The authors have no acknowledgements.

注：動物の使用を含むすべての手順は現地の法律に従って行われます。私たちの仕事はシドニー現地保健地区動物福祉委員会によって承認されています。許可された手順は、プロジェクトのライセンスのドキュメントに記載されており、すべての回で、動物の福祉を確保するためのガイドラインに従っています。作業が行われている国の動物実験に関する法律の遵守を確認してください。 EM固定剤を調製するための1プロトコル<ol><li>固定液100mlを、磁気撹拌機で絶えず撹拌しながら、三角フラスコに蒸留水25ミリリットル、65ºCの熱パラホルムアルデヒド粉末2gを添加することによって、パラホルムアルデヒドを準備します。 注：グルタルアルデヒド、パラホルムアルデヒドおよびカコジル酸ナトリウム緩衝液は、すべての有害物質であり、常に換気フード内で処理する必要があります。 </li><li> 2分間65ºCの熱は、その後冷却します。必要に応じて、水酸化ナトリウム溶液を加えるdropwISE完全に透明な溶液に、撹拌しながら。 注：水酸化ナトリウムは危険です、取り扱いに注意して。 </li><li> EMグレードの株式のグルタルアルデヒドの10ミリリットル、50の0.2Mカコジル酸ナトリウム緩衝液mlのショ糖の2グラムを追加し、マグネチックスターラーで攪拌します。 1.0 M CaCl 2を2ミリリットルを追加します。 </li><li>蒸留水で100mlにソリューションを構成するに先立ってpHを7.4に調整します。最終的な固定液は、2.5％のグルタルアルデヒド、2％パラホルムアルデヒド、2ミリモルのCaCl 2、0.1 Mカコジル酸緩衝液中の2％スクロースを含有します。総浸透圧が440ミリオスモル/ Lであることを確認してください。準備の24時間以内に使用してください。 </li></ol>肝臓の2灌流固定<ol><li> 37ºC - 35ウォーム灌流緩衝液および固定液。放血バッファおよび固定液の正確な温度は、組織の整合性を確保するのに役立ちます。 </li><li>を50mg / kgのケタミンおよび5mg / kgのキシラジンの単回腹腔内注射で動物を麻酔し。 </li><li>動物は、下になったら後肢の引っ込めと尾のピンチによって評価深麻酔は、Yの切開は、肝臓と門脈を露出させるために、動物の腹部の平滑末端のはさみで作られています。 注：その機器が容易にSEMで見られます微生物や一般破片と試料の汚染を回避するために、クリーンであることを確認してください。 </li><li>他のより遠位の肝臓に、肝臓に門脈の周りに1近位に2つの非常に緩い縫合糸を結びます。 </li><li> （マウス用のラットで18 G、22 G）適切なサイズIVカニューレとカニューレを確保する.Tighten縫合糸を門脈にカニューレを挿入。 </li><li>リン酸緩衝生理食塩水で灌流を開始37ºCに加温し、そしてH 2 Oの10cmの圧力で行われます。 PBSの5〜20mlを、通常肝臓に放血するために必要とされます。 注：気泡は、彼らが塞栓と圧力に二次的アーティファクトを誘導するようにカニューレに接続されたチューブを介して肝臓に入力できるようにしないでください。肝細胞の損傷、SI灌流圧が高すぎるnusoidsとLSECの開窓が発生します。 </li><li>バッファは肝臓から自由に出ることができるように、腹部と胸部大静脈を切断。これは、のLSECに高い背圧による損傷を防止します。 </li><li>肝臓は血液の自由になると、EM固定剤を含むPBSを交換し、肝臓が硬化して非常に薄いされるまで、約5分間灌流。 </li><li>灌流を中止し、1に固定された肝臓をカット-鋭いメスを用いて2mmの3ブロック。 </li><li> 4ºCで72時間 - 24 EM固定剤でポストフィックス組織。 注：一般的に、underfixationはoverfixation以上のアーティファクトを引き起こします。 </li><li> 4ºCで保存、0.1Mカコジル酸ナトリウム緩衝液に接尾期間以下の固定液に変更します。 注：この方法で保存された試験片は（12ヶ月まで）かなりの時間のために保存されます、しかし、タイムリーな二次固定と検査は最良の結果を得るために推奨されています。 </li></ol> 3.