The financial support by DFG (German research foundation, project CZ 55/30-1) for parts of this work is gratefully acknowledged.

1.実験のセットアップを組み立て<ol><li> 近位側の準備 <ol><li>配置し、平行光ビームを提供するレーザーを修正 - またはファイバの出射端面での視準光学系とファイバ結合レーザーを使用しています。 </li><li>参照物体ビームにレーザビームを分割する偏光ビームスプリッタ（PBS）を置きます。 （先端側）の参照光と物体光のパワーがほぼ同じになるまでマウントその回転にHWPを回転させることにより、半波長板（HWP）の方向に回します。リファレンスと物体光の両方にスクリーンを置くことによって、これを確認してください。参照ビームの偏光は、偏光に敏感な空間光変調器（SLM）に収まるようにPBSの向きを選択します。 </li><li> 2つのビームに基準ビームを分割する基準ビームにビームスプリッタ（BS）を置きます。 BS1からのこれら二つのビームはそれぞれ、OM1とOM2を渡すことができるような光変調器（OM）を配置します。&lt;/李&gt; <li>二つのミラーを採用したBS2でOM1とOM2を通過する二つのビームを結合します。両方のビームが空間的に分離されるように、ビームスプリッタとミラーを調整します。 </li><li>慎重に両ビームの入射方向が最初でBS3とBS4を無視して、SLMの画素面に垂直であることを確認するために、BS5を揃えます。最初は、何もSLM上に表示されていない、 すなわち 、それは（ステップ2を通じて）、キャリブレーションが終了するまで、ミラーのように作用します。 </li><li>位置と二つのレンズ（L）との間の距離相補型金属酸化膜半導体（CMOS）カメラでSLM面の鮮明な画像を得るためにケプラー望遠鏡を構成し調整します。 L1とL2の正しい方向を見て、収差を最小化するために（平坦な側面が互いに対向しています）。 </li></ol></li><li> 遠位側の準備 <ol><li> 2つのビームに物体光を分割し、2ミラーを採用しBS8でそれらを組み合わせることBS7を使用してください。ここでも、ビームスプリッタとマイルを調整両方のビームが空間的に分離されるようにrrors。 </li><li>顕微鏡対物レンズ（OBJ）にそれらを目指してBS9を使用して両方のビームを偏向。マルチモードファイバ（MMF）の先端にOBJ2焦点を当てます。 L3および電荷結合素子（CCD）カメラを採用したMMFからの後方反射を観察することによりピントを確認してください。 </li></ol></li><li> 接続する近位および遠位側 <ol><li> MMF採用OBJ1を出た物体光からの光を平行。 </li><li>最初は直線偏光子（LP）を無視し、BS6を使用してオブジェクトビームを分割します。 BS3とBS4の両方で参照光ミラーを採用して、両方の物体光を兼ね備えています。ビームスプリッタとミラーを調整するように小さな角度（1°未満）と交差するSLMでの基準と物体光の重なりの各ペア、。 </li><li>リファレンスのパワーと物体光は、ステップ1.1.2によると、HWPの向きを回転させることによりほぼ等しいことを確認してください。 </li><li>干渉pをチェックCMOSカメラでattern（オフアクシスホログラム）、それに応じて交差角度を調整します。干渉縞の間隔はおおよそのCMOSカメラの2つの画素の大きさに等しくなるまで、角度を大きくします。 </li><li>カメラ画像は異なる縞を示すように、CMOSカメラ画像における干渉パターンの最大のコントラストを得るためにオブジェクトと参照光の偏光に一致するようにLPの向きを調整します。 </li></ol></li></ol> 2.システムのキャリブレーション<ol><li> SLMとCMOSとの画素の関係をキャリブレーション <ol><li>参照光の一方のみを用いて全SLMを照射し、他の参照をブロックし、ビームをオブジェクト。 </li><li> CMOSカメラとSLMの画像をキャプチャします。 </li><li> 例えば PCでグラフィックソフトウェアとマウスカーソルを使用して、CMOSのカメラ画像におけるSLMの左上隅の座標を取得します。これらのピクセル座標は原点として使用しますSLMに関する。 </li><li>すべてのビームブロックを削除します。 </li></ol></li><li> 信号経路のキャリブレーション <ol><li>参照光2と物体ビーム2の両方をブロックします。 </li><li> CMOSカメラとホログラムの画像をキャプチャします。角スペクトル法19を使用して記録されたホログラムの位相を評価します。ビーム1の対応する領域での位相反転を計算します。 </li><li>ビームフォーマのブロックを削除し、現在参照ビーム1と物体光1の両方をブロックします。 </li><li> CMOSカメラとホログラムの画像をキャプチャします。再び角スペクトル法を用いて記録されたホログラムの位相を測定します。ビーム2の対応する領域で反転位相を計算します。 </li><li>すべてのビームブロックを削除します。 </li></ol></li></ol> 3.信号を送信します<ol><li>物体光をブロックします。 </li><li>一緒ビーム1,2の対応する領域で計算反転位相画像をステッチして表示SLM上の画像全体は、典型的には、コンピュータグラフィックスポートを使用します。 </li><li> OM1とOM2を活性化することによって、入力信号1及び2の変調を開始します。 </li><li> CCDカメラの出力信号1と2を観察します。 </li></ol>

