R. v. の仕事は、ロシュ社員 (2014-2017) によって支えられました。

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S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Sibarita, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Deconvolution Microscopy. 95, 201-243 (2005).</li><li>Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging. , 453-467 (2006).</li><li>Chenouard, N., 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=Objective+comparison+of+particle+tracking+methods.">Objective comparison of particle tracking methods.</a> Nat Methods. 11 (3), 281-289 (2014).</li><li>Foret, L., 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+general+theoretical+framework+to+infer+endosomal+network+dynamics+from+quantitative+image+analysis.">A general theoretical framework to infer endosomal network dynamics from quantitative image analysis.</a> Curr Biol. 22 (15), 1381-1390 (2012).</li><li>Daneman, R., Zhou, L., Kebede, A. A., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+are+required+for+blood-brain+barrier+integrity+during+embryogenesis.">Pericytes are required for blood-brain barrier integrity during embryogenesis.</a> Nature. 468 (7323), 562-566 (2010).</li><li>Bell, R. 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=Pericytes+control+key+neurovascular+functions+and+neuronal+phenotype+in+the+adult+brain+and+during+brain+aging.">Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging.</a> Neuron. 68 (3), 409-427 (2010).</li><li>Ben-Zvi, 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=Mfsd2a+is+critical+for+the+formation+and+function+of+the+blood-brain+barrier.">Mfsd2a is critical for the formation and function of the blood-brain barrier.</a> Nature. 509 (7501), 507-511 (2014).</li><li>Stewart, P. 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著者は、ロシュ社によって支払済の雇用の下で。

上記で説明したプロトコルでは、BBB の高分解能顕微鏡の脳浮遊セクション、免疫蛍光染色、画像取得、解析パラメーターの準備について説明します。このメソッドは、最近抗体配信プラットフォーム3BBB の17日間で内因性の IgG の輸送、周皮細胞損失18時に BBB の不均一性のローカリゼーションを調査するために使用されています。プロトコルのさまざまなステップは、実験の特定の目標に合わせて変更できます。まず、厚い (100 μ m) セクションの使用は免疫染色、取り付けの手順の中で自分の取り扱いを容易します。キャピラリーおよび NVU 断面の世代、毛細血管、神経血管系ユニットの三次元再構成のためができます。ティッシュ セクション内にある抗体の普及率が異なる場合があり、いくつかの抗体の汚損、coverslip の近くに組織の浅層に制限することができます。プロトコルは透過のステップの間に洗剤の濃度や抗体、組織浸透を改善するために透過ステップの長さを増やすことによって変更できます。第二に、光散乱と屈折の不一致から収差のため組織 (表面の下の通常 20 に 30 μ m) の内でより深い画像を取得する際に画質が可能性します。この問題を克服するために、組織のイメージより大きいボリュームにこのプロトコルで新しい手法をクリア組織とアクティブな抗体浸透20を結合できます。第三に、デコンボリューション、イメージの軸の解像度を改善するために画像取得後実行されます。このプロトコルで使われるブラインド等化アルゴリズムの選択は、点広がり関数の事前計算する必要はありません、(i) その使いやすさに基づいていた (ii) イメージ品質21、および (iii) mIgG 展示品の不足を改善するため堅牢性実装後細胞内構造を。サンプルで可視化された細胞内構造、によって他のデコンボリューション アルゴリズムは高画質であります。以下参照21,22は、利点と追加画像デコンボリューション アルゴリズムの制限に関する広範な議論を提供します。最後に、画像解析ソフトウェア パッケージの使用は、3 つの次元で毛細血管と細胞内構造の分割を許可しました。明らかに画像解析はプロトコル、たとえば参照23で説明されている代替のパッケージに記載されているソフトウェアに制限されない、セグメント イメージに使用することができます。BBB で細胞内構造の分析のための別のソフトウェア プログラムの適合性は、画像の領域分割の精度を評価することによって経験的に確かめられる必要があります。
このメソッドは、固定サンプルに基づいている、BBB 全体ないトランスサイトーシスのダイナミクスについて直接的な情報をすることはそれ。ただし、ことができますと組み合わせることは経時実験24、たとえば静脈内投与の速度論を再構築した後異なる時点で Bec 内蓄積を測定興味の分子を注入して細胞内の輸送。このアプローチの利点は、18、現在生体のライブ イメージング技術にアクセスされるに示すように、脳深部領域の分析のためことができますです。プロトコル間の重要なステップはティッシュの固定を注意深く監視します。固定 4 %pfa が免疫原性細胞内オルガネラと内因性または末梢投与の免疫グロブリン17 (図 1 b) を大幅に削減します。このプロトコルの制限は、蛍光抗体法に適した高品質抗体 (すなわち、低い非特異染色、低交差) するという条件です。このような試薬が利用、興味のすべての蛋白質の細胞内局在を調べるメソッドを適用できます。たとえば、プロトコルは、脳血管内皮細胞17リソソームを識別するために使用されました。このプロトコルは、共焦点顕微鏡に基づいているので、横方向の解像度は回折による制限は、約 175 に 250 より小さい構造を解決することはできませんする必要があります注意も nm (図 2)。
以前の研究では、共焦点顕微鏡25,26を使用して単位の細胞構成の詳細な分析を実行しています。ただし、透過電子顕微鏡19,27,28の使用にほとんど依存 BBB の細胞内輸送を調査します。このメソッドでは、細胞内構造の最高の水平解像度を提供しています、電子顕微鏡低スループットと挑戦的な手法のままです。また、EM によって視覚化することができますを別の分子標的の数は非常に限られました。このプロトコルでは、BBB で細胞内の輸送を調査するアクセス可能な代替手段を提供しています。5 ~ 6 日で脳画像解析するコレクションからの完全な手順を実行できます。適切な抗体が利用可能な場合、蛍光は同じサンプル内で複数の細胞のタイプ/分子の同時検出を可能します。またこのプロトコルは29空間分解能の限界を克服する超解像顕微鏡技術を組み合わせることができます。全体的に、上記で説明したプロトコルは単位内の興味の蛋白質の細胞内局在変化の定量化を可能です。異なる遺伝的または薬理学的摂動の応用機能生体内でBBB の細胞内の構造とトランスポート機能を調査できるようになります。

