このプロジェクトは、NIH / NINDS（＃1R01 - NS54252）、NSF（＃0905041、＃0079874）、テキサス先端技術プログラム（＃ATP - 000512〜0146 - 2001＃ATP - 000512〜0261 - 2001）、テキサス州で賄われていた＆M研究財団、テキサスA＆M大学のコンピュータ科学と工学科、および3Scan。我々は、技術相談やKESMイン​​ストルメンテーションのサポートのためのバーナードメサ（マイクロスターテクノロジーズ）に感謝いたします。 KESMの設計と実装の主要部分は2007年に亡くなったブルースH.マコーミック、によって行われた。

1。試料の準備：ゴルジ - コックス<ol><li>このプロトコルは、密接にMayerichらのプロトコルに従います。7。 </li><li> C57BL/6Jマウスは、イソフルラン吸入麻酔を使用して、深く麻酔し、断頭。 </li><li>脳は、ゴルジ - コックスの固定液（1％クロム酸カリウム、1％重クロム酸カリウム、及び脱イオン水の1％塩化第二水銀）に削除され、配置されます。 </li><li>脳は10〜16週間、室温で、暗所でゴルジ - コックスのソリューションに残されている。 </li><li>脳は、暗所で一晩脱イオン水で洗浄されています。 </li><li>リンスの脳は、室温で暗所で7〜10日間脱イオン水で5％水酸化アンモニウム溶液中に浸漬される。この長い準備時間は、黒い沈殿物が完全に組織に形成するチャンスがあったように、脳全体の浸透性を確保することでした。 </li><li>脳は4時間とt室温で脱イオン水で再び洗浄する室温で50％、70％、冷蔵庫（4℃）で、85％、95％（3の変更）、および100％（4〜5変更）：段階的なエチルアルコール、一連の脱水鶏。脳は、24時間ごとに溶液中に残っている。 </li><li>脱水の脳は、1:2のアラルダイトからアセトンの比率、1:1、2:1、そして最終的ににあるアラルダイト - アセトン混合物、続いて（3〜4の変化、一日ごとに）アセトンに入れられ冷蔵庫に100％アラルダイト（3の変更、一晩たびに）、（4℃）。 </li><li>最後に、処理された脳は、100％アラルダイトに包埋し、℃で3日間（アボットとソテロ2で参照されたい埋め込 ​​みプロトコル）を60に加熱される。 標本はLRホワイトに埋め込まれている場合、検体は重合前に標準的な手順である各一晩冷蔵庫に保管されているLRホワイトソリューションの3つの変化を通じて、最後の100％エタノール溶液から転送されます。 3つの変更が完全に浸透を確保するために行われている。試料はtです。60密閉容器で重合される新鮮なLRホワイトに転送鶏°適切な重合のための時間と温度の必要量である24時間C、。 </li><li>図。 1は、アラルダイトに埋め込まれているゴルジ - コックス染色した脳を示しています。 </li><li>硬化試料ブロックは、エポキシ樹脂を用いて金属試料のリングに搭載されており、ブロックの側面は、（図2参照）、必要に応じてトリミングされます。 </li></ol> 2。試料の準備：ニッスル<ol><li>マウスは、ケタミンとキシラジン（1.7mgをケタミン/ 20グラムの体重当たり0.26ミリグラムのキシラジン）腹腔内投与を使用して、深く麻酔ですし、transcardially部屋の250mLのに続いて、室温の液（pH7.4）リン酸緩衝生理食塩水50mLを用いて灌流中立温度10％液（pH7.4）緩衝ホルマリン。そして最終的に希釈していないインドのインク3.0 CCと。 </li><li>生理食塩水と固定液で全身の血流は、心臓血管系から血液をクリアするとtiを修正する必要がある<html> ssues。墨汁灌流は完全に心臓血管系の血管系を埋めるために必要です。 </li><li>その結果、脳は、傾斜エチルアルコール（25％-100％）の一連の後、脱水して、上記1.8から1.9のプロトコールに従って、アラルダイトプラスチックに埋め込まれています。 LR白埋め込むメディアの場合、上記の1.10プロトコルが続いている。 </li></ol> 4。試料の調製：一般種、一般的な臓器<ol><li>一般的なケースの場合、固定のいずれか10％中性緩衝ホルマリンあるいは4％パラホルムアルデヒドで行うことができます。 </li><li>小さな組織のボリュームの場合、代わりに血流の拡散は固定と染色をお勧めします。小さな生物や臓器の場合には、また、染色は、脱水工程中に行うことができます。 </li><li>ソリューションの浸透のための時間が全体のマウスの脳よりもはるかに小さい臓器または生物のために削減できるものの、埋め込みプロトコルでは、上記と同じです。 </li></ol>jove_title&quot;&gt; 5。KESMのセットアップおよびイメージング<ol><li>舞台をオンに、ポンプの電源を切り、カメラをオンにする、照明をオンにします。 </li><li>ナイフと客観を上げる。 </li><li>試料のリングを取り付けます。 </li><li>測定試料の寸法とKESMステージコントローラ2アプリケーションの構成ファイルを作成します。 </li><li> KESMステージコントローラ2を起動し、ステージを初期化する。 </li><li>下のナイフ/客観、ポンプの電源を入れます。 </li><li>光学電車の中で焦点調節ノブを回すと、ナイフエッジに相対カメラの視野を調整することによって、客観的とカメラのピントを合わせる。 </li><li>イメージングを開始するには[戻る]を押してください。 </li><li>図を参照してください。 3-8。技術的な詳細については、9、7を参照してください。 </li></ol> 6。画像の処理とデータの準備<ol><li>設定ファイルのフォルダ（00000、00001、等）の下でデータのフォルダにKESMスタックプロセッサの実行ファイルをコピーします。 </li><li>照明のアーティファクトを削除し、すべてのバックグラウンド強度を正規化するKESMスタックプロセッサを実行するイメージ。すべてのデータのサブフォルダに対して手順を繰り返します。 </li><li> KESMの脳アトラスのWebサーバに設定し、アップロードとファイルの設定データからマルチスケールのタイルを準備します。 </li><li>図を参照してください。 9。 </li></ol> 7。データの可視化と解析<ol><li> KESMブレインアトラスを用いて可視化。に移動し<a href="http://kesm.cs.tamu.edu">http://kesm.cs.tamu.edu</a>と適切なアトラスを選択してください。 <ol><li> Googleマップのようにナビゲートします。 </li><li> Googleマップのように拡大および縮小。 </li><li> [ - ] zの深さを増加または減少させる別の深さに移動するには、どちらかの[+]またはをクリックし、プルダウンメニューから、各ステップでどのように深く関わって選択し、。 </li><li>プルダウンメニューを使用してオーバーレイの数を調整します。 </li><li>プルダウンメニューを使用してオーバーレイの間隔を調整します。 </li><li>図を参照してください。 11。 </li></ol></li><li> MeVisLabを使用して可視化。 <ol><li> MeVisLabを起動します。 </li><li>新しいプロジェクトを作成します。 </li><li>以下では、新しいモジュールはTYによって作成することができますコマンドボックスにモジュール名でping。 </li><li>モジュールを作成する[Compose3Dfrom2DFiles]、および目的のフォルダへ移動。ファイルの文字列を入力し、[GetFileList]をクリックします。選択した画像がメモリに収まることを確認してください。 [Create3D]ボリュームを作成する]をクリックします。 </li><li>モジュール[SetWorldMatrix]、および&quot;スケール&quot;を設定の作成の下に適切なボクセルのサイズ（0.6、0.7、10X 0.45NA目標は1.0）にX、Y、Zを&quot;小学校の変換&quot;。リンク[Compose3Dfrom2DFiles]へ[SetWorldMatrix]。 </li><li>マトリックスを設定し、&quot;進む&quot;、&quot;無視&quot;、&quot;無視&quot;に&quot;マトリックス組成&quot;の下に変換します。 </li><li>モジュール[Arithmetic1]を作成し、&quot;（マックス- IMG）反転&quot;に&quot;関数&quot;を設定。 リンク[SetWorldMatrix]へ[Arithmetic1]。 </li><li>モジュールを作成する[View3D]と[Arithmetic1]を接続します。 </li><li> View3Dでは、マウスの右ボタンを押している間、しきい値を調整するためにマウスを動かす。あなたは、地域、変化の方向（マウスの左ボタンを押している間、マウスを動かす）、変更照明（ボリュームrenderinを切り抜くことができますなど、背景をgまたはMIP）をオン/オフ</li><li>図を参照してください。 10。 </li></ol></li></ol> 8。代表的な結果： ここで、我々は、全脳データと詳細を提示する。イチジク。 12月15日ショー全脳インドインク、ゴルジ体、およびニッスルデータは1、4、3を設定します。 <img alt="図1" src="/files/ftp_upload/3248/3248fig1.jpg" /> 図1、固定染色し、そして埋め込 ​​まれたマウスの脳<img alt="図2" src="/files/ftp_upload/3248/3248fig2.jpg" /> 図2組み込み、マウスの脳標本は、標本のリングに取り付け。 <img alt="図3" src="/files/ftp_upload/3248/3248fig3.jpg" /> 図3。ナイフエッジスキャン顕微鏡（KESM）。 KESMの写真は、その主要なコンポーネントがマークで示されています：（1）高速ラインスキャンカメラ、（2）顕微鏡対物、（3）ダイヤモンドナイフのアセンブリと光のコリメータを、（4）SPEcimenタンク（水浸イメージングのための）、（5）三軸の精度エアベアリングステージ、（6）白色光顕微鏡の照明、組織切片の除去のための（7）水ポンプ（バックで）、（8）ステージ制御と画像取得のためのPCサーバ、（9）花崗岩のベース、および（10）花崗岩の橋。 <img alt="図4" src="/files/ftp_upload/3248/3248fig4.jpg" /> 図4。KESMのイメージングの原理。 KESMの操作のプリンシパルが示されている。目標とナイフは（広い5ミリメートル試料は20nmと1-5の移動速度の分解能での位置決めステージの移動（実線矢印）に貼付しながら、所定位置に保持される、とダイヤモンドナイフに対してこすりれる10Xの目的のために）、ナイフ（実線矢印）上を流れる薄セクションを生成する。ラインスキャン画像は、ナイフの非常に先端付近で行われます。 <img alt="図5" src="/files/ftp_upload/3248/3248fig5.jpg" /> 図5。KESMカメラと客観的な焦点をコントロール。 <img alt="図6" src="/files/ftp_upload/3248/3248fig6.jpg" /> 図6。KESMナイフのアセンブリとコントロール。 <img alt="図7" src="/files/ftp_upload/3248/3248fig7.jpg" /> 図7。初期観測ポートを介して焦点。 <img alt="図8" src="/files/ftp_upload/3248/3248fig8.jpg" /> 図8。KESMステージコン ​​トローラ2アプリケーション（スクリーンショット）。 <img alt="図9" src="/files/ftp_upload/3248/3248fig9.jpg" /> 図9。KESMスタックプロセッサのアプリケーション（スクリーンショット）。 <img alt="図10" src="/files/ftp_upload/3248/3248fig10.jpg" /> 図10。MeVisLabアプリケーション（スクリーンショット）。 <img alt="図11" src="/files/ftp_upload/3248/3248fig11.jpg" /><br/&gt; 図11 KESM脳アトラス：Webベースインターフェース（スクリーンショット）。 <img alt="図12" src="/files/ftp_upload/3248/3248fig12.jpg" /> 図12。KESM全脳水墨データ。 KESMのデータスタックの体積の可視化は、血管のデータセットとして使用されています。 （a）のクローズアップ、血管データの。幅〜100ミクロン。 （BD）全体のマウスの脳の血管系の3つの標準ビュー（高解像度のデータから間引き）。幅〜10mmの。 <img alt="図13" src="/files/ftp_upload/3248/3248fig13.jpg" /> 図13。KESM全脳マウスゴルジデータ。 <img alt="図14" src="/files/ftp_upload/3248/3248fig14.jpg" /> 図14。KESMゴルジデータの詳細。 <img alt="図15" src="/files/ftp_upload/3248/3248fig15.jpg" /> 図15。KESM全脳ニッスルデータ。 （A）クローズアップNのISSLデータ。 〜300 UM 3。全マウスの脳ニッスルのデータ（高解像度のデータからサブサンプリング）の（BD）3つの標準ビュー。断面には、内部構造の明確な見解のために示され。