ニードルPerfusion 注：ニードル固定は、直接生検肝組織に固定液を注入することを含む灌流固定の変形です。目的は、それらを放血し、固定液に組織ブロックのすべてを露出させるために、血管を通ってフラッシュする固定剤のためのものです。ケアは、圧力傷害18,19を回避するために低い注入圧力を保つように注意しなければなりません。 <ol><li>動物または被験体から肝臓の小片を除去し、血液や破片を除去するために生理食塩水ですすいでください。 </li><li>ファインゲージの針（典型的にはインスリン注射器）を取り付けたシリンジに通常の生理食塩水を描画します。 </li><li>血液が組織の外にフラッシュされるまで、肝臓に非常にゆっくりと生理食塩水を注入します。複数回の注射が必要な場合があります。 </li><li>ファインゲージの針（典型的にはインスリン注射器）を取り付けた別のシリンジにEM固定剤を描画します。 </li><li>それはハードで色は黄褐色になるまで肝臓に非常にゆっくりと固定液を注入します。頻回注射sが必要とされ得ます。 </li><li>鋭いメスを用いて2mmの3ブロック- 1に固定された肝臓をカット。 72時間4℃ - 24のためのEM固定剤でインキ​​ュベートします。二次固定を開始する前に、0.1Mカコジル酸ナトリウム緩衝液中で3回洗浄します。 </li></ol>走査型電子顕微鏡4.準備<ol><li>洗浄サンプル、0.1Mカコジル酸ナトリウム緩衝液中で3回。脂質を修正するには、ポストは2時間、0.1Mカコジル酸ナトリウム緩衝液中の2％四酸化オスミウムのサンプルを修正します。 注：グルタルアルデヒドと四酸化オスミウムの交差反応し、SEMのアーティファクトを生成するように完全な洗浄は非常に重要です。 </li><li>脱水を開始するために、エタノールの濃度を増加さでサンプルをすすぎます。 5分間、50％エタノールで洗い流し、その後、70％エタノールで5分間3回、 90％のエタノールで5分間、3回すすぎ、 100％エタノールで5分間2回リンス。そして最後に100％エタノール（分子篩）中で10分間2回リンス。 </li><li>サンプルからすべてのエタノールを除去<html>と換気フードにヘキサメチルジシラザン（HMDS）と交換し、10分間のままにします。組織サンプルからHMDSを削除し、蒸発させます。 注：したがって、最終脱水工程のために使用する前に室温に到達させるときに冷たいHMDSは非常に吸湿性です。 </li><li>必要に応じて、すぐにサンプルをマウントするか、SEMのためにマウントする準備ができるまでデシケーターに入れます。 </li></ol> 5.取り付け注：正しい試料取付は、SEMで分析のために利用可能な明確に線引き正弦波の数を最大化します。 <ol><li>金属SEMスタブのベースにラベルを付け、スタブの先頭に両面導電性カーボンテープを適用します。 </li><li>後続のSEMのための最良の正弦波で表面を識別するために、解剖顕微鏡を用いて試料を可視化。スタブのカーボンテープ表面に上向きにその表面を置きます。 </li><li>スティック片をしっかりとスタブに、標本は今スパッタコーティングの準備ができています。 </li></ol>ove_title &quot;&gt; 6。塗装注：スパッタコーター敷地内に試料を導電性金属（金、プラチナ）の緻密な膜で試料を塗布し、電子ビームによる損傷から保護します。コー​​ティングが厚すぎると、目的の構造が隠されてもよいです。 <ol><li>回転および45秒間自動スパッタコーティングにモードを設定する自動微細スパッタコーターの真空チャンバ内に配置スタブ。しかし、金にも適しており、細かいディテールのためにプラチナコーティングを使用してください。 </li><li>被覆するために、正弦波の表面を注意していないながら、取り付けられ肝臓検体の外部表面にカーボン塗料の薄い層を適用します。 </li><li>標本はプラチナでコーティングされており、炭素塗料が乾燥した後、彼らは、SEMの観察の準備ができています。サンプルをデシケーター中で保存することができます。 </li></ol>前記走査型電子顕微鏡を使用して<ol><li>場所は、走査型電子顕微鏡に標本や真空に顕微鏡を返します。