1。イメージングセットアップ LOTは、小型·軽量の携帯型アーキテクチャ8に組み立てることができるし、あるいは断面の撮像能力を持つoptofluidic顕微鏡として9本報告では、しかし、我々は静的断層に向かってベンチトップ型の実装の ​​ための基本的なイメージングセットアップについて説明します。サンプル。 <ol><li> 照明モジュール：LOTでは、このような発光ダイオード（LED）のように部分的にコヒーレント光源を利用することができます。実験的な柔軟性を高めるために、我々は、キセノンランプ（コーナーT260、ニューポート（株））とモノクロを使用していました。モノクロは、 例えば 450から650 nmの中心波長の周り〜1月10日波長スペクトル幅と出力を提供するために調整した。この部分的にコヒーレントな出力は、システムへの部分的コヒーレント光を実現するマルチモード光ファイバ（Thorlabs AFS105/125Y）に結合されている。 光ファイバは、（Thorlabs電動回転ステージに搭載されているPRM-1Z8は、照明の角度を変更するには）Thorlabs TDC001コントローラによって駆動されます。接続された光源と電動ステージは、与えられた角度で光源の面内シフトを達成するために使用された二次元リニアXYステージ（ニューポートILS50CCニューポートMFA-PPDコントローラによって駆動される）上にマウントされています。 </li><li> 検出：Lensfree光トモグラフィーは、検出器アレイが大幅に画像取得ステップまたはデータ処理を変更せずにアプリケーションの要件に応じて選択することができるようなスケーラブルなテクノロジーです。本稿では、2.2μmのピクセルサイズ（IDSイメージング、UI-1485LE-M）で、5メガピクセルを有するCMOSセンサアレイを採用しています。検出器は検出器のアクティブ領域の上に直接置かれた試料の広い視野（ 例えば 24ミリメートル2）ホログラフィック画像を記録するために使用されます。デュアル軸断層撮影法のセットアップが実装される7である場合、検出器はすべきLSOは光軸に垂直な平面で（一緒に検出器付き）のサンプルを回転させるために水平方向（光学テーブルに対して）回転ステージ（ 例えば Thorlabs RP-01）に搭載される7。 </li></ol> 2。試料調製 lensfree光トモグラフィーは、画像などの細胞や微生物などのオブジェクトの様々なことができますが、我々はCの三次元顕微鏡法を行うことにより、基本原則を説明しますelegansのサンプル。 <ol><li>メスやヘラを使用して、Cを含むペトリ皿から寒天の小片を取る虫の文化。各次元に沿って数mmの立方体の部分は線虫の何百ものが含まれています。 </li><li> 1ミリリットルの脱イオン（DI）水を含有するポリプロピレン製のバイアル中に寒天の小さなチャンクを配置します。 </li><li>穏やかに30秒〜1分間ボルテックスし、ワームはDI水に寒天片から這い出しまで10〜15分間待ちます。 </li><li>するために一時的に、ワームを固定する5から10 mMのレバミゾール溶液（Sigma-Aldrich）を1 mlを加え、10分間待ちます。 </li><li>ピペット5月10日バイアルの底からのサンプルの添加、および2つのカバースリップの間にサンドイッチ。このサンプルは、一時的に固定化されたワーム（ 例えば 50から100ワーム）の多数を含む、データ収集を開始するには、検出器上に配置することができます。 </li></ol> 3。データ収集ここでは、LabVIEWインタフェースを開発したカスタムを使用して自動化された典型的なLOT実験のための画像取得の手順をまとめたものです。 <ol><li> 0°は、光源の垂直方向の位置に対応して-50℃に回転ステージの初期角度を調整します。 </li><li>ホームポジションであるXYステージ（0,0）の初期位置を調整します。 </li><li>最高の画像が任意のsなしでできるだけ明るくなるような、そのダイナミックレンジを活用して、検出器の露光時間を調整するaturatedピ​​クセル。 </li><li>回転ステージの角度を変えずに、9枚の画像をキャプチャします。各画像については、各画像は以前のものと比較してピクセルの約4分の1だけシフトするような3x3の正方形のグリッド内の新しい位置にXYステージを移動します。 6μ6グリッド、 例えばそれぞれの角度で複数の画像を、取得、オブジェクトの種類と信号対雑音比（SNR）に応じて分解能を向上することがあります10 </li><li>ホームポジションであるXYステージ（0,0）の初期位置を調整します。 </li><li> +50℃に到達するまで、2°刻みで回転ステージの角度を大きくします。各インクリメントした後、それぞれの新しい角度で、すなわち 、手順3-5。角度の増分は、取得時間と画像解像度の最適化に応じて細かいまたは粗いことができます。 </li><li> （デュアル軸断層撮影方式が利用される場合はオプションの手順）検出器は90°でマウントされている水平方向のステージを回転させます。その後、手順1〜6を繰り返します。 </li></ol> 4。データ解析第3節の手順1〜6に続いて、459の画像のセットは、照明の51の異なる角度の各9つのサブピクセルシフトした画像が含まれ、取得されます。まず、9枚の各セットは、デジタル角度ごとに、高分解能（HR）投影ホログラムを得るために、ピクセルの超解像アルゴリズム10を使用して、処理されます。それは、画素超解像はlensfree画像の空間分解能ではなく、回折限界ではなく、画素サイズの制限を克服することを指すことに留意すべきである。画素超解ホログラムは、その後デジタル51投影画像を得るために7月10日再建されています。 51投影画像のこのセットは、ImageJの（オープンソースの画像解析ソフトウェア）のTomoJ、プラグインを使用して、バックと予測されています。11この逆投影操作は、サンプルの三次元画像（断層像）を出力します。ここで実装されていませんが、文献に記載されたデュアル軸断層撮影方式も利用することができます。 7。この方式では、断層の2番目のセットは、サンプルに関する詳細な空間情報を提供し、軸方向の分解能を向上させる第1回転軸、に関して直交する軸に沿って光源を回転させることによって得られる。 5。代表的な結果大lensfreeの視野（FOV）光トモグラフィーを図1に示されています。試料は検出器アレイの上に直接配置されているように、オブジェクトのホログラム画像は、さらに大きいのアクティブ領域と新興の検出器アレイを使用して増やすことができます24ミリメートルの2 FOV、以上記録することができます。 検出器アレイのピクセルサイズが記録されたホログラフィック画像の解像度を制限していますが、画素超解像技術は、この問題を軽減します。 図2に示す画素超解ホログラムに沿って照明の3つの異なる角度のサブミクロンの空間分解能を提供する再構成画像（ すなわち投影画像）に設定しています。 投影画像は、検体の断層像（ すなわち、3次元画像）を計算する（ 例えば、フィルタ逆投影）の断層画像再構成技術を使用して組み合わせることができます。 図3は、ワームの前を通してxy平面上で3スライス画像を示し、ここで咽頭チューブは、約5μmの外径がこのほぼ円筒状の構造から予想されるZ =8μmのを通じて、スライスでのみ表示されます。また、xz平面における断面像（ 図3上部パネルの挿入図）を明確に咽頭の正常な3Dイメージングを実証し、ワームや咽頭チューブの内側（矢印で示される）の境界を示しています。 <img src="/files/ftp_upload/4161/4161fig1.jpg" alt="図1" /> <strオング&gt;図1は、lensfree光トモグラフィー（LOT）垂直照明のセットアップを使用して記録されたフルフィールド·オブ·ビュー（24ミリメートル2）ホログラフィックイメージを示しています。 LOTの大きな撮像面積のおかげで、多くのワームが同時に単一のデータ取得ステップで撮像することができます。 <img src="/files/ftp_upload/4161/4161fig2.jpg" alt="図2" /> 図2は、超解ホログラム（左パネル）とデジタル再構成された投影画像（右パネル） でのピクセルを示しています。つの異なる照明の角度で線虫ワーム（大きな視野からトリミングされている）。各投影画像は、フィルタ逆投影操作で3次元構造の計算を可能にする別の視野角を、についての情報が含まれています。 <img src="/files/ftp_upload/4161/4161fig3.jpg" alt="図3" /> 図3は、Cの計算された断層を示しています。 虫ワームOfは図2。 （上段）は、z = 3μmで全体のワームのスライス画像を示しています。挿入図はワームの前から断面画像を示しています。はめ込みのスケールバーは50μmである。 （下段）lensfree光トモグラフィの光学切片能力を発揮し、ワームの前までの3スライス画像を表示します。図全体の矢印は、ワームの咽頭チューブ上の同じ点を示しています。顕微鏡像（X40、0.65-NA）も視覚的に比較するために提供されています。

