This presentation was supported by a Clinical Innovator Award to JLC from the Flight Attendant Medical Research Institute and by the United States Environmental Protection Agency. Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through Cooperative Agreement CR83346301 with the Center for Environmental Medicine, Asthma, and Lung Biology at The University of North Carolina at Chapel Hill, it has not been subjected to the Agency&#8217;s required peer and policy review, and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

フリーズフラクチャー/フリーズ·エッチングのための生物試料の調製<ol><li>そのような1時間0.1 Mリン酸緩衝液中の2％グルタルアルデヒド+ 2％パラホルムアルデヒドのような従来のEM固定剤製剤を使用する。生体組織の主要な固定を行う。注：それは前に固定せず標本のいくつかのタイプを凍結することが望ましいかもしれないが、ユニバーサル血液媒介病原体の予防措置は、検体がヒトの組織で構成され、適切な固定を義務付ける。 </li><li>一次固定に続いて、わずか1時間、0.2Mのスクロースを補充した同じ緩衝液中の試料をすすぐ。 </li><li> 1時間を超えない、0.1 Mリン酸緩衝液中の25％グリセロールからなる凍結防止剤溶液に試料を移す。 </li></ol>凍結破砕の前に2。凍結と標本の保存<ol><li> （ 図4適切な大きさの金や銅試料スタブを選択して、処理中の試料について形状</st栄&gt;）。 </li><li>金属試料スタブの上に試料の位置の小さな断片が細かいグレードの鉗子および/または小ゲージの注射針を使用した。 <ol><li>少しろ紙とスタブの端から液体を抜き取ることにより、一貫性のような接着剤、粘着性の試料の液体含有量を減らします。 </li><li>単一レプリカの場合、スタブ上で組織のマウンドを作成するには、小さなゲージの注射針を使用しています。ダブルレプリカの場合は、それを正確にスタブを介して別のスタブと位置を反転し、サンドイッチ（ 図5A）を作成する試料面上に軽く置きます。 2つのスタブ間から外に標本を押さないでください。 </li></ol></li><li>液体窒素で2つの絶縁デュワー容器を埋める。注：最初の容器は市販のシリンダ（ 図5B）からプロパンガスの小容量を液化するために使用される小さなウェルを含む金属ポストを収容する。第二の容器は短いため、パーティションのストレージ/保持容器であるLY液体窒素下で凍結した標本を維持する。 <ol><li>プロパンウェル内に配置シリンダーノズルを用いて、シリンダーバルブを開き、ガスがそれを液化する最初の容器のウェル内に流れることを可能にする。注：注意！プロパンは触れた場合の傷害を凍結原因かもしれません、爆発され、窒息されています。唯一のそのような使用は、低温に対する防護服を使用し、また、外部からの発火源を避けるように注意しながら、十分な換気を維持するために認定された化学フード内でプロパンを使用してください。 </li><li>可能な限り迅速に、試料が細かいグレードの鉗子でマウントしてからすぐに液体窒素の第二の保持容器（ 図5B）に転送数秒間最初の容器内で液化ガスにそれを突入拾う。 </li><li>標本が凍結されると、直前まで、液体窒素下で店がフリーズフラクチャープラントへ転送します。拡張のための大規模なストレージ液体窒素デュワー容器にスタブを転送必要に応じて、しかし気にストレージはスタブが解凍させないように注意してください。 </li></ol></li></ol>凍結破砕装置と試料の処理の3。