&lt;/ LI&gt; <li>低倍率で開始することによって試料の可視化のための標準的な顕微鏡の操作手順を使用して、徐々に倍率を増加させます。 </li><li>顕微鏡は、距離、スポットサイズと非点収差を操作するための適切に調整されていることを確認。 </li><li>固定の整合性をチェックします。 LSECは、隙間からほとんどが無料で、30測定開窓が含まれている場合 - これは、一般的に試料作製に成功したことを示す250 nmの直径を。 </li><li>正弦波は、ギャップの完全または開窓を欠いている場合には、試料調製は成功していないか、重大な病状があります。この場合には、非常に微細なかみそりの刃でサンプルをスライスし、切断面は、重ね塗りし、正常に固定された正弦波を分析しました。 </li><li>約15,000〜20,000倍の倍率で良好な定着と開窓が明確に（表示されている場合に典型的には、少なくとも10開窓を含むとデブリのない平らなLSECの表面を選択）。 </li><li>肝臓あたり少なくとも二つのブロックを使用して、ブロックの異なる領域から、肝臓あたり少なくとも10正弦波の画像を保存します。 15,000で10顕微鏡写真をサンプリング - 開窓の定量化は、肝臓全体の代表であるために、動物あたり20,000倍の倍率は、一般的に十分です。 </li></ol> 8.分析注：NIHから無償でダウンロードすることができますImageJソフトウェアは、開窓径と周波数（定量化するために利用される<a href="http://imagej.nih.gov/ij">www.imagej.nih.gov/ijを</a> ）。 <ol><li>画像を開くJと（ 画面は、1ショット ）画像に埋め込 ​​まれたスケールバーを使用してスケールを設定します。 </li><li> ImageJの中のポリゴンツールを使用して、有窓とdefenestrated領域を含むLSECの全体の平坦な領域の周りにトレース。これは誤って気孔率を膨らまれるように、単にふるいプレートをトレースしません。 obscuriかもしれない細胞表面にあるデブリのいずれかの大規模な作品を除外NGの開窓。 </li><li> 、開窓径を測定ラインツールを選択することにより、各開窓の最大直径を通る線をトレースして、ラインを測定するために「M」を押し、「D」永久に画像上に線を描画する（描きます）。この行は、開窓の直径として定義されます。線の長さは、現在のImageJ（ スクリーンショット2）の結果ボックスで利用できるようになります。 </li><li> 250 nmのオーバーLSEC細胞質内で大きく穴であるすべてのギャップを測定し、同様に開窓。ギャップはギャップがまた、疾患および毒性の結果として発生する可能性がありますが、多くの場合、貧しい灌流または固定を反映した工芸品です。ギャップのデータは、後の分析における開窓パラメータの計算から除外されます。 </li><li>円形でないギャップの面積は、ギャップの周りトレースとImageJの上での面積を計算することによって計算されるべきです。 </li><li> furtheためのExcelスプレッドシートにはImageJに結果ボックスからデータを貼り付けR解析。 </li></ol> 9.計算<ol><li> （ギャップは≥250 nmであるギャップを含まない）、すべての開窓径の平均開窓径=平均。 </li><li>開窓面積=πR2、R、半径は、ギャップを含まない、個々の穿孔の直径（R = D / 2）から計算されます。 </li><li>気孔率（％）=（Σ（πR2）/分析総面積-ギャップのΣ（面積=））（μmの2））×100。 </li><li>開窓周波数=ギャップの（面積）（μmの2） - Σ分析開窓/（総面積の合計数。 </li></ol>開窓データの10プレゼンテーション注：開窓データの定量化を含め、可能な限りの資料は、以下の情報を含める必要があります。 <ol><li>使用境界の直径は、典型的には、（開窓を定義するために何を確認するステートメントと開窓直径、NM 250）、開窓周波数（数個/μm2）と空隙率（％） - 50との間。 </li><li>ギャップが分析に含まれているかどうかを確認するステートメント、開窓直径肝臓、ブロック、イメージおよび穿孔の数の度数分布のグラフは、分析に含まれるべきです。 </li></ol>