<ol>
	<li>Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+imaging+of+whole+small+animal+brains+with+the+knife-edge+scanning+microscope.">High-throughput imaging of whole small animal brains with the knife-edge scanning microscope.</a> Neuroscience Meeting Planner. Program No. 504.2, (2008).</li><li>Abbott, L. C., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+catecholaminergic+innervation+in+weaver+and+normal+mouse+cerebellar+cortices.">Ultrastructural analysis of catecholaminergic innervation in weaver and normal mouse cerebellar cortices.</a> Journal of Comparative Neurology. 426, 316-329 (2000).</li><li>Choe, Y., Abbott, L. C., Miller, D. E., Han, D., Yang, H. -. F., Chung, J. R., Sung, C., Mayerich, D., Kwon, J., Micheva, K., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+imaging,+analysis,+and+integration+of+mouse+brain+networks.">Multiscale imaging, analysis, and integration of mouse brain networks.</a> Neuroscience Meeting Planner. Program No. 516.3, (2010).</li><li>Choe, Y., Han, D., Huang, P. -. S., Keyser, J., Kwon, J., Mayerich, D., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+submicrometer+scans+of+mouse+brain+microstructure:+Neurons+and+vasculatures.">Complete submicrometer scans of mouse brain microstructure: Neurons and vasculatures.</a> Neuroscience Meeting Planner. Program No. 389.10, (2009).</li><li>Choe, Y., Abbott, L. C., Han, D., Huang, P. S., Keyser, J., Kwon, J., Mayerich, D., Melek, Z., McCormick, B. H., Ravi Rao, A., Cecchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy:+High-throughput+imaging+and+analysis+of+massive+volumes+of+biological+microstructures.">Knife-edge scanning microscopy: High-throughput imaging and analysis of massive volumes of biological microstructures.</a> High-Throughput Image Reconstruction and Analysis: Intelligent Microscopy Applications. , 11-37 (2008).</li><li>Choe, Y., Abbott, L. C., Ponte, G., Keyser, J., Kwon, J., Mayerich, D., Miller, D., Han, D., Grimaldi, A. M., Fiorito, F., Edelman, D. B., McKinstry, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+out+the+octopus+connectome+at+submicron+resolution+using+the+knife-edge+scanning+microscope.">Charting out the octopus connectome at submicron resolution using the knife-edge scanning microscope.</a> BMC Neuroscience. 11, 136-136 (2010).</li><li>Mayerich, D., Abbott, L. C., McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy+for+imaging+and+reconstruction+of+three-dimensional+anatomical+structures+of+the+mouse+brain.">Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain.</a> Journal of Microscopy. 231, 134-143 (2008).</li><li>Mayerich, D., Kwon, J., Sung, C., Abbott, L. C., Keyser, J., Choe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+macro-scale+Transmission+Imaging+of+Microvascular+Networks+Using+KESM.">Fast macro-scale Transmission Imaging of Microvascular Networks Using KESM.</a> Biomedical Optics Express. 2, 2888-2896 (2008).</li><li>McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Brain+Tissue+Scanner.">Development of the Brain Tissue Scanner.</a> Technical Report. , (2002).</li><li>Sporns, O., Tononi, G., K&#246;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+connectome:+A+structural+description+of+the+human+brain.">The human connectome: A structural description of the human brain.</a> PLoS Computational Biology. 1, 42-42 (2005).</li></ol>

