは、原子間力顕微鏡(AFM)を用いて画像化した。同様の実験もコントロールとして機能化されたビーズに対して行った。また、超解像蛍光顕微鏡法によってビリオンを画像化し、Alexa 647で標識されたVSV-G抗体を用いて、単一のビリオンのエンベロープの画像を作成した。高解像度蛍光イメージングは、単一分子の局在化を利用して、画像13〜15を作成する。fPALMバイ平面イメージングは、光軸15,16に沿って平面および50nm解像度の20nmを有する単一分子の局在化を可能にする。この2平面技術は、本研究に存在する超解像度蛍光画像を画像化するために使用された。同様の結果を持つ代替技術は、STORM13,17,18です。
この論文で概説した手順によってガラスに固定されたVSVのAFMおよび超分解能蛍光像の両方が、最小非特異的相互作用19を有するガラスへのビリオンの特異的結合を示した。ここでは、AFMおよび超解像度蛍光イメージング実験用のサンプル調製プロトコルを紹介します。簡単に言うと:きれいなガラスカバーリップはPLL-g-PEGおよびPLL-g-PEG-ビオチンの混合物を吸着することによって機能する。この薄膜は、コーティングされた表面に15〜25%の機能化ビオチンを提供するように設計される典型的な。カバーリップはさらに四量体アビジンでインキュベートされる。ビオチン化されたウイルス抗体は、ビリオンのユニークな結合部位を作成するために使用されます。抗体の結合は2つの方法で行うことができる。
最初の方法はAFMのために最適化されていますが、超解像蛍光イメージングには適しています。この方法では、アビジン被覆表面は、ウイルス接着の前にビオチン化抗体で処理される。固定化されたビリオンは、エンベロープを覆い、ビリオンの超解像蛍光イメージングを可能にするために、Alexa 647標識抗体で処理されます。
2番目の方法は、アビジン処理面にウイルスを付着させる前に、ビオチン化抗体およびAlexa 647標識抗体を用いてウイルスを溶液中で攻撃することです。この方法は、ウイルスエンベロープの回復が重要である超解像蛍光イメージング実験に最適化されています。この方法は、ウイルス表面に均一な抗体被覆を可能にする利点を有する。ビオチン化抗体は、ウイルスエンベロープの外側に高濃度の蛍光標識を維持しながら、アビジン処理面に対するウイルスの十分な吸着を可能にする比率でAlexa 647標識ウイルス抗体と混合することができる。これらのAlexa 647標識ウイルス抗体はビオチン化されておらず、その過剰はバックグラウンドノイズを低減するために洗い流され得る。

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			<td height="84" style="height:84px;width:216px;">Glass coverslips (fPALM)</td>
			<td style="width:71px;">Electron Microscopy Services</td>
			<td style="width:119px;">72225-01</td>
			<td style="width:167px;">25 mm Coverslips</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%)&#160;</td>
			<td style="width:71px;">SuSoS&#160;</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Stock: 0.5 mg/ml in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NeutrAvidin biotin-binding protein</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A2666</td>
			<td style="width:167px;">Stock: 0.25 mg/ml in PBS</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Alexa Fluor 647 goat anti-rabbit IgG (H+L)&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A21245</td>
			<td style="width:167px;">1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">&#945;-VSV-G&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">ab34774</td>
			<td style="width:167px;">1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer</td>
		</tr>
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			<td height="21" style="height:21px;">Gluox</td>
			<td style="width:71px;">Sigma</td>
			<td>G2133-250KU&#160;</td>
			<td>Glucose oxidase type seven from Aspergillius&#160;</td>
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			<td height="21" style="height:21px;">Catalase</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C40-100mg&#160;</td>
			<td>Catalase from Bovine liver&#160;</td>
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			<td height="21" style="height:21px;">MEA</td>
			<td style="width:71px;">Sigma</td>
			<td>30070-10G&#160;</td>
			<td>Cysteamine (MEA</td>
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			<td height="21" style="height:21px;">Stock Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>50 mM Tris-HCl (pH 8.0) + 10 mM NaCl + 10% glucose</td>
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			<td height="21" style="height:21px;width:216px;">NTE</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>10 mM Tris pH 7.4, 100 mM NaCl, 66 mM EDTA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Slide-A-Lyzer, mini dialysis unit</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>10,000 MW</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microscope Cover Glass: 35 CIRCLE #1</td>
			<td style="width:71px;">Fisherbrand</td>
			<td style="width:119px;">35 CIRCLE #1</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

sample preparation for single virion atomic force microscopy and super-resolution fluorescence imaging

ガラス表面へのビリオンの固定化は、単一のビリオンイメージングにおいて重要なステップです。ここでは、単一分子イメージングアッセイから、ガラス表面に特異性を持つ単一のビリオンの接着を可能にする技術を紹介します。この調製物は、PLL-g-PEGとPLL-g-PEG-ビオチンの混合物でガラスの表面をグラフトし、アビジンの層を追加し、最終的にビオチン化されたウイルス特異的抗体の付着を通じてビリオンアンカーを作成することに基づいています。この手法は、原子間力顕微鏡(AFM)や超解像蛍光イメージングなど、さまざまな実験に応用してきました。このサンプル調製方法は、表面へのビリオンの制御接着をもたらす。