<ol>
	<li>Cogger, V. C., Le Couteur, D. G., Arias, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Liver: Biology and Pathobiology. , 387-404 (2009).</li><li>Fraser, R., Dobbs, B. R., Rogers, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+and+the+liver+sieve:+the+role+of+fenestrated+sinusoidal+endothelium+in+lipoprotein+metabolism,+atherosclerosis,+and+cirrhosis.">Lipoproteins and the liver sieve: the role of fenestrated sinusoidal endothelium in lipoprotein metabolism, atherosclerosis, and cirrhosis.</a> Hepatology. 21, 863-874 (1995).</li><li>Wisse, W., De Zanger, R. B., Charels, K., Van Der Smissen, P., McCuskey, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+sieve:+considerations+concerning+the+structure+and+function+of+endothelial+fenestrae,+the+sinusoidal+wall+and+the+space+of+Disse.">The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse.</a> Hepatology. 5 (4), 683-692 (1985).</li><li>Reilly, J. N., Cogger, V. C., Fraser, R., Le Couteur, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+feeding+and+fasting+on+fenestrations+in+the+liver+sinusoidal+endothelial+cell.">The effect of feeding and fasting on fenestrations in the liver sinusoidal endothelial cell.</a> Pathology. 42 (3), 255-258 (2010).</li><li>Tian, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+serotonin+receptor-2B+rescues+small-for-size+liver+graft+failure+in+mice.">Activation of serotonin receptor-2B rescues small-for-size liver graft failure in mice.</a> Hepatology. 53 (1), 253-262 (2011).</li><li>Cogger, V. C., Mitchell, S. J., Warren, A., de Cabo, R., Le Couteur, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Loss+of+Responsiveness+to+2,5-Dimethoxy-4-Iodoamphetamine+in+Liver+Sinusoidal+Endothelial+Cells.">Age-Related Loss of Responsiveness to 2,5-Dimethoxy-4-Iodoamphetamine in Liver Sinusoidal Endothelial Cells.</a> J Gerontol A Biol Sci Med Sci. 69 (5), 514-518 (2013).</li><li>Reilly, J. N., Cogger, V. C., Le Couteur, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Old+age+is+associated+with+ultrastructural+changes+in+isolated+rat+liver+sinusoidal+endothelial+cells.">Old age is associated with ultrastructural changes in isolated rat liver sinusoidal endothelial cells.</a> J Electron Microsc. 59 (1), 65-69 (2010).</li><li>Le Couteur, D. G., Rattan, A. E., Le Couteur, D. G., de Cabo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Calorie Restriction, Aging and Longevity. , 191-216 (2010).</li><li>McLean, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+pseudocapillarization+of+the+human+liver.">Age-related pseudocapillarization of the human liver.</a> J Pathol. 200 (1), 112-117 (2003).</li><li>Le Couteur, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudocapillarization+and+associated+energy+limitation+in+the+aged+rat+liver.">Pseudocapillarization and associated energy limitation in the aged rat liver.</a> Hepatology. 33 (3), 537-543 (2001).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+acute+oxidative+stress+on+the+ultrastructure+of+the+perfused+rat+liver.">The effect of acute oxidative stress on the ultrastructure of the perfused rat liver.</a> Pharmacol Toxicol. 89 (6), 306-311 (2001).</li><li>Horn, T., Christoffersen, P., Henriksen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcoholic+liver+injury:+defenestration+in+noncirrhotic+livers-a+scanning+electron+microscopic+study.">Alcoholic liver injury: defenestration in noncirrhotic livers-a scanning electron microscopic study.</a> Hepatology. 7 (1), 77-82 (1987).</li><li>Jamieson, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+liver+sinusoidal+endothelium+in+a+baboon+model+of+type+1+diabetes.">Alterations in liver sinusoidal endothelium in a baboon model of type 1 diabetes.</a> Diabetologia. 50 (9), 1969-1976 (2007).</li><li>Fraser, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+perfusion+pressure+damages+the+sieving+ability+of+sinusoidal+endothelium+in+rat+livers.">High perfusion pressure damages the sieving ability of sinusoidal endothelium in rat livers.</a> Br J Exp Pathol. 61 (2), 222-228 (1980).</li><li>Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electron+microscopic+study+of+the+fenestrated+endothelial+lining+of+rat+liver+sinusoids.">An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids.</a> J Ultrastruct Res. 31 (1), 125-150 (1970).</li><li>Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultrastructural+characterization+of+the+endothelial+cell+in+the+rat+liver+sinusoid+under+normal+and+various+experimental+conditions,+as+a+contribution+to+the+distinction+between+endothelial+and+Kupffer+cells.">An ultrastructural characterization of the endothelial cell in the rat liver sinusoid under normal and various experimental conditions, as a contribution to the distinction between endothelial and Kupffer cells.</a> J Ultrastruct Res. 38 (5), 528-562 (1972).</li><li>Fahimi, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+and+immersion+fixation+of+rat+liver+with+glutaraldehyde.">Perfusion and immersion fixation of rat liver with glutaraldehyde.</a> Lab Invest. 16 (5), 736-750 (1967).