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The authors have nothing to disclose.

実験（プロトコルにおけるステップ1）の組立は、互いに対する光学部品の徹底的な位置合わせを必要とします。最も重要な側面は、高PBRを確保するためにSLM上に参照光の矩形発生です。 二つ以上の送信信号の設定を高めるために、追加のビームスプリッタを使用することができます。別の方法として、光ファイバベースの実装は、よりコンパクトで、システムはバイオ?...

マルチモードファイバ（MMF）を介して複数の光信号の伝送は、通信技術1及びバイオフォトニクス2に明らかです。通信技術において、空間分割多重（SDM）は、複数のシングルモードファイバと比較して、限られた空間の高い利用の恩恵を受け、将来のデータ転送アプリケーション用の光ファイバの伝送容量を高めるために実行可能な解決策であると考えられています3。バイオフォトニクスでは、生物学的サンプルは、MMF内視鏡4を透過する光によって操作されています。例えば、MMFの内視鏡を使用して、個々のニューロンの独立した光制御は、脳5におけるニューロンネットワークを研究するために、光遺伝学のために重要です。しかし、MMF入力ファセットに投影される光は、outpuへの伝播中に歪みによるモードの混合分散の対象となりますMMFのトンファセット。その結果、光の伝搬は、信号伝送が困難な作るれ、変更されます。 波面整形方法6は 、 図7に示すように 、空間光変調器（SLM）を使用して、散乱媒体に適用し、光による伝播8時の散乱による歪みの補償を可能にしています。光フィードバック9を使用して出力を最適化する反復アプローチがあります。これらのアプローチは、変調器要素の多くに対応するために多数の反復の必要性と高い自由度のかかるかなりの時間です。別のアプローチは、完全にその送信行列10によって記述MMF内の歪みを決定することです。送信されるモードの数が多い場合、これは、同様に時間がかかるであろう。対照的に、デジタル光位相共役（DOPC）があると考えられています迅速かつ有利ここで、ほんの数焦点がMMFの出力面で生成されなければならないからです。位相共役アプローチは、生体組織12、13、14を介して集束またはイメージングのために実証されています。 これまで、DOPCは、単一の時間信号のみ15、16のために使用し、そしてMMF 17を通る光の透過のために適用しました。複数の独立した信号のDOPCのアプローチがなされていません。我々は、単相のみのSLM 18を用いて 、各信号のために整形する個々の波面を使用して複数の光信号の独立した伝送を提供する強化されたDOPC方法を開発しました。この目的のために、SLMは各信号に対して1つを送信する、領域に分割されます。提案された実験は、 図1に示されていますキャリブレーションは、実際の送信前）に行われる場合、）（b）に発生します。 <img alt="図1" src="/files/ftp_upload/55407/55407fig1.jpg" /> 図1：実験セットアップ。 BS =ビームスプリッタ、CCD =電荷結合素子、OM =光変調器、CMOS =相補型金属酸化膜半導体、HWP =半波長板、L =レンズ、LP =直線偏光子、MMF =マルチモードファイバ、OBJ =顕微鏡対物レンズ、 PBSは=偏光ビームスプリッタ、SLM =空間光変調器（位相のみ） - （a）のキャリブレーションおよび（b）の伝送にのみ関連ビームが描かれている<a href="http://ecsource.jove.com/files/ftp_upload/55407/55407fig1large.jpg" target="_blank">。この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>spatial light modulator</td>
			<td>Holoeye</td>
			<td>PLUTO&#8208;VIS&#8208;016</td>
			<td></td>
		</tr>
		<tr>
			<td>CMOS camera</td>
			<td>Mikrotron</td>
			<td>MC4082</td>
			<td></td>
		</tr>
		<tr>
			<td>diode&#8208;pumped solid state laser</td>
			<td>Laser Quantum</td>
			<td>torus 532</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>IDS</td>
			<td>U3&#8208;3482LE&#8208;M</td>
			<td>CMOS camera; suitable as well</td>
		</tr>
		<tr>
			<td>lens 1</td>
			<td>Qioptiq</td>
			<td>G063204000</td>
			<td></td>
		</tr>
		<tr>
			<td>lens 2</td>
			<td>Qioptiq</td>
			<td>G063203000</td>
			<td></td>
		</tr>
		<tr>
			<td>lens 3</td>
			<td>Thorlabs</td>
			<td>AC508&#8208;180&#8208;A&#8208;ML</td>
			<td></td>
		</tr>
		<tr>
			<td>multimode fiber</td>
			<td>Thorlabs</td>
			<td>M14L02</td>
			<td></td>
		</tr>
		<tr>
			<td>beam splitters</td>
			<td>Thorlabs</td>
			<td>BS013</td>
			<td>9x</td>
		</tr>
		<tr>
			<td>polarizing beam splitters</td>
			<td>Thorlabs</td>
			<td>PBS251</td>
			<td></td>
		</tr>
		<tr>
			<td>mirrors</td>
			<td>Thorlabs</td>
			<td>PF10&#8208;03&#8208;P01</td>
			<td>5x</td>
		</tr>
		<tr>
			<td>microscope objectives</td>
			<td>Thorlabs</td>