血液脳関門 (BBB) は連続の細胞バリア アストロ サイト, ペリサイト, ニューロン, と内皮細胞によって形成される1血液循環から中枢神経系 (CNS) を分離します。BBB 輸送の規制は、脳のホメオスタシスの維持に重要な役割を果たしているし、脳血管内皮細胞 (Bec) の特殊な性質により媒介されます。Bec と基底率が低いトランスサイトーシスとタイトな上皮の存在は、血液媒介分子、2それぞれの傍細胞と transcellular の輸送を制限します。最近では、脳の3,4への治療上の大きな分子の配信を強化する Bec のトランスサイトーシス経路が利用されています。ただし、BBB にトランスサイトーシスのメカニズムまだされていない完全に特徴づけられる5,6。
広範な作業は行われている体外Bec7,8,9,10,間での細胞内輸送を調節する細胞および分子メカニズムを解読する11が、複雑なアーキテクチャと単位 (NVU) の生理機能を要約するこのようなシステムが失敗します。その一方で、研究生体内で12,13 BBB で輸送料金の詳細な定量的情報を提供が輸送の細胞内メカニズムに洞察力を提供しています。したがって、NVU の細胞および細胞内のコンポーネントを調査生体内でおよび前のヴィヴォ非常に挑戦的な14のまま。技術の限られた数だけは、NVU の細胞内細胞レベル下の構造の分析に適しています。ほとんどの研究は、電子顕微鏡を使用してが、この手法が適切なティッシュの準備と検体の取扱いに必要な複雑なプロトコルによって制限されます。したがって、脳のサンプルの処理、分析、および、NVU の細胞内細胞内コンパートメントの定量化を促進する高分解能共焦点顕微鏡に基づく方法を設けています。
ここでは、BBB と NVU は細胞および細胞内レベルでの定量的イメージングを実行するマウス脳浮遊セクションを利用するプロトコルについて述べる。我々 はテストし、イメージを作成し、3 つの次元で NVU を再構築する抗体の数を検証します。さらに、このプロトコルでは、脳の毛細血管内の細胞小器官の最大光の回折限界の解像度で画像ができます。一緒に画像解析マウス神経変性疾患モデルでたとえば、異なる実験条件下で BBB に高分子の細胞内輸送を調査するこのプロトコルを使用することができます。

