A. OZCANは感謝して科学者やエンジニアのための大統領早いキャリア賞（PECASE）の支援を認める陸軍研究局（ARO）若手研究者賞、全米科学財団（NSF）のキャリア賞、海軍研究局（ONR）若手研究者賞と米国立衛生研究所（NIH）所長、国立衛生研究所のオフィスからディレクターズ新イノベーター·アワードDP2OD006427。

このセクションでは、我々の携帯電話ベースの広視野蛍光顕微鏡13と光流体イメージングサイトメトリープラットフォーム14のための実験プロトコールを紹介します。我々は、これらのイメージングプラットフォームをテストするために蛍光ビーズおよび蛍光標識白血球を使用します。 広視野蛍光顕微鏡オプト流体イメージングフローサイトメーター基づき携帯電話のA.準備カメラ付き携帯電話やコンパクトフォトカプラ流体アタッチメントアドオン：携帯電話ベースの広視野蛍光顕微鏡またはフローサイトメーターは、2つの主要な部分から構成されます。 1。カメラ付き携帯電話紹介する手法は、ほぼすべてのカメラ付き携帯電話にも適用可能であるが、我々は、これらのデバイスのためのベースとしてソニーエリクソン相野を選択しました。この携帯電話は、それと〜4.65ミリメートルの焦点距離（f 1）を内蔵しており、レンズにインストール〜8メガピクセルのRGBのCMOSセンサーを持っています。 </p&gt; 2。広視野蛍光顕微鏡用オプト流体アタッチメント光学添付ファイルは、オートデスクが設計されており、ABSplus熱可塑性材料を使用してディメンション·エリートの3次元プリンタで印刷されます。この印刷プロセスでは、モデルとサポート材料がプリンタ内に押出ヘッドで加熱され、モデリングベース上の層ごとに堆積される。この手順が完了すると、支持材料は、所望のプロトタイプの堅牢な3Dモデルを残して、溶解させることができる我々の光学アタッチメントの設計では、LED（〜470nmの中心波長、Digikeyを）、プラスチックフィルター（＃NT54-46、エドムンドで構成されていますオプティクス）、サンプルトレイと、平凸レンズF 2 = 15ミリメートル（＃NT45-302、エドモンド·オプティクス）。すべてのLEDとプラスチックフィルターを簡単にフルオロフォアのスペクトルに基づいて変更することができます。このオプト流体アタッチメントを組み立てるための手順は次のとおりです。 <ol><li>その具体的なレンズホルダ内の添付位置にレンズを配置します。 </li><li>フィルタトレイの上にプラスチック製のフィルターを置き、添付ファイルの中にスライドさせ、またはテープ携帯電話のカメラレンズの前にプラスチックフィルター。 </li><li>添付ファイルに、LEDカセットを挿入します。 </li><li>サンプルトレイにサンプルスライドガラスに置きます。添付ファイルにサンプルトレイをスライドさせます。サンプルに向かってLEDが直面している。 </li><li>余分なレンズが直接、携帯電話のカメラレンズに触れているような、携帯電話上に添付ファイルをクリップ。 </li><li> LEDをオンに添付ファイルでスイッチを使用します。 </li><li>画像その &quot;ナイトモード&quot;を使用して携帯電話のカメラユニットとの利害のサンプル。 </li></ol> 3。蛍光イメージングサイトメトリー用オプト流体アタッチメントまれな事象の検出のための液体試料を大量に選別する必要があるとき、optofluidicフローサイトメトリー装置が好まれることがあります。私たちは、広視野の蛍光顕微鏡の設計を変更し、フローサイトメーター、ワットに変換することができますここに、PDMSベースのマイクロ流体チャネルは絶えず撮像ボリュームを通して液体試料を提供する目的で使用されています。光学アタッチメントもオートデスクによって設計され、ディメンション·エリートの3次元プリンタで印刷されます。また、LEDは（〜470nmの中心波長、Digikeyを）、プラスチックフィルター（＃NT54-46、エドモンド·オプティクス）、サンプルトレイ、非球面レンズ（f = 4.5ミリメートル）（製品＃C230TME -で構成されています。 Thorlab）。このオプト流体アタッチメントを組み立てるための手順は次のとおりです。 <ol><li>添付ファイルに非球面レンズを配置します。 </li><li>フィルタトレイの上にプラスチック製のフィルターを置き、添付ファイルの中にスライドさせ、またはテープ携帯電話のカメラのレンズの前にプラスチック製のフィルター。 </li><li>同じ光流体アタッチメントにマイクロ流体チャネルをスライドさせます。 </li><li>余分なレンズが直接、携帯電話のカメラレンズに触れているような携帯電話に添付ファイルをクリップ。 </li><li> LEDをオンに添付ファイルでスイッチを使用します。 </li><li>マイクロ流体を接続ICは、シリンジポンプへのチャネルと一定流量でマイクロ流体デバイスに液体試料を提供します。 </li><li>携帯電話のカメラのビデオモードを使用して、マイクロ流体チャネルを流れる蛍光細胞/粒子のムービーをキャプチャします。 </li></ol> B.サンプルの調製 4。蛍光マイクロ粒子試料の調製<ol><li> 10μmの直径（：製品番号F8834励起/発光580nm/605nm、緑のビーズ：赤のビーズ製品番号F8836：励起/発光505nm/515nm）で蛍光ビーズは、Invitrogen（カールスバッド、カリフォルニア州）から購入する。 </li><li>緑色蛍光ビーズ、DI水の40μlの赤色蛍光ビーズを10μl10μlを混合します。 </li><li> Place10マイクロピペットを用いてスライドガラス上でこのビーズ混合物μlとサンドイッチ構造を作るために、その上に別のスライドガラスを入れた。 </li><li>サンプルトレイにこのサンドイッチ構造を挿入して、携帯電話の添付ファイルの中にスライドさせます。 </li&gt; </ol> 5。蛍光標識白血球の調製<ol><li> SYTO16核酸の蛍光標識キット（＃S7578、ライフテクノロジー）とリン酸を取る食塩水（PBS）は冷蔵庫からバッファリングされ、室温にそれらをもたらす。 </li><li> EDTA採血管から1.5 mlのポリスチレンチューブ（＃05-408-129、フィッシャー·サイエンティフィック）に200μlの全血検体を転送します。 </li><li> 200μlの全血検体に1ミリリットル赤血球溶菌バッファー（＃R7757、Sigma-Aldrich）を追加し、よく混合します。 </li><li> 5分後、溶解された血液試料を遠心分離し、上澄み液を除去します。 </li><li> 200μlのPBSバッファーに白血球細胞ペレットを再懸濁し、穏やかに混ぜる。 </li><li>白血球細胞試料に5μlの1mMのSYTO16溶液を加える。 〜30分間、暗い環境でのアルミニウム箔とインキュベートしたサンプルを包む。 </li><li>再度サンプルを遠心分離します。上清を除去し、標識された白血球細胞ペレットが再PBS緩衝液に懸濁した。 </li><li>カバースリップに白血球液体試料を標識した5から10μlを置き、試料の上に第2のカバースリップを置く。 </li><li>それは携帯電話蛍光顕微鏡を用いて挟まれたサンプルサンプルトレイにスライドして画像を挿入します。 </li></ol>代わりに<ol start="10"><li>また、ビデオモードで携帯電話のカメラを使って流れる細胞の蛍光顕微鏡映画を捉えながら、継続的に、自動化されたシリンジポンプを使用してマイクロ流体チャネルを介して蛍光標識された白血球をお届けします。我々はまた、携帯用の電池式シリンジポンプあるいは重力が微小流体チャネルを通る流れを駆動するために利用できることを強調すべきである。 </li></ol>

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博士OZCANは、計算イメージングと顕微鏡ツールの実用化を目指しているベンチャー企業の創設者でもある。

我々は、携帯電話のカメラに軽量かつコンパクトな光流体添付ファイルを使用して携帯電話ベースの広視野の蛍光顕微鏡と光流体イメージングフローサイトメトリーでの我々の最近の結果を発表しました。このプラットフォーム技術を用いて、我々は、微小粒子と全血サンプル中の標識された白血球を含む蛍光オブジェクトをイメージした。したがって、このコンパクトで費用対効果の高い?...

