This work was supported by DARPA grant number 84389.01.44908, an NSF CAREER award (DBI-1253293), an NIH exploratory/developmental research grant (CA195709), and NIH New Innovator Awards (HD080351, DP2-AR068129-01), and a New Directions grant from the UCSF resource allocation program.

1. SU8マスター製作<ol><li>設計ソフトウェアを使用して、3層の製造のためのマイクロ流体構造を設計し、10μmの分解能で回路基板フィルム上のベンダーによって印刷されたデザインを持っています。デバイス設計の詳細は、添付文献6に記載されているチャネルの形状は、 図1に示されている。層は、各製造層8の機能を連結するために役立つ位置合わせマークを含むべきです。 </li><li>スピンコーター上の予め清浄3インチ径のシリコンウェハを置き、チャックに固定するために真空をオンにします。 80ミクロンの層の厚さを提供し、1750 rpmで、その後30秒間、500rpmで20秒間ウェハとスピンの中心にSU8-3050の1ミリリットルを適用します。 </li><li>ウェハを取り外し、30分間135℃のホットプレート上で焼きます。ウエハは、次のステップに進む前に室温まで冷却します。 </li><li>コリメートさ190メートル下の1 番目のレイヤーマスクに塗布されたウェハを公開W、365 nmのは、3分間のLED。露光後、次のステップに進む前に、室温まで冷却、1分間135℃のホットプレート上にウエハを配置します。 </li><li>スピンコーターにウェハを置き、チャックに固定するために真空をオンにします。 40ミクロ​​ンの追加の厚さを提供する層で、その結果、5,000 rpmで、その後30秒間、500rpmで20秒間ウェハとスピンの中心にSU8-3050の1ミリリットルを適用します。 </li><li>次のステップに移動する前に室温に冷却後、ウエハを取り出して、30分間、135℃のホットプレート上で焼きます。 </li><li> 1.3にパターニングジオメトリ上に2 番目のレイヤーマスクの位置を合わせ、3分間LED nmのコリメート190ミリワット、365に塗布されたウェハを公開します。露光の後、4分30秒135℃のホットプレート上の場所は、次のステップに進む前に、室温まで冷却。 </li><li>スピンコーターにウェハを置き、チャックに固定するために真空をオンにします。その後、500rpmで20秒間ウェハとスピンの中心に30をSU8-3050の1ミリリットルを適用します秒は1000rpmで、100μmの追加の厚さを提供する層が得られます。 </li><li>次のステップに移動する前に室温に冷却後、ウエハを取り出して、30分間、135℃のホットプレート上で焼きます。 </li><li> 1.3にパターニングジオメトリ上に第3層マスクの位置を合わせ、3分間LED nmのコリメート190ミリワット、365に塗布されたウェハを公開します。露光の後、9分間135℃のホットプレート上の場所は、次のステップに進む前に、室温まで冷却。 </li><li> 30分間のプロピレングリコールモノメチルエーテルアセテートの撹拌浴に浸漬して、マスクを作成します。 1分間135℃のホットプレート上でイソプロパノールとベークでウエハを洗浄します。ポリジメチルシロキサン（PDMS）成形用の100ミリメートルペトリ皿に開発されたマスターを配置します。 </li></ol> 2. PDMSデバイスの製造<ol><li>プラスチック製のカップに硬化剤5gでシリコーンベースの50グラムを組み合わせることにより、1 PDMS：10を準備します。攪拌棒を取り付けた回転工具で内容を混ぜます。 DEG30分間、デシケータ内部に混合物として、または全ての気泡が除去されるまで。 </li><li>マスターの上に3ミリメートルの厚さを与え、さらにガス抜きのために戻ってデシケーターに配置するためにPDMSを注ぎます。一旦全ての気泡が除去され、80分間80℃でデバイスを焼きます。 </li><li>パターン化された面を上にして清浄な表面上メスと場所を使用して、金型からデバイスをカットし、0.75ミリメートル生検パンチで流体入口と出口をパンチ。 120μmから220μmの背の高い形状は、デバイスの側からアクセス可能であるように、デバイスを切断しなければなりません。 </li><li>プラズマは、300 Wのプラズマクリーナーで20秒間1ミリバールのO 2プラズマで3インチのガラススライドにより予め洗浄2インチと共に、機能面を上にして、デバイスを扱います。スライドガラスのプラズマ処理面上にPDMSデバイスのパターン形成された側を配置することによってPDMSデバイスを結合。 80℃のオーブンにデバイスを配置し、40分間組み立てられた装置を焼きます。 </li><li>チャンネルをレンダリングフッ素化された表面処理液を用いてデバイスを洗い流すために、注射器を用いて、疎水性。 10分、溶媒を蒸発させるために直ちに80℃でデバイスを焼きます。 </li></ol>光学部品の調製<ol><li> 105μmのコア、125μmのクラッド、NA = 0.22の光ファイバの最後の5ミリメートルから絶縁体を除去することにより、レーザ励起光ファイバを準備します。 </li><li> 2 回目の同一の繊維3.1を繰り返すことにより、2 番目のカラーレーザー励起ファイバーを準備します。 </li><li> 200μmのコア、225μmのクラッド、NA = 0.39の光ファイバ用の3.1を繰り返すことにより、蛍光信号を収集するための光ファイバを準備します。 </li><li>先端が平らな面で終わっていない場合は、ファイバスクライブと端を再切断する - 全ての繊維の先端を点検します。 </li><li> 50 mWのにレーザーファイバーカプラを接続し、405 nmのレーザー、レーザーに105ミクロンコアファイバの1を添付してください。レーザパワーのセンサーでストリッピング終了を指示メータは、レーザパワーを最大化するためにレーザカプラの微調整を使用します。 </li><li> 50ミリワット、473 nmのレーザーのために3.5を繰り返します。 </li><li>レーザー光を遮断し、放出される蛍光を送信するために、レンズチューブを使用して、PMT上446/510/581/703 nmのクワッドバンドパスフィルタをマウントします。光はPMTに到達する前にフィルタを通過するように、ファイバカプラを取り付けます。 </li><li> 3.3から3.7に組み立てられたユニットに光ファイバを接続します。 </li></ol> 4.オフライン混合乳剤の生成<ol><li> 60ミクロン×60ミクロンのオリフィスを有するフロー焦点マイクロ流体dropmaker装置を得ます。 </li><li> 2％のイオン性フッ素系界面活性剤9とHFE 7500油と1ミリリットル注射器を埋めます。 </li><li> PBS中で1ミリリットルフルオレセインの次の組み合わせでシリンジ（FITC）およびデキストランコンジュゲートの青色色素（CB）のシリーズを埋める：1 nMのFITC / 10 nMのCB、10 nMのFITC / 10 nMのCB、1 nMのFITC / 100 nMでCB、および10nMのFITC / 100 nmのCB。 </li><li>反転マイクロ​​のステージにdropmakerデバイスをマウント対処とシリンジポンプに水と油の注射器。カップルPE-2のチューブを使用して、デバイスへの注射器。 </li><li>油のための500μL/ hrであり、水溶液のための250μL/時でシリンジポンプを実行して、4.3からソリューションを含む80ミクロン液滴の混合物を作成します。水性試料の種類ごとに、交差汚染を除去するために、新鮮なdropmakerデバイスを使用します。空のシリンジに各色素の組み合わせからエマルジョンの〜200μLを収集します。 </li><li> 4滴型は単一収集シリンジに収集された後、繰り返し注射器を回転させることによってエマルションを混合します。 </li><li>繰り返し水溶液はPBS中0.1、1、10、及び100nMのFITCを含有する4.2〜4.6ステップ。 </li></ol> 5.光ファイバの挿入<ol><li> &lt;100マイクロ秒のシャッタースピードの可能なデジタルカメラと接続された倒立顕微鏡のステージ上のステップ2で作製したマイクロ流体チップを置きます。 </li><li>側から慎重に作業し、inse主流路にを通して穿刺しないように注意しながら、最も遠い上流120μmのチャネルに473 nmのレーザーに結合された繊維を室温。 </li><li> 300ミクロンの繊維間隔を提供し、最下流120μmでハイサイドチャネルに405 nmのレーザーに結合された光ファイバを挿入します。 </li><li> 2レーザー励起繊維に通常の220μmの背の高いチャネルにPMT結合繊維を挿入します。 </li></ol>混合エマルジョンの6蛍光検出<ol><li> PE-2のチューブで検出装置のスペーサーオイル入口に​​2％のイオン性フッ素系界面活性剤とHFE 7500を充填した5ミリリットル注射器を取り付けてください。 </li><li> PE-2のチューブを使用してデバイスの液滴再注入入口に垂直に配向されたシリンジポンプやカップルでの混合FITC / CBエマルジョンを含む注射器をマウントします。廃棄物容器にデバイスの出口からPE-2チューブの長さを接続します。 </li><li>総理の両方まで、千マイクロリットル/時間でポンプのそれぞれを実行して、デバイス石油や液滴は、定期的にデバイスに結合し、下流に流れるされるように見られています。 </li><li>スペーサーオイルが検出領域を通って移動する液滴との間に有意な間隔を提供し、6000μL/ hrで、100μlの/時の液滴で実行されるように流量を調整します。 </li><li> 、レーザーをオンにするデータ収集プログラムを起動し、ベースラインのノイズ・フロアを100倍以上ある信号を提供するためにPMTのゲインを調整します。二重ピークのすべてが単一の、直線的にスケーリングされたtimetraceにはっきりと見えるようにレーザーパワーを調整します。 </li><li>少なくとも20kHzでPMT電圧timetraceの60秒を取得します。 MATLABなどのコンピューティングのプログラムにデータをインポートし、カスタム・コンピューティング・プログラム・スクリプトを使用して記録ダブレットのピークの高さを測定します。 注：これは、補足情報として提供されます。 </li><li>繰り返しは、405 nmレーザーをオフにし、4.7で作成されたFITC-のみの混合エマルジョンを使用して6.2から6.7を繰り返します。 </li></ol>

