著者は全く確認応答がありません。

1。マイカやスライドの準備<ol><li>場所番号1.5（0.17ミリメートル）は、染色ラックにカバースリップ。 </li><li> 60℃で2％の洗剤中の30分間の超音波処理</li><li>脱イオン水で20回洗浄する。 </li><li>ピンセットを用いてスライドを削除して、圧縮空気や窒素を用いたドライ吹く。 </li><li>マイカシートをハサミやカミソリの刃を使用して10×10ミリメートル角に切る。 </li><li>カミソリの刃を使用して、2月3日より薄いリーフレットに各マイカピースをカット。 注：このステップでは、鋭い刃を使用する必要があります。 </li></ol> 2。マイカ組み立てと装着室<ol><li>エタノールで、顕微鏡ガラススライドを清掃してください。 </li><li>光学接着剤を用いてガラススライドに、ステップ1.6に切断した雲母の接着剤リーフレット。低粘度の接着剤は、より良いドロップを広げて、雲母を接着することをお勧めします。 </li><li> 、UVランプ下で10分間硬化させる。 NOTE：接着剤は、380nmから350の範囲の最大吸収とのrequ推奨されるエネルギーでUV光によって硬化される完全硬化のための赤外発光ダイオードは、4.5 J / cm 2である。しかしながら、異なる光源は、（接着剤供給業者によって提供される資料を参照）は、この工程のために使用することができる。 </li><li>スコッチテープで最初の数層を除去することにより、クリーンな雲母表面を露出。 </li><li>雲母表面上に光学接着剤の小滴（〜20μm）を配置します。 注：このステップでは、低自家蛍光のレベルの高粘度の接着剤をお勧めします。我々の経験は、低（ステップ2.2）の組み合わせを用いて、高粘度の接着剤は著しくマイカ分割（ステップ2.7）の有効性を増加させることを示した。 </li><li>ゆっくりと1分間沈降させ、気泡を避けて、接着剤のドロップにしたばかりのきれいにカバースリップを配置。 </li><li> 、UVランプ下で10分間硬化させる。 注：このステップの後、スライドガラス、マイカ、カバースリップのサンドイッチは、（数週間まで）比較的長期間保存することができる。雲母表面が新鮮であることを確認するために、単に実際のSLBの準備の前に次のステップに進みます。 注：接着剤は、380nmから350の範囲の最大吸収をUV光で硬化させ、完全な硬化に必要なエネルギーが推奨さ4.5J/cm 2である。しかしながら、異なる光源は、（接着剤供給業者によって提供される資料を参照）は、この工程のために使用することができる。 </li><li> exactoナイフを使用して、ビデオのように優しく側のガラススライドからカバースリップをマウント解除。ほとんどの場合、マイカの薄くて平らな層がカバーガラスに付着したままになります。スライドガラスに付着雲母（ステップ2.4​​から繰り返す）再利用することができる。 </li><li>マイカ層がまだカバーグラスに接着され、ステップ2.8での分裂の際に、完全に除去されなかったことを確認するために、肉眼で、または解剖顕微鏡で表面品質を確認してください。ピンセットで細かい傷を作ったり、針を解剖すると、マイカとは検出異なる一貫性を持っている接着剤を区別するのに役立ちます。 </li><li> 1.5ミリリットルからゴム製のシールを取り外しバイアルキャップと光を用いて表面に逆さまにキャップを接着接着剤やマニキュア、UVランプで硬化、または空気が、それぞれ、10分を乾燥させます。 注：サンプルはAFMイメージングとの同時光学（TIRFMまたは共焦点）のために準備されている場合は、1.5ミリリットルバイアルキャップは、顕微鏡のステージ上でのAFMヘッドをマウントするには小さすぎる可能性があります。その場合、キャップは、AFMヘッドを取り付けるのに適した大きい直径を有する任意のプラスチック製のO-リングによって置換され得る。実験は、雲母表面を損傷することなく、キャップを除去する必要が場合には、シリコーングリースではなく、接着剤またはマニキュアを用いることもできる。 </li></ol> 3。支持された脂質二重層（SLB）の形成<ol><li>表面室に新たに調製したリポソーム溶液を配置します。 SLBの形成に必要な最小量は、約30μlのである。 注：リポソームおよびSLBの形成の詳細については、このようなバッグらなどのような公開されたプロトコルを参照してください、2014 15。 </li><li>目的のプロトコルを使用して、SLBの形成を進める。インキュベーション中とイメージングは​​、チャンバは、熱ブロックまたはそれらの溶融温度以上使用されている脂質を維持するために必要な温度を維持するために加熱された顕微鏡ステージに配置することができる。 </li></ol>