<table style="border-collapse:collapse;width:665pt" width="886">
	<colgroup>
		<col style="width:223pt" width="297" />
		<col style="width:98pt" width="130" />
		<col style="width:93pt" width="124" />
		<col style="width:251pt" width="335" />
	</colgroup>
	<tbody>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rat monoclonal antibody clone ER-MP12 against CD31/PECAM</td>
			<td class="xl18" style="width:98pt" width="130">Novus Biologicals</td>
			<td class="xl18" style="width:93pt" width="124">MCA2388</td>
			<td class="xl18" style="width:251pt" width="335">Labels Brain Endothelial Cells 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rat monoclonal antibody against Podocalyxin</td>
			<td class="xl18" style="width:98pt" width="130">R&#38;D Systems</td>
			<td class="xl18" style="width:93pt" width="124">MAB1556</td>
			<td class="xl18" style="width:251pt" width="335">Labels Lumen of capillaries 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rabbit polyclonal antibody against CollagenIV</td>
			<td class="xl18" style="width:98pt" width="130">Biotrend</td>
			<td class="xl18" style="width:93pt" width="124">BT21-5014-70</td>
			<td class="xl18" style="width:251pt" width="335">Labels Basement membrane of capillaries 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rabbit IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Goat polyclonal antibody against CD13</td>
			<td class="xl18" style="width:98pt" width="130">R&#38;D Systems</td>
			<td class="xl18" style="width:93pt" width="124">AF2335</td>
			<td class="xl18" style="width:251pt" width="335">Labels pericytes 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-goat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl22" height="42" style="width:223pt;height:31.5pt" width="297">Mouse monoclonal antibody clone G-A-5 against GFAP coupled to Cy3</td>
			<td class="xl18" style="width:98pt" width="130">Abcam</td>
			<td class="xl18" style="width:93pt" width="124">ab49874</td>
			<td class="xl18" style="width:251pt" width="335">Labels Astrocytes 
			Use at dilution 1:100</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rabbit polyclonal antibody against Iba-1</td>
			<td class="xl18" style="width:98pt" width="130">Wako</td>
			<td class="xl18" style="width:93pt" width="124">019-19741</td>
			<td class="xl18" style="width:251pt" width="335">Labels Microglia 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rat monoclonal antibody ABL93 gainst LAMP2</td>
			<td class="xl18" style="width:98pt" width="130">Fitzgerald</td>
			<td class="xl18" style="width:93pt" width="124">10R-CD107BBMSP</td>
			<td class="xl18" style="width:251pt" width="335">Labels Lysosomes 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl23" height="42" style="width:223pt;height:31.5pt" width="297">donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor594</td>
			<td class="xl18" style="width:98pt" width="130">LifeTechnologies</td>
			<td class="xl18" style="width:93pt" width="124">A21203</td>
			<td class="xl21">Use at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor488</td>
			<td class="xl18" style="width:98pt" width="130">LifeTechnologies</td>
			<td class="xl18" style="width:93pt" width="124">A21202</td>
			<td class="xl21">Use at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">goat polyclonal antibody against mouse IgG (H+L) AlexaFluor555</td>
			<td class="xl18" style="width:98pt" width="130">LifeTechnologies</td>
			<td class="xl18" style="width:93pt" width="124">A21422</td>
			<td class="xl21">Use at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Filter paper</td>
			<td class="xl18" style="width:98pt" width="130">Sigma-Aldrich</td>
			<td class="xl18" style="width:93pt" width="124">WHA10334347</td>
			<td class="xl18" style="width:251pt" width="335">Whatman prepleated qualitative filter paper 
			Grade 0858 1/2, grained</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="21" style="width:223pt;height:15.75pt" width="297">Cyanoacrylate glue</td>
			<td class="xl18" style="width:98pt" width="130">Roth Carl</td>
			<td align="right" class="xl18" style="width:93pt" width="124">258.1</td>
			<td class="xl18" style="width:251pt" width="335">Roti Coll 1</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="21" style="width:223pt;height:15.75pt" width="297">Fluorescent Mounting medium</td>
			<td class="xl18" style="width:98pt" width="130">Dako</td>
			<td class="xl18" style="width:93pt" width="124">S3023</td>
			<td class="xl18" style="width:251pt" width="335">Dako fluorescent mounting medium</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Adult mice</td>
			<td class="xl18" style="width:98pt" width="130">The Jackson Laboratory</td>
			<td class="xl18" style="width:93pt" width="124">000664</td>
			<td class="xl18" style="width:251pt" width="335">C57BL/6J male or female mice 
			between 9 and 19 months of age</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="21" style="width:223pt;height:15.75pt" width="297">Vibratome</td>
			<td class="xl18" style="width:98pt" width="130">Leica Biosystems</td>
			<td align="right" class="xl18" style="width:93pt" width="124">14047235612</td>
			<td class="xl21">Leica VT100S vibrating blade microtome</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Laser Scanning Confocal microscope</td>
			<td class="xl18" style="width:98pt" width="130">Leica Microsystems</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl18" style="width:251pt" width="335">Leica TCS SP8 X with HyD detectors 
			and White light laser</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Image processing software</td>
			<td class="xl18" style="width:98pt" width="130">Leica Microsystems</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl21">Leica Application Suite AF version 3.1.0 build 8587</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Image analysis software</td>
			<td class="xl18" style="width:98pt" width="130">Bitplane scientific software</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl21">Imaris version 7.6.5 build 31770 for x64</td>
		</tr>
		<tr height="99" style="height:74.25pt">
			<td class="xl18" height="99" style="width:223pt;height:74.25pt" width="297">Fluorescence SpectraViewer</td>
			<td class="xl18" style="width:98pt" width="130">ThermoFisher Scientific</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl25" style="width:251pt" width="335">https://www.thermofisher.com/ch/en/home/life-science 
			/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html</td>
		</tr>
	</tbody>
</table>