<ol>
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The authors have nothing to disclose.

光ファイバ検出は、流体チャネルに対する光ファイバの位置合わせを必要とします。我々のデバイスは、多層フォトリソグラフィーで作製ガイドチャネルを利用しているので、互いに対するマスクの配置が非常に重要です。ファイバガイドチャネルは、流体チャネルに近づきすぎている場合、流体の漏れの可能性があります。ガイドチャネルが離れすぎて配置又はずれている場合、検出ファ?...

液滴マイクロフルイディクスは、キャリアオイル1に懸濁水性液滴の多数の実験を区画化することにより、高スループット生物学のためのプラットフォームを提供します。液滴は、単一細胞分析2、デジタルポリメラーゼ連鎖反応（PCR）3のように変化させるような用途に使用され、進化4酵素されています。彼らの明るい信号と高速時間応答がキロヘルツのレートでサブナノリットルの液滴の体積を検出すると互換性があるとして蛍光アッセイは、液滴マイクロ流体の検出の標準モードです。多くのアプリケーションは同時に、少なくとも2色のための蛍光検出を必要とします。例えば、私たちの研究室では、一般的に、アッセイの結果を一つの検出チャネルを使用した実験をソートPCR活性化液滴を行い、アッセイ陰性滴可算5にするために、二次背景染料を使用しています。 液滴マイクロ流体のための典型的な検出局はBAです落射蛍光顕微鏡上のsed、およびサンプルに集中するために、顕微鏡に自由空間レーザからの励起光を導入するために、複雑な光の操作スキームを必要とします。蛍光を液滴から出射された後、各検出チャンネル一光電子増倍管（PMT）は、波長帯を中心に利用するように、放出された蛍光を光がフィルタリングされます。落射蛍光顕微鏡ベースの光検出システムは、それらの費用、複雑さに参入障壁を提供し、メンテナンスを必要としました。光ファイバは、ファイバが手でマイクロ流体デバイスに挿入することができるので、ミラーベースの光経路の必要性を除去し、簡略化された堅牢な検出スキームを構築する手段を提供し、光路は、光ファイバコネクタを使ってインターフェースされることを可能にします。 本稿では、光学ファイバのアレイを利用することにより、多色蛍光検出を実行するためのコンパクトなモジュラー方式の組み立てと検証を記述しますダ単一の光検出器6。光ファイバは、個々のレーザに結合され、規則的な空間的オフセットでL字型の流路に垂直に挿入されます。蛍光収集ファイバは、励起領域に平行に配向され、単一のPMTに接続されています。液滴が異なる時間にレーザービームを通過するため、PMTによって記録されたデータは一時的にそのユーザが液滴がそれぞれ別個のレーザ光により励起された後に放出される蛍光とを区別することを可能にするオフセットを示しています。この時間的なシフトは、ダイクロイックミラーとバンドパスフィルタのシリーズを使用して、別個のPMTへの光を分離する必要がなくなります。検出器の有効性を検証するために、我々は、異なる色や濃度の染料をカプセル化液滴集団で蛍光を定量します。システムの感度は、単一色の蛍光検出のために調査、および0.1 nMの、200倍感度インプレッションまでの濃度で液滴を検出する能力を示してい文献7に報告され、最近の光ファイバベースの手法と比較してovement。