</li><li>Wisse, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+methods+for+electron+microscopy+of+human+and+other+liver.">Fixation methods for electron microscopy of human and other liver.</a> World J Gastroenterol. 16 (23), 2851-2866 (2010).</li><li>Vreuls, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jet-fixation:+a+novel+method+to+improve+microscopy+of+human+liver+needle+biopsies.">Jet-fixation: a novel method to improve microscopy of human liver needle biopsies.</a> Hepatology. 59 (2), 737-739 (2014).</li><li>Furrer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+reverts+age-related+capillarization+and+failure+of+regeneration+in+the+liver+through+a+VEGF-dependent+pathway.">Serotonin reverts age-related capillarization and failure of regeneration in the liver through a VEGF-dependent pathway.</a> Proc Natl Acad Sci U. S. A. 108 (7), 2945-2950 (2011).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OxLDL+induced+injury+and+defenestration+of+HLSECs+via+LOX-1.">OxLDL induced injury and defenestration of HLSECs via LOX-1.</a> J Mol Endocrinol. , (2014).</li><li>May, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+model+for+conditional+induction+and+rescue+of+portal+hypertension+reveals+a+role+of+VEGF-mediated+regulation+of+sinusoidal+fenestrations.">A transgenic model for conditional induction and rescue of portal hypertension reveals a role of VEGF-mediated regulation of sinusoidal fenestrations.</a> PLoS One. 6 (7), e21478 (2011).</li><li>Funyu, J., Mochida, S., Inao, M., Matsui, A., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+can+act+as+vascular+permeability+factor+in+the+hepatic+sinusoids+through+upregulation+of+porosity+of+endothelial+cells.">VEGF can act as vascular permeability factor in the hepatic sinusoids through upregulation of porosity of endothelial cells.</a> Biochem Biophys Res Commun. 280 (2), 481-485 (2001).</li><li>Venkatraman, L., Tucker-Kellogg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD47-binding+peptide+of+thrombospondin-1+induces+defenestration+of+liver+sinusoidal+endothelial+cells.">The CD47-binding peptide of thrombospondin-1 induces defenestration of liver sinusoidal endothelial cells.</a> Liver Int. 33 (9), 1386-1397 (2013).</li><li>Wack, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinusoidal+ultrastructure+evaluated+during+the+revascularization+of+regenerating+rat+liver.">Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.</a> Hepatology. 33 (9), 363-378 (2001).</li><li>Morsiani, E., Mazzoni, M., Aleotti, A., Gorini, P., Ricci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+sinusoidal+wall+permeability+and+liver+fatty+change+after+two-thirds+hepatectomy:+An+ultrastructural+study+in+the+rat.">Increased sinusoidal wall permeability and liver fatty change after two-thirds hepatectomy: An ultrastructural study in the rat.</a> Hepatology. 21 (2), 539-544 (1995).</li><li>Straub, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic-stimulated+liver+sinusoidal+capillarization+in+mice+requires+NADPH+oxidase&#8211;generated+superoxide.">Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase&#8211;generated superoxide.</a> J Clin Invest. 118 (12), 3980-3989 (2008).</li><li>Barber&#225;-Guillem, E., Arrue, J. M., Ballesteros, J., Vidal-Vanaclocha, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+in+endothelial+cells+of+developing+rat+liver+in+the+transition+from+fetal+to+postnatal+life.">Structural changes in endothelial cells of developing rat liver in the transition from fetal to postnatal life.</a> JJ Ultrastruct Mol Struct Res. 97 (1-3), 197-206 (1986).</li><li>Jamieson, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caloric+restriction+reduces+age-related+pseudocapillarization+of+the+hepatic+sinusoid.">Caloric restriction reduces age-related pseudocapillarization of the hepatic sinusoid.</a> Exp Gerontol. 42 (4), 374-378 (2007).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperlipidemia+and+surfactants:+the+liver+sieve+is+a+link.">Hyperlipidemia and surfactants: the liver sieve is a link.</a> Atherosclerosis. 189 (2), 273-281 (2006).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+oxidative+stress+on+the+liver+sieve.">The effects of oxidative stress on the liver sieve.</a> J Hepatol. 41 (3), 370-376 (2004).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structured+illumination+microscopy+of+liver+sinusoidal+endothelial+cell+fenestrations.">Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations.</a> J Struct Biol. 171 (3), 382-388 (2010).</li><li>Braet, F., de Zanger, R., Seynaeve, C., Baekeland, M., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+atomic+force+microscopy+study+on+living+skin+fibroblasts+and+liver+endothelial+cells.">A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells.</a> J Electron Microsc. 50 (4), 283-290 (2001).</li><li>Monkemoller, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+fenestrations+in+liver+sinusoidal+endothelial+cells+by+optical+localization+microscopy.">Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy.</a> Phys Chem Chem Phys. 16 (24), 12576-12581 (2014).</li></ol>