運用<ol><li>真鍮のホルダーにチャンバ内に検体をロード<ol><li>冊子型ダブルレプリカ標本ホルダーにゴールドの試料スタブをロードするか、液体窒素下でデバイスをクランプし、試料室（ 図6）に配置するために準備ができるまでそこに維持する。 </li><li>チャンバーは高真空（約2×10 -6ミリバール）を達成しており、ステージがチャンバーベント、ポンプの電源を切り、液体窒素温度まで冷却し、可能な限り迅速に試料台に取り付けられた試料スタブを配置している場合。 </li><li> 、ポンプの電源を入れ、高真空を再確立し、ミクロトームアームおよびBRIに試料台を-100℃までの温度と直接液体窒素を調整する電子ステージとアーム温度制御を使用液体窒素温度にngの。 </li></ol></li><li> 破壊過程 <ol><li>高真空、液体窒素温度で-100ºCとミクロトーム腕のステージ温度を達成すると、リモートで、それによって試料マウント（凍結fracture_movie1.mov）上の検体を破砕ダブルレプリカ試料ホルダーを開きます。 </li><li>エッチング後の骨折のために意図標本の場合には、（ すなわち、骨折）を剃ることミクロトーム腕にクランプで維持カミソリの刃を使用してステップ3.2.1の代わりに、試験片の表面（凍結etch_movie2.movを）。 </li><li>加え凍結エッチング工程が実行される場合には、数分に1ための破断面上に冷却したミクロトームアームを位置決めする。注：この機動は破断面から（ すなわち、エッチング）水を昇華されます。この手技の効果は、FRAに加えて、水の昇華によって真の細胞表面、細胞外マトリックス、および/または分子集合体を明らかにすることである影は、膜の脂質二重層を通過する対向する。 </li></ol></li><li> 電子銃を用いた金属シャドウイングとレプリカ支援蒸発 <ol><li>白金/炭素型電子銃や抵抗をアクティブにし、それが30°の角度から破断面の上に白金​​/カーボンの薄層（約2nm）を蒸発させる - 45°。 20秒 - これは典型的には約15を必要とします。 </li><li>ステップ3.3.1のように、炭素の電子や抵抗銃をアクティブにして、レプリカに支援を与えることを真上（90°）から破砕試料表面に炭素の薄い層を蒸発させる。 20秒 - これは典型的には約15を必要とします。 </li></ol></li><li>レプリカの検索<ol><li>レプリケーションのシャドーイングと炭素の安定化が完了すると、真空ポンプの電源を切り、チャンバーベント、試料が装置からマウント取り外します。 </li><li>細かいグレードのピンセットを使用すると、doubleから各金試料スタブを削除レプリカブックレット/クランプと静かにスポットプレート（ 図7）で水面上にスタブを下げる前に、数秒間に解凍することができます。注：接着性試料材料を含有する炭素/白金レプリカが水面上に浮遊する。 </li><li>細いワイヤループまたは微細グレードの鉗子と、1〜数時間のために50％硫酸中の5％重クロム酸ナトリウムを含む別のスポットプレートに移すことによって保持された従来の銅電子顕微鏡グリッドのいずれかを使用してレプリカを操縦。注：この浴はレプリカから実際の生体試料材料を消化する。完全な強度の家庭用漂白剤は、重クロム/硫酸溶液の代わりに用いてもよい。 </li><li>消化が完了すると、きれいな水の表面に戻って複製を転送し、標準的な銅の電子顕微鏡グリッド上に取り出す。 （注：レプリカは、消化中に小片に断片化するかもしれないが、でも非常に小さなレプリカが、実質的な情報を含んでいてもよく、RETRであるべき彼らは穏やかな取り扱いに年間安定残る市販のグリッドボックス内のグリッドをサポートしています。）お店のレプリカをievedと検討した。 </li></ol></li></ol>フリーズフラクチャー/エッチレプリカの4微細構造の検討<ol><li> 80 kVの - 典型的には50から加速電圧での透過型電子顕微鏡での表示のレプリカ。 </li><li>標準のTEM膜上または高解像度デジタルカメラで、関連する画像を記録します。 </li></ol>