			<td>RMS20X</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>half wave plates</td>
			<td>Thorlabs</td>
			<td>WPH10M&#8208;532</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>linear polarizer</td>
			<td>Thorlabs</td>
			<td>LPVISB050&#8208;MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>optical modulators</td>
			<td>Thorlabs</td>
			<td>MC2000B&#8208;EC</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>linear and rotation stage for CMOS camera</td>
			<td>Thorlabs</td>
			<td>XYR1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>fiber connector</td>
			<td>Thorlabs</td>
			<td>S120&#8208;SMA</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>reducing ring for microscope objectives</td>
			<td>Qioptiq</td>
			<td>G061621000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>xy adjustment for objective adapters</td>
			<td>Qioptiq</td>
			<td>G061025000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>z translation mount for fiber adapter</td>
			<td>Thorlabs</td>
			<td>SM1Z</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>rods for fiber alignment to objectives</td>
			<td>Qioptiq</td>
			<td>G061210000</td>
			<td>8x</td>
		</tr>
		<tr>
			<td>mounts for lenses 1 and 2 plus two phantom mounts</td>
			<td>Qioptiq</td>
			<td>G061047000</td>
			<td>4x</td>
		</tr>
		<tr>
			<td>rail carriers for objective and lens mounts</td>
			<td>Qioptiq</td>
			<td>G061372000</td>
			<td>6x</td>
		</tr>
		<tr>
			<td>rail for rail carriers</td>
			<td>Qioptiq</td>
			<td>G061359000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td colspan="2">adapter for CCD camera to 1 post</td>
			<td></td>
			<td>in-house</td>
		</tr>
		<tr>
			<td>adapter for laser to 4 posts</td>
			<td></td>
			<td></td>
			<td>in-house</td>
		</tr>
		<tr>
			<td>mount for lens 3</td>
			<td>Thorlabs</td>
			<td>LMR2/M</td>
			<td></td>
		</tr>
		<tr>
			<td>mounts for half wave plates</td>
			<td>Thorlabs</td>
			<td>RSP1D/M</td>
			<td>2</td>
		</tr>
		<tr>
			<td>mounts for mirrors</td>
			<td>Thorlabs</td>
			<td>KM100</td>
			<td>5x</td>
		</tr>
		<tr>
			<td>mount for linear polarizer</td>
			<td>Thorlabs</td>
			<td>RSP05/M</td>
			<td></td>
		</tr>
		<tr>
			<td>mounts for beam splitters and SLM</td>
			<td>Thorlabs</td>
			<td>KM100PM/M</td>
			<td>11x</td>
		</tr>
		<tr>
			<td>clamping arms for beam splitters and SLM</td>
			<td>Thorlabs</td>
			<td>PM4/M</td>
			<td>11x</td>
		</tr>
		<tr>
			<td>posts for mounts, rail carriers and adapters</td>
			<td>Thorlabs</td>
			<td>TR75/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>holders for posts</td>
			<td>Thorlabs</td>
			<td>PH50/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>pedestals for holders</td>
			<td>Thorlabs</td>
			<td>BE1/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>clamping forks for pedestals</td>
			<td>Thorlabs</td>
			<td>CF125</td>
			<td>29x</td>
		</tr>
	</tbody>
</table>

