Name: optical control of living cells electrical activity by conjugated polymers
Uploaded: 2016-01-28T14:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Optical Control of Living Cells Electrical Activity by Conjugated Polymers at JoVE.com

The work was supported by EU through project FP7-PEOPLE-212-ITN 316832-OLIMPIA, 
Telethon - Italy (grants GGP12033 and GGP14022), Fondazione Cariplo (grant ID 2013-0738).

光活性基板の作製<ol><li> 20グラム/ LのP3HT濃度でクロロベンゼン中：PCBM溶液（1 / wの1：）P3HTを準備します。 60℃で少なくとも4時間、マグネチックスターラーを用いて溶液を混合します。各基板を用意するために、溶液の150μlの量を考えてみましょう。 </li><li>クリーンITO被覆ガラススライド超音波処理器で脱イオン水、アセトンおよびイソプロパノールの連続したバス付き（R S = 10Ω/□、18×18ミリメートル2、厚さ170ミクロン）（ステップごとに10分）。窒素銃でカバーガラスを乾燥させます。 10分間、100Wの電力でプラズマクリーナーでサンプルを入れてください。 </li><li>スピンコーティングにより洗浄された基板上に活性層の薄膜を堆積させます。 <ol><li>ダイヤモンドチップを用いて、ITO被覆された基材の同一の横方向寸法に顕微鏡用スライドガラス（厚さ≈1 mm）を切断します。ホットプレートからPCBM溶液と、それは周囲温度まで冷却してみましょう：P3HTを削除してください。 </li><li> 2-STを設定EPSスピンコータープログラムは、i）800 rpmで30秒; ⅱ）1,600 rpmで30秒。スピンコーターのチャック上にスライドを配置し、それを修正するために真空を開始します。上を向いITO被覆側で、スライドした後、洗浄した基板上に、アセトンのドロップを入れてください。過剰のアセトンを取り除くためにスピンコーターの回転を開始します。 </li><li> （1.3.2で説明したのと同じプロトコルを使用する）基板上にPCBM溶液と、再びスピンコーターを開始：P3HTの150μLを入れてください。 </li><li>スピンコーターが終了した後、サンプルを除去し、慎重にスライドガラスからコーティングされた基板を取り外します。アセトンに浸した綿棒クリーンルームでの基板の裏面を清掃してください。 注：使用するまで光活性基板は暗箱で数日間保存することができます。より長い期間のために、不活性ガスでグローブボックスに保管してください。 </li></ol></li></ol>細胞培養培地および電気生理学ソリューションの作製<ol><li>完全増殖培地（CGM）のための準備をHEK-293細胞：ダルベッコのイーグル培地（DMEM）、10％ウシ胎児血清（FBS）を補充していない熱不活性化を、100 U / mlペニシリンおよび100μg/ mlストレプトマイシンを改変。 4℃で0.2μmのろ過システムや店舗を有する媒体を滅菌します。 </li><li> 135のNaCl、5.4のKCl、5 HEPES、10グルコース、1.8のCaCl 2、1のMgCl 2：超純水（MM中の濃度）で細胞外溶液を準備します。 NaOHでpHを7.4に調整します。 4℃で0.2μmのろ過システムや店舗で溶液を滅菌します。 </li><li>超純水で細胞内液を調製し（MM中の濃度）：12のKCl、125 K-グルコン酸、1のMgCl 2、0.1のCaCl 2、10 EGTA、10 HEPES、10 ATP-のNa 2。 ATP-のNa 2は、熱に敏感であることに注意してください、最後に追加します。 NaOHでpHを7.4に調整します。 -20℃で0.2μmのろ過システム、1ミリリットルチューブ中のアリコートとストアで溶液を滅菌します。 </li></ol> 3.メッキHEK-293細胞アクティブ基板上<ol><li>ガラスビーカーアルミニウムまたは閉じたペトリ皿で​​覆い、殺菌のため2時間120℃のオーブンに入れて光活性基板を配置します。以降、この瞬間から、無菌状態ですべての操作を実行します。無菌フード内のサンプルを入れて、彼らがクールダウンしましょう​​。 </li><li>コー​​ト接着層と光活性基板は、表面疎水性を減少させると、細胞接着を促進します。 <ol><li> P35ペトリ皿または6マルチウェルプレート内のアクティブ基板を配置します。共役ポリマー層の疎水性を考えると、サンプルを修正して解決策を閉じ込めるためにも、ポリジメチルシロキサン（PDMS）を作ります。 <ol><li>粘性スラリーを得るために、ガラス棒で1割合：10にPDMSと触媒を混合します。 </li><li>各ウェルの半分をカバーするのに十分な量を分配する、6マルチウェルプレート中のスラリーを注ぎます。 </li><li> RTでPDMS重合を待ちます。 </li><li> obtaのため途中で硬化PDMSをカットポリマー試料の同じ次元の正方形ining。 </li><li>少なくとも30分間、70％エタノール - 水混合物中に浸漬することによってPDMSウェルを滅菌します。 </li></ol></li><li>よくPDMSを使用している場合は、ウェルの細胞培養後、PDMSを除去する際に、全層の剥離を防止するために、先の尖ったピンセットでも全体の国境に沿って光活性層を傷つけます。 </li><li>フィブロネクチン溶液（PBS中の2mg / L）を用いて、各サンプルの表面全体を覆​​います。 PDMSはよく（：15x15 mm 2のウェルのクリアな表面）を使用する場合、サンプルあたり500〜750μlの間の容積は、通常は十分です。よくPDMSがなければ、フィブロネクチン溶液約2mlを完全にサンプルを浸すために必要です。溶液を添加しながら、基板が浮いていないことに注意を払います。少なくとも1時間、37℃でサンプルをインキュベートします。 </li><li>ガラスピペットを用いてフィブロネクチン溶液を除去し、2mlのPBSで1回洗浄。 </li></ol></li><li> PL完全増殖培地中/ cm 2に15,000細胞の密度で、フィブロネクチン処理サンプルのHEK-293細胞を食べました。よくPDMSが使用されている場合はそれ以外のCGM 2〜3mlのが必要であり、各サンプルのCGMの750-1,000μLの容量を使用してください。 24〜48時間37℃で細胞を、5％CO 2でインキュベートします。 </li></ol> 4.光刺激プロトコルおよび電気生理学的測定<ol><li>熱化するために37℃の水浴中で細胞外溶液を入れ、 、細胞内液を解凍する34ゲージの針で1mlシリンジにそれを入れて、ATPの分解を回避するために氷と接触してそれを維持します。 </li><li>慎重にPDMSを取り除くだけでなく存在する場合、インキュベーターから細胞で被覆された基板を取ります。ガラスピペットを用いて、増殖培地を除去し、細胞外液でサンプルをすすいでください。細胞の剥離を避けるために、ゆっくりと進みます。 </li><li>外solutioと電気ステーションのサンプルホルダーにデバイスを入れてnおよび基準電極。 （理想的な抵抗値が範囲2-4MΩである）プラーとパッチクランプ用の新鮮なガラスマイクロピペットを準備します。細胞内溶液でピペットの半分を記入し、マイクロマニピュレータにマウントします。 </li><li>デバイス上のパッチにする健康な細胞を探します。光活性基板の望ましくない励起を回避するために、顕微鏡照明装置の前に長波長通過フィルタ（例えば、カット波長λ= 750nmの可視で吸収性ポリマーに使用することができる）を配置し、撮像のためにIR照射を用います。 </li><li>選択したセルにパッチを適用し、顕微鏡（ 図1）の蛍光励起経路を介して試料に光を提供することにより、光刺激のための目的のプロトコルを適用します。パッチクランプのプロトコルの詳細な説明は、参照20で見つけることができます。 <ol><li>マイクロメートルマニピュレータを用いて細胞膜に近接してパッチピペットを置きます。位置決め時、overpressur適用先端に付着した汚れを避けるために、ピペットに電子。ピペットは、セルの上に数ミクロンであるべきです。 </li><li>パッチ管理ソフトウェア上のピペット抵抗を制御して細胞膜に向かってピペットを下げ始めます。ピペット抵抗は1MΩ程度増加しているときは、ピペットと細胞膜との間のギガシールを形成するために、穏やかな吸引を適用しながら、ピペットからの過度の圧力を削除する（ すなわち、測定された抵抗は1GΩ以上の値に増やす必要があります） 。 </li><li>シールの安定化を助けるために（HEK293細胞のために-40 mVでの電位を使用することができます）、予想される細胞静止電位に近いピペットに負の電位を適用します。パッチアンプおよび/またはパッチ・制御ソフトウェア上の相対的なコマンドを使用して、ピペット容量による容量トランジェントを補正してください。 </li><li>短いと、強い吸引パルスを印加することによって、細胞質への電気的アクセスを可能にするために、膜を破りますピペットへ。細胞膜電位を追跡するために、（セル内に注入電流なしで、すなわち、I = 0）電流クランプモードにパッチアンプを設定します。セル電位が安定しているかどうかを確認するために数分を待ちます。 </li><li>細胞に所望の照明プロトコルを適用し、膜電位への影響を記録します。 注：照明は、顕微鏡のアーキテクチャに応じて、底部または上部のいずれかからのサンプルに送達することができます。 <ol><li>井戸活性材料によって吸収された励起波長を選択します。 P3HTのため：PCBMブレンド、450〜600 nmの範囲の波長を使用することができます。適当な光励起密度は10〜100の範囲ミリワット/ mm 2としています。 注：光がオフにされるまで長時間照明で（ミリ秒以上のパルスの数百）持続的な過分極が観察されている間（10〜20ミリ秒以下）の光の短いパルスは、細胞の一過性脱分極が生じます。 </li></ol></li&gt; </ol></li></ol>

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The authors have nothing to disclose.