The authors have nothing to disclose.

正確かつ再現肝類洞内皮の状態を測定する能力は、これらの非常に特殊化した細胞の生物学を理解する上で重要なステップです。このような構造化照明顕微鏡32などの新しい技術、原子間力顕微鏡33とD-STORM（直接確率光学Reconstrucion顕微鏡）34は、 インビトロでこれらの細胞の形態に関する重要な情報を付与しますが、SEMはにその構造を視覚化し、測定す?...

肝類洞内皮細胞（のLSEC）は、高度の肝正弦波の壁を覆う内皮細胞を分化されます。のLSECを非diaphragmedある穿孔で穿孔され、経細胞は、直径50〜250 nmの細孔。のLSECの表面の最大20％が篩板1-3（ 図1）と呼ばれる数十〜数百のグループに通常は穿孔によって覆われています。開窓は非常に効率的限外濾過システムを作成し、血液と肝細胞との間にプラズマとnanosubstratesの転送を可能にします。開窓は動的構造である - それらのサイズおよび/または数の両方は、種々の生理学的状態、薬物、および疾患に応じて変更することができます。例えば、開窓は摂食状態4よりも絶食が大きいです。 2ジヨードアンフェタミンは開窓の数を増加させ、5,6および細胞あたり穿孔のサイズおよび数の減少は、加齢および多くの疾患状態7-13で起こります</sup&gt;。穿孔のサイズと数の正確な測定は、LSECの形態は、種々の疾患、毒性、および生理学的状態に影響される方法を理解するために重要です。肝機能に及ぼす影響。および開発するための治療的介入1を開窓を調節。 開窓の研究は困難です。開窓の直径は、両方の無傷の肝臓組織または培養のLSECに電子顕微鏡を用いて、先にのみ観察が可能であった、従来の光学顕微鏡の解像度の下にあります。走査型電子顕微鏡（SEM）は、最も頻繁に、SEMは、数千の内皮表面と測定の大部分の観察を可能にするために開窓サイズ、周波数及び気孔率（開窓によって穿孔さLSEC膜の割合）を研究するために使用されていますそうでない場合は数十開窓の何千もの。その有用性にもかかわらず、結果がために、SEMベースの研究から報告されますこのような開窓大きさ、数、周波数および多孔性などのLSECのパラメータは、文献（ 表1）に大きく異なります。 開窓とふるい板は、契約、ブレーク拡張または試料調製中に合体壊れやすい構造であり、このように慎重な処理は、それらの整合性を維持するために必要とされます。上昇灌流圧14;固定液およびバッファ15の誤った浸透圧;不十分な固定または固定時間。とポストの固定、脱水、乾燥の速度は超微細構造の保存（ 図2）と干渉アーティファクトを生成することができるSEMのための処理のすべての領域です。開窓の損失（「窓外放出 &#39;）と開窓収縮が減少し開窓径および細胞間隙率で、その結果、貧困層の固定の結果として発生する可能性があります。 SEM分析のための試料の保存性を向上させる方法は、先に15-17に記載されていると説明されますここでは、試料の保存性を改善する方法についての追加のヒントを。試料保存の主な目標は、LSECの表面を可視化することができるように、正弦波から血液を除去し、高圧または遅延固定のいずれかからLSECの損傷を回避するためです。門脈を介して固定液の全肝灌流は、肝臓固定するための好ましい方法です。詳細に説明したように他の場所で16,18灌流が低圧で行われなければならない（例えば、H 2 0 10 cm）のLSECに圧力関連灌流遺物や損傷を回避するためには、典型的には、細胞膜における内などの大きなギャップを明らかに。他の箇所19で詳細に説明したようしかし、合理的な固定は、多くの場合、ヒトおよび動物の肝生検の針灌流を使用して得ることができます。血液試料からフラッシュされるまで、この技術は、直接組織に固定液を注入することを含むと組織がしっかりと固定されています。電子顕微鏡用試料の固定はPERFする必要があります肝臓極めて迅速自己分解プロセスの結果として生じる微細構造の変化を防止するための血流の停止後、できるだけ速やかにormed。 我々は、アーチファクトの混入を最小限に抑え、かつ開窓の測定を標準化する画像解析の方法をも提供します。多孔性および開窓周波数について顕微鏡写真用正弦波の選択の変化、アーティファクトの画像解析、およびセル面積の測定は、公表された結果の主な相違につながっています。開窓とデータ表示のための最小要件の評価および測定のための標準化されたアプローチは、明らかに以前4,10,20-31文献で ​​扱われていません。