<ol>
	<li>Giocondi, M. -. 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=Surface+topography+of+membrane+domains.">Surface topography of membrane domains.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1798, 703-718 (2010).</li><li>Garcia-Saez, A. J., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+analysis+of+membrane+dynamics.">Surface analysis of membrane dynamics.</a> Biochim Biophys Acta. 1798, 766-776 (2010).</li><li>Plochberger, B., 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=Cholesterol+slows+down+the+lateral+mobility+of+an+oxidized+phospholipid+in+a+supported+lipid+bilayer.">Cholesterol slows down the lateral mobility of an oxidized phospholipid in a supported lipid bilayer.</a> Langmuir. 26, 17322-17329 (2010).</li><li>El Kirat, K., Morandat, S., Dufr&#234;ne, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+analysis+of+supported+lipid+bilayers+using+atomic+force+microscopy.">Nanoscale analysis of supported lipid bilayers using atomic force microscopy.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1798, 750-765 (2010).</li><li>Szmodis, A. W., Blanchette, C. D., Longo, M. L., Orme, C. A., Parikh, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermally+induced+phase+separation+in+supported+bilayers+of+glycosphingolipid+and+phospholipid+mixtures.">Thermally induced phase separation in supported bilayers of glycosphingolipid and phospholipid mixtures.</a> Biointerphases. 5, 120-130 (2010).</li><li>Strulson, M. K., Maurer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+printing+for+creation+of+patterned+lipid+bilayers+on+tetraethylene+glycol+self-assembled+monolayers.">Microcontact printing for creation of patterned lipid bilayers on tetraethylene glycol self-assembled monolayers.</a> Langmuir. 27, 12052-12057 (2011).</li><li>Satriano, 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=Plasma+oxidized+polyhydroxymethylsiloxane--a+new+smooth+surface+for+supported+lipid+bilayer+formation.">Plasma oxidized polyhydroxymethylsiloxane--a new smooth surface for supported lipid bilayer formation.</a> Langmuir. 26, 5715-5725 (2010).</li><li>Bag, N., Sankaran, J., Paul, A., Kraut, R. S., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+and+limits+of+camera-based+fluorescence+correlation+spectroscopy:+a+supported+lipid+bilayer+study.">Calibration and limits of camera-based fluorescence correlation spectroscopy: a supported lipid bilayer study.</a> Chemphyschem: a European Journal of Chemical Physics and Physical Chemistry. 13, 2784-2794 (2012).</li><li>Singh, S., Keller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+of+supported+planar+membrane+bilayers.">Atomic force microscopy of supported planar membrane bilayers.</a> Biophysical Journal. 60, 1401-1410 (1991).</li><li>Przybylo, M., 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=Lipid+Diffusion+in+Giant+Unilamellar+Vesicles+Is+More+than+2+Times+Faster+than+in+Supported+Phospholipid+Bilayers+under+Identical+Conditions.">Lipid Diffusion in Giant Unilamellar Vesicles Is More than 2 Times Faster than in Supported Phospholipid Bilayers under Identical Conditions.</a> Langmuir. 22, 9096-9099 (2006).</li><li>Scomparin, C., Lecuyer, S., Ferreira, M., Charitat, T., Tinland, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+in+supported+lipid+bilayers:+influence+of+substrate+and+preparation+technique+on+the+internal+dynamics.">Diffusion in supported lipid bilayers: influence of substrate and preparation technique on the internal dynamics.</a> Eur Phys J E Soft Matter. 28, 211-220 (2009).</li><li>Oreopoulos, J., Yip, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+scanning+probe+and+total+internal+reflection+fluorescence+microscopy.">Combined scanning probe and total internal reflection fluorescence microscopy.</a> Methods. 46, 2-10 (2008).</li><li>Shaw, J. E., Slade, A., Yip, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+in+situ+total+internal+reflectance+fluorescence/atomic+force+microscopy+studies+of+DPPC/dPOPC+microdomains+in+supported+planar+lipid+bilayers.">Simultaneous in situ total internal reflectance fluorescence/atomic force microscopy studies of DPPC/dPOPC microdomains in supported planar lipid bilayers.</a> J Am Chem Soc. 125, 11838-11839 (2003).</li><li>Skaug, M. J., Faller, R., Longo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+anomalous+diffusion+with+lipid+bilayer+membrane+structure+using+single+molecule+tracking+and+atomic+force+microscopy.">Correlating anomalous diffusion with lipid bilayer membrane structure using single molecule tracking and atomic force microscopy.</a> J Chem Phys. 134, (2011).</li><li>Bag, N., Yap, D. H., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+diffusion+in+model+and+live+cell+membranes+characterized+by+imaging+fluorescence+correlation+spectroscopy.">Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy.</a> Biochimica et Biophysica Acta. , (2013).</li><li>Sbalzarini, I. F., Koumoutsakos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feature+point+tracking+and+trajectory+analysis+for+video+imaging+in+cell+biology.">Feature point tracking and trajectory analysis for video imaging in cell biology.</a> J Struct Biol. 151, 182-195 (2005).</li><li>Linkert, M., 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> J Cell Biol. 189, 777-782 (2010).</li><li>Schutz, G. J., Schindler, H., Schmidt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+microscopy+on+model+membranes+reveals+anomalous+diffusion.">Single-molecule microscopy on model membranes reveals anomalous diffusion.</a> Biophys J. 73, 1073-1080 (1997).</li><li>Matysik, A., Kraut, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrackArt:+the+user+friendly+interface+for+single+molecule+tracking+data+analysis+and+simulation+applied+to+complex+diffusion+in+mica+supported+lipid+bilayers.">TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers.</a> BMC Research Notes. 7, (2014).</li></ol>