<ol>
	<li>Schmitt, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+(OCT):+a+review,+J.+Sel.+Top.">Optical coherence tomography (OCT): a review, J. Sel. Top.</a> Quant. Elect. 5, 1205-1215 (1999).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+zebrafish+early+embryonic+development+by+scanned+light+sheet+microscopy.">Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.</a> Science. 322, 1065-1069 (2008).</li><li>Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-S&#248;rensen, J., Baldock, R., Davidson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Projection+Tomography+as+a+Tool+for+3D+Microscopy+and+Gene+Expression+Studies.">Optical Projection Tomography as a Tool for 3D Microscopy and Gene Expression Studies.</a> Science. 296, 541-545 (2002).</li><li>Sung, Y., Choi, W., Fang-Yen, C., Badizadegan, K., Dasari, R. R., Feld, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+diffraction+tomography+for+high+resolution+live+cell+imaging.">Optical diffraction tomography for high resolution live cell imaging.</a> Opt. Exp. 17, 266-277 (2009).</li><li>Debailleul, M., Simon, B., Georges, V., Haeberle, O., Lauer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holographic+microscopy+and+diffractive+microtomography+of+transparent+samples.">Holographic microscopy and diffractive microtomography of transparent samples.</a> Meas. Sci. Technol. 19, 074009 (2008).</li><li>Charri&#232;re, F., Pavillon, N., Colomb, T., Depeursinge, C., Heger, T. J., Mitchell, E. A. D., Marquet, P., Rappaz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+specimen+tomography+by+digital+holographic+microscopy:+Morphometry+of+testate+amoeba.">Living specimen tomography by digital holographic microscopy: Morphometry of testate amoeba.</a> Opt. Exp. 14, 7005-7013 (2006).</li><li>Isikman, S. O., Bishara, W., Mavandadi, S., Yu, S. W., Feng, S., Lau, R., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens-free+optical+tomographic+microscope+with+a+large+imaging+volume+on+a+chip.">Lens-free optical tomographic microscope with a large imaging volume on a chip.</a> Proc. Nat. Acad. Sci. 108, 7296-7301 (2011).</li><li>Isikman, S. O., Bishara, W., Sikora, U., Yaglidere, O., Yeah, J., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-portable+Lensfree+Tomographic+Microscope.">Field-portable Lensfree Tomographic Microscope.</a> Lab Chip. 11, 2222-2230 (2011).</li><li>Isikman, S. O., Bishara, W., Zhu, H., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optofluidic+tomography+on+a+chip.">Optofluidic tomography on a chip.</a> App. Phys. Lett. 98, 161109 (2011).</li><li>Bishara, W., Su, T. W., Coskun, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lensfree+on-chip+microscopy+over+a+wide+field-of-view+using+pixel+super-resolution.">Lensfree on-chip microscopy over a wide field-of-view using pixel super-resolution.</a> Opt. Exp. 18, 11181-11191 (2010).</li><li>Messaoudi, C., Boudier, T., Sorzano, C. O. S., Marco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TomoJ:+tomography+software+for+three-dimensional+reconstruction+in+transmission+electron+microscopy.">TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy.</a> BMC Bioinformatics. 8, 288 (2007).</li><li>Heng, X., Erickson, D., Baugh, L. R., Yaqoob, Z., Sternberg, P. W., Psaltis, D., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optofluidic+Microscopy:+A+Method+for+Implementing+High+Resolution+Optical+Microscope+On+A+Chip.">Optofluidic Microscopy: A Method for Implementing High Resolution Optical Microscope On A Chip.</a> Lab on a Chip. 6, 1274-1276 (2006).</li></ol>