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The author has nothing to disclose.

その導入および商業的入手次の年では、フリーズフラクチャー/エッチングの手順は、広く、生物学的膜構造の調査のために利用した。実際、膜の構造の専門分野のいくつかの最高の視点がフリーズフラクチャー/エッチ製剤において得られている。これらの研究は、膜の構造組織の理解に貢献しただけでなく、構造と機能がどのように関連しているかについての洞察を提供していないだけ。 <...

概念と生物学的標本の凍結破砕処理の実用化は、半世紀以上前スティア1によって導入された。初期の装置は、作業内蔵ユニット1内に異種のコンポーネントを流用。元の装置は遠隔操作、高真空の維持、および透過型電子顕微鏡による検査に適したレプリカを生成するために、炭素、金属の蒸発（ 図1および図ための重要な必要性に対応するために、市販の器具に変更し、精製した2）。 典型的な器具は、試料台及びミクロトームアーム調節性液体窒素スループットを有する（ 図1）と高真空チャンバから構成されている。室内には、試料ステージに90°の角度やプラティのための他に位置炭素の蒸発を安定化するための2つの電子銃、1を収容45°（ 図2） -調節可能な角度、通常は15°で、シャドーイングNUM /カーボン。ユニットへの電力は、温度調整及び電子銃制御を調節する真空ポンプおよび電子パネルを操作するために適用される。 本来のウイルスの改善されたイメージングを達成するために考え出され、凍結破壊は、細胞膜及びその特殊化2,3の検査および分析のための技術として、さらに多くの人気を得た。実際に、この手順では、細胞や組織内の構造/機能の関係を解明に不可欠であったと、これらの研究の多くは、細胞生物学および分子生物学4-9に古典的な貢献として立っている。フリーズフラクチャー法の開発のための主要な目標と原理は、従来の生物学、電子顕微鏡で使用される化学固定および処理に由来する電子顕微鏡の分解能で観測アーティファクトを制限することであった。ここでの目標は、化学物質を制限することです固定は、十分な速度で、頻繁に氷の結晶の形成やその他の冷凍アーティファクトを制限するために、凍結防止剤の存在下で試料を凍結すると。さらに最近では、この技術は、ナノ粒子とナノ材料の検査のための分子生物学者と材料科学の研究者からの関心の復活を発見した。 凍結破砕し、凍結エッチ画像が3次元キャラクタを示し、時には走査型電子顕微鏡写真を間違わある。しかし、フリーズフラクチャー製剤は、透過型電子顕微鏡によって検査され、高解像度の形態学的研究への主要な貢献は、細胞膜の構造/機能要素の独自の表現である。凍結破砕処理および/またはグリセロールなどの凍結保護剤を用いて氷の結晶化を制限するために十分な速度で、細胞および組織を凍結することによって開始される。標本を真空と担当者の下で破断しているLICAは破断面上に炭素と白金の蒸発によって生成される。オリジナルの標本は、標準のEM標本グリッド上に検索されるレプリカから消化される。フリーズフラクチャー画像の別の一般的な誤解は、それらが細胞表面を描写することである。フリーズフラクチャーの基本的な前提は、生体膜の破壊過程（ 図3）によって脂質二重層を通して分割されるということですが。生体膜のこのプロセスは、2つの破断面を、外に隣接する二分子膜のリーフレットの半分を明らかに細胞質、PF-面に隣接する膜の半分の編成を明らかにする一つを生じる環境、EF-顔。真の細胞表面は、フリーズフラクチャー画像に表さなく破壊手順に従って凍結エッチングの後に添加ステップが使用される場合にのみ表示されていない。効果的に表面detaiを明らかにするために以前に破断試験片をエッチングするためにlは、試験片を急速にかつunetchablecryoprotectantずに凍結されている必要があります。根本的な特徴を明らかに骨折した試料表面からの水のエッチングは、試料を保持するステージとの間の温度差を生成する試料ステージ上に冷却ミクロトームアームと、表面から昇華させる冷却水をミクロトームアームを位置決めすることによって達成される。水が凍結エッチング操作中、骨折片の表面から昇華した場合、実際の細胞表面、細胞外マトリックス、細胞骨格構造、および分子集合体の側面は、高解像度で明らかにすることができる。このように凍結破砕し、凍結エッチングは互換用語ではなく、むしろ特定の研究の必要性に応じて必要または望ましくないかもしれない後者は段階的なプロセスを反映している。 フリーズフラクチャー/凍結エッチ手順に従って、破断面が向けられevaporaに供されるレプリカへのサポートおよびイメージングのコントラストを提供するために、炭素と白金のコートを電性。白金/炭素イメージング蒸発は、単方向または回転式であってもよく、抵抗または電子銃のいずれかによって達成される。既知の角度からの単方向シャドウイングは、通常は30° - 45°、特定の形態学的計算を行うのに有用である。ディープエッチングが施された試験片は、一般的に陰に回転式であり、これらの検体の結果画像は、写真的に評価のために逆になっている。 フリーズフラクチャー/凍結エッチング技術の歴史だけでなく、現在の目標は、より従来型の透過型電子顕微鏡の手続きに関連している化学固定および処理試料アーティファクトを制限することである。しかしながら、この技術は、三次元ディテールを付与するため、生物学、材料科学における形態計測データの取得を容易にする能力において実質的な利点を提供し、そしてnanotechnology標本。骨折、凍結および凍結エッチ手順は複雑かつ多面的であり、その応用のいくつかの側面がカスタマイズされています。このプレゼンテーションでは、プロセスの主な機能の調査ビューを提供しており、読者は細部に対処し、特定の研究ニーズに合わせてプロセスをカスタマイズするために、包括的に公開されたプロトコル10、11と呼ばれている。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Balzers Freeze-fracture/freeze-etch plant</td>
			<td style="width:71px;">Balzers</td>
			<td style="width:119px;">BAF400T</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard buffers</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">standard aldehyde fixatives</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium dichromate</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sulfuric acid</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Disposable supplies for Platinum/Carbon Evaporation</td>
			<td style="width:71px;">Technotrade International</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Liquid nitrogen</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Freon</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Disposable supplies for electron microscopy</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transmission electron microscope</td>
			<td style="width:71px;">Carl Zeiss Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy

自然の中での試料、典型的には、生物学的またはナノ材料、骨折し、凍結し、カーボン/プラチナ &quot;キャスト&quot;透過型電子顕微鏡による検査のために意図を生成するために複製されることにより、フリーズフラクチャー/凍結エッチングは、プロセスについて説明します。試験片を液体窒素で試料のその後の破砕と、氷晶形成を制限するために凍結保護剤の存在下で、多くの場合、超高速凍結速度が施され、高真空下で高温に冷却した。生じた破断面には鋳造、表面の三次元詳細を与える角度から炭素と白金の蒸発によって複製され、安定化される。この技術は、細胞膜とその専門分野の研究に特に啓発証明していると、関連する細胞機能に対する細胞の形の理解にかなり貢献してきました。本稿では、実行するための機器の要件や技術的なプロトコルを調査凍結骨折、関連した用語と、破断面の特性、フリーズフラクチャー画像の解釈のための従来の手順のバリエーション、および判定基準。この技術は、広く細胞生物学の多くの分野での超微細構造の調査のために使用され、分子、ナノテクノロジーのための新たなイメージング技術として有望であり、材料科学の研究されています。

フリーズフラクチャーの読影の鍵前提は、破断面が規則PF-面（プラズマ破断面）とEF-面（外破断面）（ 図によっていわゆる二破断面を付与する膜の脂質二重層を通過することである3）。 PF-faceが細胞の細胞質およびEF面に隣接する膜脂質二重層の半分が細胞外環境に隣接した膜脂質二重層の半分である。フリーズフラクチャー法は、膜構造の調査のために特に有用で...

Watch this Scientific Journal Video about Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy at JoVE.com

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

基本的な技術および透過型電子顕微鏡による検査のための生体試料とナノ材料のフリーズフラクチャー処理の改良が記載されている。この技術は、生体膜の超微細構造の特徴と専門性を明らかにするため、材料科学、ナノテクノロジー製品の超微細構造レベル次元の空間データを取得するための好ましい方法である。

生物学電子顕微鏡でのフリーズフラクチャー/フリーズエッチングの基本技術要素

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

fundamental-technical-elements-freeze-fracturefreeze-etch-biological

Research

JoVE Journal

Biology

33.0K ビュー.  The University of North Carolina at Chapel Hill. 基本的な技術および透過型電子顕微鏡による検査のための生体試料とナノ材料のフリーズフラクチャー処理の改良が記載されている。この技術は、生体膜の超微細構造の特徴と専門性を明らかにするため、材料科学、ナノテクノロジー製品の超微細構造レベル次元の空間データを取得するための好ましい方法である。 

ビデオ: Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

の電子顕微鏡のための胚の調製ショウジョウバエ胎児心臓の管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

生物学

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

の初期発生のタイムラプス顕微鏡線虫（Caenorhabditis elegans）

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

分裂酵母セクシャルライフサイクルの顕微鏡観察

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

マリンTubewormでライブ光学および電子顕微鏡技術を用いた石灰化イベントのキャラクタリゼーション

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

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

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

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

走査型透過電子トモグラフィーにより太い生物学的サンプルの調製と観察

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

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 ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

生体組織試料のシリアルブロック顔走査電子顕微鏡(SBF-SEM)

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

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.

極低温電子断層撮影の最適化戦略

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

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. 
These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.