<table>
	<tbody>
		<tr>
			<td>Photomasks</td>
			<td>CadArt Servcies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3&#34; silicon wafers, P type, virgin test grade</td>
			<td>University Wafers</td>
			<td>447</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3035</td>
			<td>Microchem</td>
			<td>Y311074</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>Microchem</td>
			<td>Y111072</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>Krayden</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringes</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>27 gaugue needles</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>PE 2 polyethylene tubing</td>
			<td>Scientific Commodities, Inc.</td>
			<td>B31695-PE/2</td>
			<td></td>
		</tr>
		<tr>
			<td>Novec 7500</td>
			<td>Fisher Scientific</td>
			<td>98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr>
			<td>Ionic Krytox Surfactant</td>
			<td></td>
			<td></td>
			<td>Synthesis instructions in ref #10</td>
		</tr>
		<tr>
			<td>Dextran- conjugated cascade blue dye</td>
			<td>Life Technologies</td>
			<td>D-1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein sodium salt</td>
			<td>Sigma</td>
			<td>28803</td>
			<td></td>
		</tr>
		<tr>
			<td>Quad bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-446/510/581/703-25</td>
			<td></td>
		</tr>
		<tr>
			<td>PMT</td>
			<td>Thorlabs</td>
			<td>PMM02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber port</td>
			<td>Thorlabs</td>
			<td>PAFA-X-4-A</td>
			<td></td>
		</tr>
		<tr>
			<td>lens tube</td>
			<td>Thorlabs</td>
			<td>SM1L05</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch cable with 200 um core / 225 um cladding optical fiber with one stripped end and one FC/PC connector</td>
			<td>Thorlabs</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch cable with 105 um core / 125 um cladding optical fiber with one stripped end and one FC/PC connector</td>
			<td>Thorlabs</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>125 um fiber stripping tool</td>
			<td>Thorlabs</td>
			<td>T08S13</td>
			<td></td>
		</tr>
		<tr>
			<td>225 um fiber stripping tool</td>
			<td>Thorlabs</td>
			<td>T10S13</td>
			<td></td>
		</tr>
		<tr>
			<td>laser fiber adapter</td>
			<td>OptoEngine</td>
			<td>FC/PC Adapter</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm CW laser at 50 mW</td>
			<td>OptoEngine</td>
			<td>MDL-III-405</td>
			<td>Distributor for CNI lasers</td>
		</tr>
		<tr>
			<td>473 nm CW laser at 50 mW</td>
			<td>OptoEngine</td>
			<td>MLL-FN-473-50</td>
			<td></td>
		</tr>
	</tbody>
</table>

multicolor fluorescence detection for droplet microfluidics using optical fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

光ファイバの挿入を可能にするPDMSデバイスの製造は、種々の高さ（ 図1）のチャネルを作成するために、多段階のフォトリソグラフィ手順を必要とします。まず、SU-8の80μmの背の高い層は、シリコンウェハ上にスピンコートし、流体ハンドリングジオメトリを作成するために、マスクを用いてパターニングされます。次に、SU-8の追加の40μmの層がウェハ?...

Watch this Scientific Journal Video about Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers at JoVE.com

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

光ファイバを用いて、液滴マイクロフルイディクスのための多色蛍光検出

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

multicolor-fluorescence-detection-for-droplet-microfluidics-using

Research

JoVE Journal

Bioengineering

10.4K ビュー.  University of California, San Francisco. Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

ビデオ: Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

マイクロ流体液滴のプラットフォームのための蛍光検出法

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

生物工学

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

細胞融合の検出のためのCre - LOX Pの組換えのアプローチインビボでの</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

を光学的に検出<em&gt; E.大腸菌の</emメソポーラスケイ素バイオセンサーによる&gt;バクテリア

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

における損傷応答のライブイメージングや研究のためのマイクロ流体チップを使用<em&gt;ショウジョウバエ</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

連続フローMicrospotterを使用して変更表面に細胞のサブマージ印刷

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

細胞内小胞の追跡への応用：変形二光子顕微鏡での3d軌道の追跡

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

商業顕微鏡でのライブ細胞膜上の分子拡散法への高速蛍光イメージングから

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

マイクロトロイドの光共振器を用いたラベルフリー単一分子検出

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

DNA結合色素化学と液滴デジタルPCRを使用したマイクロRNA定量循環

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

マウス心臓の研究のための光シート蛍光顕微鏡

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...