AFMを使用した単一のビリオンイメージング:
上で概説したサンプル調製プロトコルは、野生型のビリオンをガラス表面に固定するのに用いた。VSVビリオンは、長さ180nm、直径80nmの弾丸形状です。この技術が適用される可能性のある様々なビリオンがあるので、この概念はビオチン化された36 nmビーズにもまた実証されています。結果として得られた AFM 実験を 図 1に示します。VSVビリオンは、非エンベロープウイルス(GPa)と比較してヤング率(100 MPa)が低い点に注意することが重要です。単一のビリオンVSVの特定の画像は、硬いカンチレバーを有する周囲条件下でタッピングモード下で得られた。その目的は、ウイルス内の余分なタンパク質密度がAFM画像内のバンプとして見えるようになる点までウイルスを変形させることを目的としている。この方法は、ウイルス空洞19内の余分なタンパク質密度を検出するために使用される。
単一のビリオン超解像度イメージング:
Alexa 647標識VSV-G抗体は、方法5Aを用いた超解像実験のために個々のVSVビリオンのエンベロープをコーティングするために使用された。テザリング密度と低い非特異的結合を示すために、サンプルの大きなスキャンを 図2Aに示します。代表的なビリオン上のウイルスエンベロープの回復は 図2B( 青色同一体面)に示されている。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51366/51366fig1highres.jpg" src="/files/ftp_upload/51366/51366fig1.jpg" style="opacity: 0.9; font-size: 14.399999618530273px; line-height: 28px;">
図 1.機能化されたPEGG表面のビーズおよびVSVのAFMイメージ投射。36 nmビオチン化ビーズ(A、B)とVSVビリオン(C)をビオチン化VSVG抗体で表面に固定したAFMスキャン。AFMはACエアトポグラフィスキャンモードで行われました。先端半径は<25nmであり、測定は力変調および光タッピング下で行った。先端は3 N /mの力定数および15 kHzの不確実性の共振頻度75 kHzを有していた。ビーズはAFMスキャン中に高さを保持していましたが、VSVビリオンは若い弾性率が大幅に小さく、高さが有意に変形します。この画像では、ビリオンは、小さなキシコンボリューション(ウイルスの先端対鈍端を決定するために使用される)を生成するタッピングモードで硬い片持ち体で特異的に画像化され、ウイルスの先端と鈍い端の間の高さの差は、ウイルス19の鈍い端で余分なタンパク質密度を検出するために使用される。
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51366/51366fig2highres.jpg" src="/files/ftp_upload/51366/51366fig2.jpg"> 図 2.PEGG機能表面上のVSVビリオンの蛍光ベースのイメージング。 A)広視野蛍光で画像化されたビオチン化抗VSVG抗体を用いてPEGG表面に固定化された組換えVSVビリオン。 B)ビオチン化された抗VSVG抗体を介してPEGG表面に取り付けられ、アレクサ647標識抗VSVG抗体で飾られたVSVのエンベロープの高分解能再構築(方法5Aはこれらの画像を作成するために使用された)。青のサーフェスは、2D 等値面投影です。左は弾丸型ウイルスのモデルを示す。
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<tr height="27">
<td height="27" style="height:27px;width:119px;">カバースリップサイズ</td>
			<td style="width:93px;">流体</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">40 mm</td>
			<td>65 μl</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">35 mm</td>
			<td>50 μl</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">30 mm</td>
			<td>36 μl</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">25 mm</td>
			<td>25 μl</td>
		</tr>
<tr height="20">
<td height="20" style="height:20px;">20 mm</td>
			<td>16 μl</td>
		</tr>
</tbody></table>表 1.カバースリップサイズに応じた流体アリコート。

Watch this Scientific Journal Video about Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging at JoVE.com

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

表面へのビリオンの付着は、超解像蛍光イメージングまたは原子間力顕微鏡(AFM)による単一のビリオンイメージングの要件です。ここでは、AFMおよび超解像蛍光イメージングでの使用に適したガラス表面へのビリオンの制御接着のためのサンプル調製方法を示す。

単一のビリオン原子間力顕微鏡と超解像蛍光イメージングのサンプル調製

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

sample-preparation-for-single-virion-atomic-force-microscopy-super

Research

JoVE Journal

Biology

9.4K ビュー.  University of Utah. 表面へのビリオンの付着は、超解像蛍光イメージングまたは原子間力顕微鏡(AFM)による単一のビリオンイメージングの要件です。ここでは、AFMおよび超解像蛍光イメージングでの使用に適したガラス表面へのビリオンの制御接着のためのサンプル調製方法を示す。

ビデオ: Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

生物学

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

細菌分裂機械の超解像イメージング

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

最適STORM超解像顕微鏡のためのテストサンプル

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

迅速な分析および蛍光顕微鏡画像の探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

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.

VirusMapperと超解像顕微鏡のためのオープンソースの単一粒子分析

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

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳類細胞における地上州枯渇超解像イメージング

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

サンプル準備および透過電子顕微鏡によるエクソソームのイメージ投射

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

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.

透過電子顕微鏡用小型サンプル準備

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

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 ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

細胞下構造の超分解能ライブ細胞イメージング

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...