顕微鏡およびフローサイトメトリーは、広く生物医学および科学研究のテクニック1月12日だけでなく、様々な種類の細胞の計数および特性評価のための臨床診断に使用されます。しかし、従来の顕微鏡やフローサイトメトリー機器は、主に定評のある中央研究所にその使用を制限する、比較的複雑で高価である。最近、私たちは約束を示している携帯電話、13,14上に集積コンパクトかつ軽量蛍光イメージングサイトメトリーや顕微鏡装置を開発したコスト効率のための資源が限られた環境に蛍光顕微鏡、フローサイトメトリーおよび関連ミクロ解析技術を翻訳世界的な健康に影響を与える様々な遠隔医療アプリケーション。 optofluidicフローサイトメトリーの設定では（ 例えば、 図1C及び1D参照）、カスタム設計のポリジメチルシロキサン（PDMS）ベースのマイクロ流体チャネルはpositioneです携帯電話のカメラユニットの前面で、dは、どこで、発光ダイオード（LED）は、チャネルのエッジに突き合わせ結合されている。一緒に液体試料の入ったこのマイクロ流体チップは、励起光を均一にマイクロ流路内の蛍光標識された試料を圧送するように導かれるように光流体平面導波路を（例えば、PDMS液-PDMS用で構成）を形成する。これらの標識されたオブジェクトからの蛍光発光、EG細胞は、さらに携帯電話のカメラユニットの直後に配置された追加のレンズを通って撮像されると携帯電話相補型金属酸化膜 ​​半導体（CMOS）イメージセンサ上にマッピングされます。蛍光発光は、励起光路に垂直に収集されているので、安価なプラスチック吸収フィルターは、散乱励起光を除去するのに十分であると蛍光イメージングに必要なまともな暗視野背景を提供することができます。我々は、画像のstatの蛍光オブジェクトも、同じようなオプト·流体設計をすることができます使用して蛍光粒子は二枚のガラスの間に挟まれているICモード（ 図1Aおよび1Bを参照）、マイクロ流体チャネルに流れるのではなく、スライドさせると、これらの蛍光粒子からの蛍光発光は、粒子計数のために携帯電話のCMOSイメージセンサによって捕捉され、特性評価。異なるアプリケーション要件、フローサイトメトリーや広視野蛍光顕微鏡に基づいて選択することができる。例えば、携帯電話のフローサイトメトリー装置は、希少な細胞や病原体の検出のための液体試料を大量に（数ml など ）をスクリーニングするために特に有用である可能性があります。 本稿では、コ​​ンパクトでコスト効果の高い光流体添付ファイルを使用して携帯電話上の蛍光顕微鏡やイメージングフローサイトメトリーツールの統合、我々の最近の結果のいくつかを確認します。これらの携帯電話ベースのマイクロ分析、イメージングサイトメトリーおよびセンシングプラットフォームは、様々な機会を提供することができます遠隔医療やポイントオブケア診断のために、特に世界のリソースが限られた地域では、世界の健康課題に対する我々の戦いに影響を与える。

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog Number </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell-phone</td>
			<td>Sony</td>
			<td>Sony Ericsson Aino</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plano-convex lens</td>
			<td>Edmund Optics</td>
			<td># NT45-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspherical lens</td>
			<td>Thorlab</td>
			<td># C230TME-A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Edmund Optics</td>
			<td>#NT54-46</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue LED</td>
			<td>Digikey</td>
			<td>#365-1201-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Battery</td>
			<td>Digikey</td>
			<td>#P032-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene tube</td>
			<td>Fisher Scientific</td>
			<td>#05-408-129</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYLGARD 184 SILICONE ELASTOMER KIT</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8834</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Green fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8836</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYTO16 nucleic acid fluorescent labeling</td>
			<td>Life Technologies</td>
			<td># S7578</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone

蛍光顕微鏡やフローサイトメトリーは、広く生物医学研究と臨床診断のツールを使用されています。しかし、これらのデバイスは、リソースが限られた設定で、それらはあまり効果的なもの、一般的には相対的にかさばるし、高価である。潜在的に、これらの制限に対処するために、我々は最近、小型·軽量、かつ費用対効果の高い光流体添付ファイルを使用して携帯電話で広視野の蛍光顕微鏡やイメージングフローサイトメトリーツールの統合を実証している。私たちのフローサイトメトリーの設計では、蛍光標識された細胞は、既存の携帯電話のカメラユニットの上方に位置しているマイクロ流体チャネルを介してフラッシュされます。電池式発光ダイオード（LED）が効果的に励起光を均一に蛍光ターゲットを励起するために導かれ、マルチモードのスラブ導波路として機能し、このマイクロ流体チップの側面に尻結合されています。携帯電話のカメラが流れる蛍光細胞のタイムラプスムービーを記録するこの映画のデジタルフレームが目的の標的ソリューション内で標識された細胞の数を数えるのに使われるマイクロ流体チャネル、。我々はこれらの蛍光標識された細胞は、 例えば、2つのスライドガラスの間に蛍光粒子を挟むと、 例えばの空間分解能を達成することができる携帯電話のカメラを使用してその蛍光画像をキャプチャすることにより、静的モードで画像も、同様の光流体設計をすることができます使用して〜 〜81 mm 2の非常に大きな視野以上10μmである。この携帯電話ベースの蛍光イメージングフローサイトメトリーや顕微鏡プラットフォームは、HIV陽性患者のモニタリングに向けたり、水を飲んで水を媒介とする寄生虫の検出のためのCD4 + T細胞の計数などのために、特にリソースが限られた設定で役に立つかもしれません。

携帯電話のカメラが流れる細胞のタイムラプス蛍光顕微鏡映画を記録しながら、ポンピング光流/励起スキーム（ 図1Cおよび1D）で、蛍光標識された細胞は、連続して、シリンジポンプを用いて、マイクロ流体チャネルに配信することができます。これらの蛍光映画はその後急速に蛍光イメージングフローサイトメータの機能を考慮して、自動的に絶対数とマイクロ流...

Watch this Scientific Journal Video about Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone at JoVE.com

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

我々は、コンパクトでコスト効果の高い光流体添付ファイルを使用して携帯電話上の蛍光顕微鏡やイメージングフローサイトメトリーツールの統合、我々の最近の結果を確認します。これらの携帯電話基にしたマイクロ分析装置は、以下のようなリソースが限られた設定で、例えば、水試料のハイスループットスクリーニングのためだけでなく、様々な細胞計数タスクを実行するように、フローサイトメトリー分析のために有用かもしれません。

携帯電話上の広視野蛍光顕微鏡と蛍光イメージングフローサイトメトリー

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

wide-field-fluorescent-microscopy-fluorescent-imaging-flow-cytometry

Research

JoVE Journal

Bioengineering

23.5K ビュー.  University of California, Los Angeles. 我々は、コンパクトでコスト効果の高い光流体添付ファイルを使用して携帯電話上の蛍光顕微鏡やイメージングフローサイトメトリーツールの統合、我々の最近の結果を確認します。これらの携帯電話基にしたマイクロ分析装置は、以下のようなリソースが限られた設定で、例えば、水試料のハイスループットスクリーニングのためだけでなく、様々な?...

ビデオ: Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

生体内イオン濃度の測定のための蛍光ナノ粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

生物工学

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

チップ上Lensless蛍光顕微鏡

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

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 ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

マルチスペクトルイメージングフローサイトメトリーによるナノ粒子と細菌の細胞内在化を分析する

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

トラクション力顕微鏡用の細胞培養表面近くのローカライズレポーター蛍光ビーズのための新しい方法

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

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 ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

マルチスペクトルイメージングフローサイトメトリーを用いてインビトロ微小核アッセイを行う自動化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

光音響フローサイトメトリーを用いた卵巣癌検出

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

蛍光色素標識リポソームのin vivo組織特異的細胞取り込みの調製、投与、および評価

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...