high-resolution confocal imaging of the blood-brain barrier: imaging, 3d reconstruction, and quantification of transcytosis

血液脳関門 (BBB) は、循環と脳の分子の輸送を調節する動的な多細胞インターフェイスです。BBB のトランスサイトーシスは、ホルモン、代謝、および治療用抗体の脳実質への配信を調整します。ここでは、レーザ走査共焦点顕微鏡と BBB で内皮細胞内の細胞小器官を可視化する画像解析による浮遊セクションの蛍光抗体法を組み合わせたプロトコルを提案する.半自動抽出法と細孔容積の定量化と表面積、数、BBB で細胞内オルガネラの強度の 3 D 画像解析ソフトウェアでデータのセットを組み合わせることが可能します。細胞内小胞とその定量化、bbb 内マウス内在性免疫グロブリン (IgG) の検出は、メソッドを説明するために使用されます。このプロトコルは、異なる分子の生体内の BBB トランスサイトーシスを制御するメカニズムの調査に潜在的適用できます。

画像の代表的な例は、ここで説明されているプロトコルから取得、マウスの脳のセクションが基底膜、アストロ サイト、ペリサイト、NVU のさまざまなコンポーネントを認識する抗体で染色した、内皮細胞 (参照してください。使用される特定の抗体の材料表) (図 1 a D、およびE)。この解像度でアストロ サイトの個々 のプロセスと最後の足は毛細血管との直接接触は、区別することが可能です。
このプロトコル細胞内構造を検出するための適合性を強調するには、脳末梢人間の反タウ mAb86 抗体16注入される動物から染色蛍光標識抗ひと抗体 (図 1B)。mAb86 は、タウ16の病理学的形を表現する具体的ターゲット ニューロンに知られています。本記述されているプロトコルを使用して、mAb86 は、回折限界解像度ニューロン (図 1B C) 内の個々 の小胞構造内で検出されました。さらに、内因性マウス IgG はペリサイト (D E 図 1および図 2) ではなく細胞内構造を血管内皮細胞内で検出されました。
脳血管系の高分解能共焦点 z スタックによる毛細血管の BBB で細胞内小胞三次元のセグメンテーションが可能です。図 2では、細胞内のレンダリングと付けられて CollagenIV とマウス IgG 陽性キャピラリーをセグメント化のプロセスの例が示しています小胞。細孔容積あたりの小胞の数の測定によって例えば分割された画像の完全なデータセットを定量化することでさまざまな条件下での細胞内輸送プロセスの変化を研究することが可能です。図 3B C pdgf bret retマウス モデルで以前に報告されたとしての周皮細胞枯渇時にそれぞれ、脳実質の mIgG に対応する mIgG 小胞数と蛍光強度の違いを示しています。17です。 同じアプローチは異なる脳領域18間 bbb 細胞内輸送の変化を分析するも最近使用されました。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56407/56407fig1.jpg"> 
図 1:複数の細胞のタイプと、血管の細胞レベル下の構造の表示ユニット単位 (AとD) とニューロン (B) に含まれている細胞内の小胞またはこのプロトコルで得られた内皮細胞 (E) の代表的なイメージ。A (上) で最大強度投影画像は、周囲の毛細血管 (緑) CollagenIV ラベル GFAP 陽性アストロ サイト (赤) の分布を示します。矢印は、アストロ サイトの個々 のプロセスをポイントします。スケール バー = 20 μ m。この解像度では、個々 のアストロ サイト プロセスと終わり足箱入り地域 (下) の拡大図のように、はっきりと見える。矢印は、アストロ サイトのエンド フィートをポイントします。スケール バー = 10 μ m。Bのイメージは、末梢注入抗体、海馬ニューロン内 (緑)、mAb86 の蓄積を示しています。矢印は、個々 の mAb86 肯定的な小胞をポイントします。スケール バー = 10 μ m。Cのグラフは単一の小胞の線プロファイル強度を示しています。小胞のサイズは強度曲線 (緑のラインとサークル) のガウス適合 (黒実線) の半値全幅から推定しました。Dの画像は、基底膜 (CollagenIV、グレー) 内 (赤) (緑)、周皮細胞に囲まれた内皮細胞の三次元再構成を示しています。下のパネルは、個々 の蛍光チャネルを表示します。スケール バー = 10 μ m。Eの画像ではなくペリサイト (正しいパネルで、グリーンの CD13) が、mIgG (赤) 細胞内小胞 (左右パネル、緑色で CD31) 血管内皮細胞内の局在化を示しています。すべてのイメージの核を染色 DAPI は青で示されます。スケール バー = 5 μ m パネル D と E を参照17から変更されています。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56407/56407fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56407/56407fig2.jpg"> 