著者らは、開示することは何もありません。

このプロトコルは、脂質二重層の沈着及び高分解能イメージングのための滑らかで薄い雲母表面を調製するための方法が記載されている。技術は、主に高品質の雲母表面を得るために重要であるガラスマイカ - ガラスのサンドイッチ（ステップ2.8）、慎重に解体に限定最小限の手動スキルを必要とします。マイカは、切断せずに光学接着剤から剥離することが可能であるので、新たに切断さ?...

本プロトコルの全体的な目標は、また原子間力と組み合わせることができる光全反射蛍光顕微鏡（TIRFM）または共焦点顕微鏡を用いて雲母支持された脂質二重層（のSLB）の高分解能イメージングのために雲母表面を調製する方法を示すことである顕微鏡（AFM）。 のSLBは、脂質クラスタリング、相分離、ペプチド、タンパク質または他の化合物1-5との二重層成分またはそれらの相互作用の動力学の多くの研究のために広く使用されるモデルである。異なる基質は、研究4,6-8の性質に応じてSLB形成（ すなわち 、ガラス、雲母、二酸化ケイ素、ポリマー）のために使用され得る。典型的な膜の研究では、このようなTIRFMおよびAFMなどの顕微鏡ベースのイメージング技術に依存している。ガラスは透明であるので、従って、TIRFMイメージングのために、ガラス表面は、典型的な選択である。ガラスの製造は比較的容易であり、結果の質は、主に前脂質小胞の沈着に清掃の徹底面で決定。その高距離分解能によるAFMは、雲母表面を必要とします。マイカは完璧な基礎の切断に近いと、ケイ酸塩鉱物である。これにより、新たに切断された雲母が偶数サブナノメートルスケールで膜9の高さの差の観察を可能にする、原子的に平坦である。 （FRAP）の光退色後の、蛍光相関分光法（FCS）、（SMT）を追跡する単一分子、および蛍光回復などの方法を用いて拡散研究は、それによって、ガラス、脂質膜のダイナミクスは、それらが堆積される表面の種類に大きく依存することが示されたマイカは、広くさまざまな結果を10,11を与えることができます。これらの違いは、膜プローブの拡散係数でなく、異なる速度で拡散粒子の別個の集団の検出、およびおそらく異なる状態間の切り替えのみならず挙げられる。 このように、同じ表面（この場合マイカで）使用されない限り、TIRFMおよびAFM技術を用いて得られた結果の直接比較は、しばしば問題となる。 TIRFMおよびAFM二層イメージングは同じ雲母表面12,13上で実施されたいくつかの研究がありますが、マイカはほとんど主な原因の取り扱いの問題により、光学顕微鏡のために使用されていません。マイカの準備をして、光学接着剤12を使用してカバースリップに接着された薄いリーフレットの中に手で切断する必要があります。しかしながら、この方法は満足な結果を達成するために、いくつかの練習を必要とする。また、得られた表面は、それらが困難な低ワーキングディスタンス、高開口数レンズを使用すること、およびしばしば波状の厚さである。 このプロトコルに記載のように調製した雲母表面が成功した高分解能イメージングのため重要となる、「うねり」を避け、非常に薄い（〜220ミクロン、170ミクロンのカバーガラスの厚さを含む）と、非常に平坦である。それらを使用することができるTIRFMまたは共焦点セットアップ用。さらに、同じサンプルを、AFMに転送することができ、さらにはTIRFM /共焦点及びAFMと同時に画像化した。これら2つの手法を組み合わせることにより、二分子膜構造14との拡散挙動の直接的な相関関係が可能になります。雲母表面が新たに切断されるので、清潔で、（ガラス洗浄プロトコルは、通常、ピラニア溶液、硫酸ナトリウム/水酸化カリウムなどの化学物質を含む）時間がかかり、再現性に乏しい、潜在的に危険な清掃手順を必要としない。また、プロトコルに記載小室、の装着未満、50μlに効果的な二重層形成に必要な小胞の体積を減少させる。最後に、表面アセンブリの全体のプロセスは時間がかかるでない（調製物は、30分未満を要する）、および従来の、雲母切断および接着が行うように、操作の熟練度が高い必要としない。