利害の衝突が宣言されません。

それはlensfreeオンチップ·ホログラフィック顕微鏡のユニークな形状が画素超解像と断層イメージングを実現するための重要なイネーブラであることを強調することが重要です。撮影した画像は、12に近づくと支離滅裂の接触イメージングのように、投影画像であると仮定されていませんが、 投影ホログラム 、透過光の回折、それが検出器に入射するまでは、デジタルホログラ...

<table border="1"><tbody><tr><td>名</td><td>コンパイ</td><td>カタログ＃ </td><td>コメント</td></tr><tr><td>リニアXYステージ</td><td>ニューポート（株） </td><td> MFA-PP </td><td>ミニチュアリニアステージ</td></tr><tr><td>電動回転ステージ</td><td> Thorlabs </td><td> PRM1Z8 </td><td>電動精密回転マウント</td></tr><tr><td>マルチモード光ファイバ</td><td> Thorlabs </td><td> AFS105/125Y </td><td>マルチモードファイバー</td></tr><tr><td>光源</td><td>ニューポート（株） </td><td> 6255 </td><td>オゾンフリーキセノンランプ</td></tr><tr><td>単色光分光器</td><td>ニューポート（株） </td><td> 74100 </td><td>基礎260 1/4メートルモノクロ</td></tr><tr><td> CMOSセンサアレイ</td><td>アプティナ株式会社</td><td> MT9P031STC </td><td> 5メガピクセルCMOSセンサー</td></tr><tr><td> C. elegansのサンプル</td><td>カロライナBiosupply </td><td> 173500 </td><td>野生型C.エレガンス </td></tr><tr><td>レバミゾール</td><td>シグマアルドリッチ</td><td> L9756-5G </td><td> Tetramisole塩酸塩</td></tr></tbody></table>

lensfree on-chip tomographic microscopy employing multi-angle illumination and pixel super-resolution

それは、異なるサイズのスケールのオブジェクトに関しては3次元（3D）構造の情報を提供することができるように断層イメージングは​​、医学で広く使われているツールでした。マイクロ波·ミリスケールでは、光学顕微鏡のモダリティは、可視光の非電離自然に増加する使用により、検索、および豊富な照明ソースのセット（例えば、レーザや発光ダイオードなど）と検出素子（などの可用性ラージフォーマットCCDとCMOSディテクタ·アレイ）​​。最近開発された光断層顕微鏡のモダリティのうち、一つは光コヒーレンストモグラフィー、光の回折トモグラフィー、光学投影トモグラフィと光シート顕微鏡を含めることができます。1月6日これらのプラットフォームは、細胞、微生物やC などのモデル動物の断面イメージングを提供します線虫 、ゼブラフィッシュとマウスの胚。 既存の3D光学イメージャは、一般的に目を制限する、比較的大型で複雑なアーキテクチャを持っているeは、高度な研究所にこれらの機器の可用性、およびラボオンチッププラットフォームとマイクロ流体チップとの統合を妨げる。別の断層顕微鏡を提供するために、我々は最近7 LOTを破棄レンズやかさばる光学部品の使用。高スループット、コンパクトでコスト効率に優れた光断層撮影法としてlensfree光トモグラフィー（LOT）を開発し、代わりにマルチアングルに依存しています照明と大型撮像ボリューム上のマイクロオブジェクトの深さ分解イメージングを達成するためのデジタル計算。 LOTは、のx &lt;1μmのX &lt;3 15〜100ミリメートルの3大撮像ボリューム上のそれぞれx、y、およびz寸法の程度、、とすることができ、特に有用&lt;1μmの空間分解能で画像生体試料缶ラボオンチッププラットフォーム。

Watch this Scientific Journal Video about Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution at JoVE.com

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Lensfree光トモグラフィーは、&lt;1μmの空間分解能を提供して三次元顕微鏡技術である×&lt;1ミクロン×15〜100ミリメートルの大型イメージングボリューム上のそれぞれx、yおよびz次元において&lt;3μmの<sup&gt; 3</supラボ·オン·チップ·プラットフォームとの統合のために特に役立ちます&gt;。

マルチアングル照明とピクセル超高解像度を採用しLensfreeオンチップ·トモグラフィー顕微鏡

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

lensfree-on-chip-tomographic-microscopy-employing-multi-angle

Research

JoVE Journal

Bioengineering

11.4K ビュー.  University of California, Los Angeles. Lensfree光トモグラフィーは、

ビデオ: Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

干渉反射イメージングセンサー（IRIS）を用いた生体分子の検出

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

生物工学

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

イオンの分離及び核酸の精製のためのオンチップ等速電気泳動

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

オンチップ·血管内皮の炎症性表現型

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

複数の色で高速構造化照明TIRF顕微鏡のガイド

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

ドライ フィルムのレジストを用いた電気化学マイクロ流体バイオ プラットフォーム: デバイス作製、チップの試金の準備、およびシステム操作

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

作製と内皮下電気抵抗を直接定量化する統合型電極を用いたオンチップ器官システムの検証

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters PDC and PDF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

メタノール・組成分析解消抑圧技術の採用によって独立式

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

CRISPR/Cas12a サッカロマイセスセレビシエの多重ゲノム編集と酵母ピクセルアートの創造

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

オープンソースマイクロプレート互換照明パネルのアプリケーション

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

光遺伝学的実験のための簡便で汎用性の高い照明システムの構築

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...