図 2: 毛細血管の血液脳関門で細胞内小胞三次元レンダリングします。説明されたプロトコル、(緑) CollagenIV 肯定的な毛細血管のマウス IgG の細胞内小胞 (赤) が高分解能共焦点画像 (A) を取得しました。左側のパネルでは、断面を持つ単一光学セクションを示しています。矢印は、脳血管内皮細胞内の個々 の mIgG 肯定的な小胞をポイントします。フル z スタックを使用して右のショー 3 D 再構成に関するパネル画像処理ソフトウェア (材料の表を参照してください)。細孔容積 (B) および個々 の小胞 (C) 3 次元レンダリングされた、画像処理ソフトを用いて定量化 (材料の表を参照してください)。すべてのイメージの核を染色 DAPI は青で示されます。スケール バー = 5 μ m.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56407/56407fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56407/56407fig3.jpg"> 
図 3: MIgG BBB の細胞内局在性の定量化.代表的なイメージ (A) (collagenIV、緑で標識) 毛細血管の三次元再構成と mIgG の分布 (赤) c57bl/6 マウスとpdgf bret ret周皮細胞枯渇マウス19で記述されています。スケール バー = 10 μ m。Bの画像は、CollagenIV マスク内で細胞内小胞のセグメンテーションを示しています。Thb e グラフは、定量化と毛細血管の体積当たり mIgG 小胞数の比較を示しています。各ポイントは、個々 の毛細血管セグメントからの測定値を表します。実線は平均値と誤差範囲は、データの標準偏差を表します。Cショーの画像、 CollagenIV マスク外 mIgG 蛍光信号。同様に、 Cのグラフは、定量化と mIgG の蛍光強度の比較pdgf bret retと c57bl/6 マウスの脳実質周皮細胞枯渇マウスです。蛍光強度の単位は、すべて c57bl/6 マウスで測定した平均 mIgG 柔強度によって正規化されました。この図は、参照17から変更されています。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56407/56407fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis at JoVE.com

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

血液-脳関門の高分解能共焦点イメージング: イメージング、三次元、トランスサイトーシスの定量化

ここで単位と血液脳関門脳浮遊セクションを使用して高解像度の画像とマウスの三次元再構成のための顕微鏡ベースのプロトコルを紹介します。このメソッドは、可視化、解析、および BBB で細胞内オルガネラの定量化のためことができます。

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

high-resolution-confocal-imaging-blood-brain-barrier-imaging-3d

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.6K ビュー.  Roche Innovation Center Basel. ここで単位と血液脳関門脳浮遊セクションを使用して高解像度の画像とマウスの三次元再構成のための顕微鏡ベースのプロトコルを紹介します。このメソッドは、可視化、解析、および BBB で細胞内オルガネラの定量化のためことができます。

ビデオ: High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

出生後の脳における神経細胞の移動の高分解能タイムラプスイメージングのための器官型スライスアッセイ

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

神経科学

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

セミ厚い脳スライスにおける高解像度蛍光イメージングへの迅速なアプローチ

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

3Dイメージングと形態素解析のための成体マウスの脳血管キャスティング

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

人間の中脳の高解像度機能的磁気共鳴画像法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高解像度<em&gt;インビボ</em&gt; 3T磁気共鳴画像を用いたヒト海馬サブフィールドのためのマニュアルセグメンテーションプロトコル

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

共焦点肉眼検査を使用した生後小脳顆粒細胞の移動のex vivoイメージング

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

ヒト皮質下の高分解能構造磁気共鳴イメージング<I&gt;インビボ</I&gt;と死後

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

網膜リボンシナプスにおける膜受容体の高分解能定量的イムノゴールド分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.