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Bath Sonicator</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">FB15051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Coverslips 24 x 50mm - No H1.5</td>
			<td style="width:135px;">Marienfeld</td>
			<td style="width:119px;">102222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DOPC</td>
			<td style="width:135px;">Avanti Polar Lipids</td>
			<td style="width:119px;">850357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Hellmanex III (detergent)</td>
			<td style="width:135px;">Hellma Analytics</td>
			<td style="width:119px;">320.003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mica V-1 Grade</td>
			<td style="width:135px;">SPI Suppliers</td>
			<td style="width:119px;">1872-CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (high viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA63</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (low viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sphingomyelin-ATTO647N</td>
			<td style="width:135px;">AttoTec</td>
			<td>AD 647N-171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">UV lamp</td>
			<td style="width:135px;">Synoptics Ltd.</td>
			<td style="width:119px;">GelVue GVM20</td>
			<td>The lamp was set to 100% power</td>
		</tr>
	</tbody>
</table>

preparation of mica supported lipid bilayers for high resolution optical microscopy imaging

サポートされている脂質二重層（のSLB）が広く膜特性（相分離、クラスタリング、ダイナミクス）およびこのような薬物またはペプチドなどの他の化合物との相互作用を研究するためのモデルとして使用される。しかしSLB特性は使用される支持によって異なります。 SLB画像化および測定のための一般的に使用される技術は、単一分子蛍光顕微鏡法、FCSおよび原子間力顕微鏡（AFM）である。 AFMは極めて平坦面（一般に雲母）を必要とするほとんどの光イメージング研究は、ガラス支持体上で実施されるため、これらの材料の電荷と平滑特性が強く拡散に影響を与えるので、これらの技術からの結果は、直接比較することはできない。残念ながら、スライドガラスに切断し、雲母の薄いスライスを接着するために必要な手先の器用さの高レベルは、SLBの準備のためのマイカの日常的な使用にハードルを提示します。これは選択の方法、そのような準備マイカになりますが表面は、多くの場合、特に小さなワーキングディスタンス、高開口数レンズを、凹凸（波線）と画像が困難になってしまう。ここでは、脂質小胞沈着およびSLBの準備のために、薄く平らな雲母表面を調製するためのシンプルかつ再現可能な方法を提示する。さらに、当社のカスタムメイドのチャンバーは、SLBの形成のための小胞の非常に小さなボリュームを必要とします。 AFM研究において使用されるものと直接比較され、高品質の脂質二重層表面の、効率的な簡単で安価な製造の全体的な手順をもたらす。

のSLBにおける蛍光脂質プローブの拡散挙動は、基質に応じて異なる。 SMT技術と組み合わせたTIRFMは、粒子の動きを可視化し、それらの拡散係数を抽出するための有益な方法である。ガラスと雲母上に支持DOPC（1,2 -ジオレオイル-sn-グリセロ-3 -ホスホコリン）二重層中に拡散するスフィンゴ-Atto647Nからプローブの単一分子信号が取り付けアニメーションの図に示されている。雲母表面は?...

Watch this Scientific Journal Video about Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging at JoVE.com

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

我々は、高解像度の顕微鏡検査のために雲母支持された脂質二重層を調製する方法を提供する。マイカは、原子スケールで透明で平坦であるが、まれに、取り扱いの困難の撮影で使用されていません。我々の調製マイカシートの偶数堆積をもたらし、二層の調製に使用される材料を減少させる。

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 ...

preparation-mica-supported-lipid-bilayers-for-high-resolution-optical

Research

JoVE Journal

Bioengineering

13.3K ビュー.  Nanyang Technological University. 我々は、高解像度の顕微鏡検査のために雲母支持された脂質二重層を調製する方法を提供する。マイカは、原子スケールで透明で平坦であるが、まれに、取り扱いの困難の撮影で使用されていません。我々の調製マイカシートの偶数堆積をもたらし、二層の調製に使用される材料を減少させる。 

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

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分解能光ファイバMicroendoscopy用その場で細胞イメージング

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

生物工学

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 ...

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

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

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 ...

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., ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

のためのツールと​​してのリポソームカプセル化された近赤外蛍光体の蛍光消光<em&gt;インビボ</em&gt;光学イメージング

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

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 ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

平面支持された二分子層​​上の単一分子蛍光顕微鏡

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

サポートされている脂質二重層中のリガンドナノクラスター配列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

切り替え可能な音響光学分解能光音響顕微鏡<em&gt;インビボ</em&gt;小動物の血管系イメージング

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.