細胞のインビトロ光刺激のために報告されたプロトコルの重要なステップは、主に、感光性ポリマーの選択、熱殺菌パラメータ、強度と光刺激の持続時間を懸念します。 P3HT：それは良い時間的および電気化学的安定性を保証するため、PCBM薄膜は、ここで選択しました。しかし、人はいないすべての光感受性ポリマーは、照射時に22具体的には、アナログ性能を提供できること...

正確な空間的及び時間的分解能で細胞活性を操作する可能性は、神経科学研究および神経学的および精神医学的障害の治療における重要な戦略を表す。1従来の方法は、近接してまたはと接触して配置された電極を使用して細胞の電気刺激に基づいています（ インビボ脳組織、単一細胞、セルラーネットワーク、脳切片）は、異なる複雑さのものとすることができる標的システム、2。過去世紀の間、パッチクランプ、金属と基板と一体型の電極の使用は、単一ニューロンのニューラルネットワークの機能メカニズムの生理学および病態生理学の詳細な画像を提供してきました。しかし、電気刺激が重要な制限を受けます。最初のものが原因容易に適合させることができない電極とそれらの固定形状の物理的寸法、一般的に貧しい空間分解能に関係しています生物学的組織のような複雑な組織化システムに関する。また、電極インピーダンスおよび刺激および記録システム間のクロストークに関連する問題は、測定値の最終的な信号対雑音比を劣化させることができる。3一方、刺激のための光の使用は、多くの制限を克服するのを助けることができます電気的なアプローチの。まず第一に、それが可能な特定の細胞型、あるいはサブ細胞区画を標的とするようになって、今までにない空間（&lt;1ミクロン）と時間分解能（&lt;1ミリ秒）を提供しています。それは目的の組織との物理的接触を回避し、記録から刺激をdisentanglesので加えて、非常に非侵襲的です。また、光の強度および波長の両方を正確に調整することができるので、多様な刺激のプロトコルを適用することができる。3,4 しかし、動物細胞の大部分は光に特定の感度を提示しません。光学stimulatioのためのいくつかの戦略nはこのように、いずれかの近く、または細胞内の光に敏感な分子メディエーターを利用し、又は細胞に近い外部に配置された光活性デバイスを使用して提案されています。前者は、可視光や赤外線（IR）光だけでなく、光切断/光異性化化合物または感光性分子アクチュエータ（光遺伝学）の遺伝子発現のいずれかを使用することによって、刺激等の内因性のメカニズムを指します。後者のクラスは、無機ナノ/マイクロ粒子または光伝導性のシリコン基板を使用して達成外因性の刺激のための技術を含む。5にもかかわらず、すべてのこれらのシステムは、明るい側面と欠点を有します。特に、可視範囲内の細胞の内因性の吸収が弱く、信頼性がなく、反応性酸素種の同時発生は、細胞に有害であり得ます。一般に、IRは、吸水による局所的な熱的加熱を誘導するために使用されるが、水の吸光係数が小さくなり、従ってSTを要求していますチョロン赤外光（数十からW / mm 2の数百）標準的な顕微鏡光学系を経由して提供することは困難であり、 生体内でのアプリケーションのための安全性の問題を引き起こすことがあります。一方、光スイッチング可能ケージング化合物は、時間制限された作用を有し、しばしば、限られた組織浸透に配信しにくいUV光を必要とします。加えて、それらは、照射された領域外の光分解により活性化化合物の拡散の問題に悩まされています。最後に、optogeneticツールは、科学者が特定の細胞亜集団とサブコンパートメントを標的とすることが許されており、急速に神経科学の研究における重要な技術の一つとして浮上しています。しかし、ウイルスベクターにより外来性DNAセグメントを挿入すると、特にヒト患者への適用の観点から、重要な安全性の問題が発生します。これらの理由から、5,6、セル光学的操作が可能な新しい材料やデバイスの研究は非常にホットな話題です。 最近、小説効率的に細胞の電気的活動の調節に光刺激を伝達することができる感光性共役ポリマーの使用に基づくアプローチが、提案されています。ポリマー光励起（CSPP）技術による細胞の刺激は、有機半導体の典型的な多くのキー可能な機能を活用する：彼らは、可視域の光に対して本質的に敏感である; 7彼らは、生体適合性柔らかく順応性であり、その機械的柔軟性は、組織との親密なインタフェースを可能にしますin vitroおよびin vivoの両方8-10。それとは別に、彼らは簡単に機能をプロービングし、センシング、よりよい生きている細胞とのインタフェースに適応するために、具体的な励起を可能にするために官能化することができる。11,12はまた、彼らは組み合わせに最適です、電子だけでなく、イオン輸送をサポート電子広告の生物学の。13,14興味深いことに、彼らは外部バイアスFを適用する必要がなくなり、太陽光発電モードで動作することができますまたは効率的な細胞光刺激。15 CSPP技術の信頼性は、以前に一次ニューロン、15,16アストロサイト、17の二次電池線 18と外植網膜組織を含む、いくつかのシステムで実証されている。本研究では16、必要なすべてのステップは、光に敏感なバイオポリマーを製造するためにインビトロ系の光刺激のためのインターフェース19は、詳細に記載されています。作用する試験事例、地域規則的ポリ（3-ヘキシルチオフェン）（RR-P3HT）、電子供与体として機能し、かつフェニルC61 - 酪酸メチルエステル（PCBM）の典型的な有機光起電性ブレンド、など電子受容体が使用されます。生物系として、ヒト胚性腎臓（HEK-293）細胞が使用されます。電気生理学的測定を介した細胞活性の相対的な記録と光刺激プロトコルの例が提供されます。 説明プラットフォーム適切に要求された細胞培養プロトコル、めっき手順及び時間を変えることによって（ただし、一般有効であり、それは容易に（適切液調製工程と、堆積パラメータを調整することによって）、他の共役ポリマーの使用に拡張することができる、異なる細胞型細胞播種および増殖）と異なる刺激プロトコル（光波長、刺激頻度および期間、光励起密度）のため。