transmission of multiple signals through an optical fiber using wavefront shaping

マルチモード光ファイバを介して複数の独立した光信号の伝送は、ファイバ内伝搬中の光の歪みを補償するために、波面整形を用いて達成されます。私たちの方法論は、光の波面を個別変調器の異なる領域で変調された単一の空間光変調器、光信号ごとに一つの領域を使用するデジタル光位相共役に基づいています。デジタル光位相共役アプローチは、繊維の波伝播挙動の完全な決意が行われる（例えば）他の波面成形アプローチよりも速くなると考えられます。それが唯一の光信号ごとに1キャリブレーションを必要とするのでこれとは対照的に、提示されたアプローチは、時間効率的です。提案手法は、通信工学における空間分割多重化のための潜在的に適切です。さらに応用分野は、特にOで、バイオフォトニクスにおける内視鏡の光配信されています生体組織内の単一の細胞が選択的に高い空間分解能と時間分解能で照明されなければならないptogenetics、。

<html>長さ2mファイバの先端側の典型的な出力信号は、 図2に示されています。所望の焦点スポット（ピーク）は原則としてDOPCの不完全性に起因する望ましくないスペックルパターン（背景）、を伴っていることに注意してください。対応するピーク対バックグラウンド比（PBR）が53に達する（単に信号1が &#39;on&#39;である）、36はそれぞれ、ここで（両方の...

Watch this Scientific Journal Video about Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping at JoVE.com

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

ウェーブフロントシェイピングを用いた光ファイバを介して複数の信号の伝送

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

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9.7K ビュー.  TU Dresden. We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

ビデオ: Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三次元光学分解能光音響顕微鏡

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

生物工学

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

シランカップリング剤を用いた石英系光バイオセンサーへの生物学的プローブの取り付け

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

低強度の超音波を用いた接着依存性のシグナルの誘導

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

ネイティブ、繊維状の長い間隔と分節ロング間隔コラーゲンのin vitro合成

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

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 ...

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.

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 ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

調整可能なナノ細孔を介してナノ粒子の転ベロシティを経由してゼータ電位の決意：例として、DNA修飾された粒子を用いました

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

ひと血清における心筋バイオ マーカーを定量化する臨時の光伝送を使用してください。

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

CRISPRコンカテマーを用いたマウス小腸オルガノイドにおける複数の遺伝子ノックアウトのためのプロトコル

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

対話型、患者固有のモデルを用いた脳深部刺激療法の神経繊維の管を対象と

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...