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Name of material</td>
			<td style="width:100px;">Company</td>
			<td style="width:93px;">Catalogue Number</td>
			<td style="width:311px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">EM grade Glutaraldehyde</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C001</td>
			<td style="width:311px;">Store stock at -20 C until needed, avoid refreeze</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Paraformaldehyde powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>158127</td>
			<td style="width:311px;">Always prepare Paraformaldehyde fresh</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:155px;">Sodium Cacodylate powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C0250</td>
			<td style="width:311px;">Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH2O, used mostly at 0.1 M by preparing 1:2 dilution</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:155px;">Calcium Chloride</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C1016</td>
			<td style="width:311px;">Prepare 1 M CaCl2by dissolving powder in dH2O</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:155px;">Osmium tretroxide</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C011</td>
			<td style="width:311px;">Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Ethanol- Absolute</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>459836&#160;</td>
			<td style="width:311px;">100 % Ethanol must be high grade and stored with Molecular Sieve</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Other grades of Ethanol</td>
			<td style="width:100px;">Labtech</td>
			<td style="width:93px;">EL5</td>
			<td style="width:311px;">Prepare graded Ethanols with dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Hexamethyldisilazane</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>52619&#160;</td>
			<td style="width:311px;">Allow to reach room temperature before use</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:155px;">Cannulas</td>
			<td style="width:100px;">Terumo</td>
			<td style="width:93px;">TSROX1832C, TSROX2225C, TSROX2419C</td>
			<td style="width:311px;">18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Conductive Carbon tape</td>
			<td style="width:100px;">ProSciTech</td>
			<td>IA0201</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Carbon Paint</td>
			<td style="width:100px;">ProSciTech</td>
			<td>I003</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Ketamine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be optained under licence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Xylazine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be obtained under licence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Molecular Sieve</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">208647</td>
			<td style="width:311px;">Removes water from the 100 % Ethanol</td>
		</tr>
	</tbody>
</table>

a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

走査電子顕微鏡による低倍率での初期の可視化は、多くの大規模な肝血管および正弦波（ 図1A）を観察するのに十分な大き露出面積と肝臓試料の平らな面を明らかにする。実装スタブの正しい肝臓ブロック配置を確保することが、この理由（ 図1B）のために避けるべきである正弦波と肝臓のグリソン鞘の鮮明な画像を得るために必須です。倍率を大きくすると、肝臓...

Watch this Scientific Journal Video about A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy at JoVE.com

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

走査型電子顕微鏡による肝類洞内皮細胞およびそれらの開窓の分析のための標準化された方法

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

a-standardized-method-for-analysis-liver-sinusoidal-endothelial-cells

Research

JoVE Journal

Biology

14.2K ビュー.  University of Sydney and Concord Hospital. The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

ビデオ: A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

生物学

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

ネズミ弁内皮細胞の単離

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

細胞外マトリックスを視覚化するために浸軟組織の電子顕微鏡をスキャンします

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

クライオ走査電子顕微鏡により凍結破砕葉組織内の光合成膜の超分子組織を学びます

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

問題のあるプラント、卵菌類、および真菌サンプルのための電子顕微鏡（SEM）プロトコルをスキャン

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

原核生物と真核生物の化学形態分析走査電子顕微鏡 (SEM) での乾燥の準備

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

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 ...

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 ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

走査型電子顕微鏡を用いたボリューム情報の標的取得のためのアレイ断層化ワークフロー

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

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, ...