<table><tbody><tr><td>rr-P3HT</td><td>Sigma Aldrich</td><td>698989-5G</td><td></td></tr><tr><td>ITO-coated substrates</td><td>Nano-CS</td><td>IT10300100</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F1141</td><td></td></tr><tr><td>chlorobenzene</td><td>Sigma Aldrich</td><td>319996</td><td></td></tr><tr><td>PCBM</td><td>Nano-C</td><td>Nano-CPCBM-BF</td><td></td></tr><tr><td>acetone&amp;nbsp;</td><td>Sigma Aldrich</td><td>270725</td><td></td></tr><tr><td>isopropyl alcohol</td><td>Sigma Aldrich</td><td>563935</td><td></td></tr><tr><td>HEK cells</td><td>LGC standards srl</td><td>ATCC-CRL-1573</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H0887</td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P5244</td><td></td></tr><tr><td>E-MEM</td><td>LGC standards srl</td><td>ATCC-30-2003</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E8008-</td><td></td></tr><tr><td>FBS</td><td>LGC standards srl</td><td>ATCC-30-2020</td><td></td></tr></tbody></table>

optical control of living cells electrical activity by conjugated polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

細胞はP3HTで簡単に培養することができる：PCBM基質、（記載のプロトコルのステップ3.2で使用されるフ​​ィブロネクチンのような）適切な接着層が堆積されることを条件とします。 P3HT：可視スペクトルの緑の部分でPCBMの光吸収ピーク;しかしながら、他の感光性共役ポリマーは、好ましい光刺激波長範囲（ 図2）によれば、選択することができます。こ...

Watch this Scientific Journal Video about Optical Control of Living Cells Electrical Activity by Conjugated Polymers at JoVE.com

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

生細胞共役ポリマーにより電気的活動の光制御

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

optical-control-living-cells-electrical-activity-conjugated

Research

JoVE Journal

Bioengineering

7.5K ビュー.  Istituto Italiano di Tecnologia. A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

ビデオ: Optical Control of Living Cells Electrical Activity by Conjugated Polymers

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

制御、細胞間相互作用における遺伝子発現パターンを分析して光遺伝学的方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

遺伝学

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

微生物光遺伝学アプリケーションのための自動照明、培養、およびサンプリングシステムの設計と実装

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

生物工学

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

神経細胞の金ナノロッド補助光刺激

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

神経科学

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

ニューロンに遺伝的にコードされた非天然アミノ酸を用いた神経タンパク質の光制御

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

哺乳類細胞における安定した光遺伝学的遺伝子回路を確実にエンジニアリングおよび制御

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

赤外線レーザー支援マイクロパターンによる細胞形状の制御

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

非遺伝的光刺激によるCa2+ 伝播を研究するための心臓微小生理学的システム

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

同時光遺伝学的変調と電気的神経記録のためのオプトロードアレイ

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

A Light-Stimulation Grid Approach to Study Cellular Optogenetic Protein

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3&#39;,5&#39;-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 &#181;m2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.