トッドハフマンは3Scan、KESM技術（米国特許＃6744572）を製造販売する営利企業となります。

KESMは、生物学的試料の大量のサブミクロンレベルの調査（〜1 cm 3）とすることができます。ボリュームのこの種は、これらの器官の構造的な組織についての前例のない量的な情報を提供し、様々な機能の計算機モデルを有効にすることができますこのような脳、肺、心臓、腎臓、などの全体の小動物の器官は、そのような器官から画像をスキャン保持するのに十分です。回路とネットワークダイナミクス、電気的性質、流体と空気の流れのダイナミクス、および筋肉のダイナミクスを含めてこれらの器官の側面、。 この記事で説明する試料調製のプロトコルとポスト解析のプロトコルがKESMへ簡単にアクセスし、結果のデータを持っている外部の研究グループを支援することが期待されています。 KESM操作プロトコルは、これらの外部の研究者がこのユニークな画像診断法の利点と限界を理解するのに役立ちます。

A correction was made to <a href="http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

A correction was made to <a href="http://http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

Erratum: Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy

高スループット、高解像度、3D顕微鏡技術の大きな進歩は、サブミクロンの分解能で神経解剖学的、大量のデータの取得を有効にしている。全脳規模なデータを生成する最初のそのような楽器の一つは、ナイフエッジの走査型顕微鏡（KESM）7、5、9、開発し、著者らの研究室でホストされている。 KESMは、複雑な神経回路網の詳細（ゴルジ体）1、4、8、血管のネットワーク（墨汁）1、4、および細胞体の分布（ニッスル）3明らかに、セクションとサブミクロンの解像度で画像全体をマウスの脳に使用されています。 KESMの使用は、マウスでも脳に限定されるものではありません。私たちは正常にタコの脳6、マウスの肺、及びラットの脳を画像化している。我々は現在、全体のゼブラフィッシュの胚に取り組んでいます。微小循環および血行動態の研究に;これらのようなデータは大幅にconnectomics研究10に貢献できるとproviによる立体学の研究に鼎正確な地上真実。 この記事では、我々は試料作製（固定、染色、および埋め込み）を含むパイプライン、、KESMの設定とセットアップ、切片およびイメージングKESMと、画像処理、データの準備、およびデータの視覚化と分析を説明します。強調は、試料作製し、得られたKESMデータの可視化/分析になります。我々は、この記事で紹介する詳細なプロトコルがKESMとその利用率を高めるために、アクセスを広げるために期待しています。

Watch this Scientific Journal Video about Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy at JoVE.com

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

脳標本の準備からデータの可視化と分析に、ナイフエッジの走査型顕微鏡を使用してシリアルのセクショニング画像への完全なプロセスが記載されている。この手法は、現在のマウスの脳のデータを取得するために使用されますが、それは他の臓器、他の種に適用可能です。

ナイフエッジスキャン顕微鏡用試料作製、イメージング、および解析プロトコル

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

specimen-preparation-imaging-analysis-protocols-for-knife-edge

Research

JoVE Journal

Bioengineering

17.5K ビュー.  Texas A&M University. 脳標本の準備からデータの可視化と分析に、ナイフエッジの走査型顕微鏡を使用してシリアルのセクショニング画像への完全なプロセスが記載されている。この手法は、現在のマウスの脳のデータを取得するために使用されますが、それは他の臓器、他の種に適用可能です。

ビデオ: Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三次元光学分解能光音響顕微鏡

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

生物工学

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子間力顕微鏡によるイメージングのための細菌の固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

ビデオレートでスキャニング共焦点顕微鏡とMicroendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

蛍光顕微鏡を用いたイメージング細胞遊走用接着剤及び可溶性グラデーションを作成する

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

単一分子蛍光顕微鏡による受容体ダイナミクスの高分解能時空間解析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

マイカの製造は、高分解能光学顕微鏡イメージングのための脂質二重層をサポート

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &amp;#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

ハイブリッドμCT-FMT撮像と画像解析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子間力顕微鏡イメージングおよびサポートされている脂質二重層のフォーススペクトロスコピー

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

相関光電子顕微鏡 (クレム) 追跡のウイルス蛋白質のイメージング関連するクライオ固定化細胞の構造

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

回転ディスク全内部反射蛍光顕微鏡による細胞の接着の形成の可視化詳細

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...