著者らは、土地、バーデン·ヴュルテンベルク州とアオイ連合ユーロぺフォンエリーゼのためダイエオリエEntwicklungに感謝-資金ZAFH光子N、無資金研究助成金のためにエリーゼのためBundesministerium Bildung undのForschung（BMBF）のために。 1792C08ならびにプロジェクト &quot;Auramiを&quot;資金調達のためのバーデン·ヴュルテンベルク·財団社。

1。細胞の播種およびインキュベーション<ol><li>シード個々の細胞、 例えば U373-MGまたはU-251-MGの神経膠芽腫細胞をスライドガラス上に100細胞/ mm 2の典型的な密度にする（ 例えば RPMI-1640、10％ウシ胎児血清を添加した培地中で4872時間のためにそれらを育てると抗生物質）で37℃、5％CO 2のインキュベーターを用いた。 </li><li> 8μM（培地中の）4、または細胞質マーカーの濃度で60分間適用例 6-ドデカノイル-2 -ジメチルナフタレン（laurdan）、膜マーカーで細胞をインキュベートし、 例えばカルセインacetomethlyesterは、少なくとも40分間印加4,5;5μMの3、または光増感膜関連の濃度は、 例えば、プロトポルフィリンIXは、その前駆体5 -アミノレブリン酸（1mMの5-ALA）を用いて4時間のインキュベーションの際に誘導した。 </li><li>蛍光顕微鏡の前に緩衝生理食塩水（PBS）培地またはリン酸で細胞を洗浄。&lt;/ LI&gt; </ol> 2。 VA-TIRFM <ol><li>正立顕微鏡、 例えばツァイスAxioplanを使用しており、文献に記載されているように、照明装置によって、そのコンデンサーを交換してください。半球状または光学的にオブジェクトをスライドします （ 図1を参照）に結合された半円筒形のガラスプリズムで3。 </li><li>その試料の表面（テレセントリック瞳孔線）上の平行ビームでガラスプリズム、さらに光学部品（ 例えば凹面鏡）による撮影後、再び結果並列コリメートされたレーザービームを使用しています。電場ベクトルが入射面に垂直偏光であるように注意してください。 </li><li>試料面に結像されると発生率（オブジェクトの線）の可変角度で試料に照射されるコンデンサーの内部に位置調整可能なミラーを使用しています。 goniometric測定3によって決定された調整可能なミラーは、それぞれの位置は入射よく定義された角度に対応するステップモータによって駆動することができる</&gt;（商標）。 </li><li> ΘC = 61.3°ガラス-水移行のための固定値として臨界角を使用して調整可能なミラーの角度位置のキャリブレーションを行います。 Θcは蛍光色素（ 例えば 1mMのフラビンモノヌクレオチド、FMN）を水に溶解させ、Θの変化に応じて可視化することができる。でミラーの位置Θ=ΘCミラー位置決めプログラムに格納されています。 </li><li>温度、ゲイン、録音時間と変速速度を含むカメラのパラメータを指定します（バック点灯アンドールIXON EMCCD、DV887DC、アンドールテクノロジー、ベルファースト、イギリス6）、設定します。 </li><li>適切な倍率と優先的に適度な開口数（ 例えば、40×/ 63×0.75 / 0.90水浸）の対物レンズを使用し、顕微鏡内のオブジェクトのスライドをローカライズします。ガラスプリズムと試料の光学的接触を確実にするためにイマージョンオイルを使用しています。の変動により、同一試料の蛍光画像を記録照明の角度（Θ≥ΘΘCとCセル-ガラス界面のための64.5°に対応）。 0.5°または1°刻みで66°-74°の角度範囲が推奨されます。蛍光検出のための適切なロングパスまたはバンドパス·フィルタを使用します。 </li></ol> 3。データ解析<ol><li>様々な角度Θと同様、カメラの設定（背景差別のために）、励起波長とガラス（N 1 = 1.52）と細胞（N 2 = 1.37）に対する屈折率で記録された画像（ 例えば 、ASCIIフォーマットで）読み込む評価プログラム（; NanoCell LabViewの）に挿入します。各パラメータセット浸透深さd（Θ）と透過率のためにTは（Θ）は自動的に計算されます。 </li><li>細胞 - 基質距離Δは式（5）（膜マーカー）または式（4）（細胞質マーカー）によると、不均質を有する個々の画像の評価に使用される画像を選択照明（顕E、G。ストライプ）が除外されることがあります。 </li><li>細胞 - 基質トポロジーの画像を計算して表示するのに適したカラーコードを選択します。 図2に示すように、個々の画素の評価プロファイルの間に、表示させることができます。これらのプロファイルは、フィットの質の指標として用いることができる。 </li><li>評価プロトコルを格納します。 </li></ol> 4。代表的な結果代表的な実験は、親切教授ヤンモレンハウアー、分子腫瘍学専攻、南デンマーク大学、オーデンセによって供給される遺伝子組み換えU251-MGの神経膠芽腫細胞を用いて行われる。それらのU251細胞のサブクローン2腫瘍抑制遺伝子でTP53またはPTENはテトオン誘導性発現系、これらの細胞が減少し腫瘍形成の可能性を発揮し、より少ない悪性とみなすことができること等を用いて過剰発現されています。抑制遺伝子のない高悪性U251-MG細胞を対照として用い、およびU251-アールMGは野生型細胞は、さらに比較のために役立つ。 （;：391 nmで、パルスエネルギー：12 PJ、繰り返し率：波長ドライバーPDL 800-B Picoquant、ベルリン、ドイツでLDH400 40 MHz）をピコ秒光パルスの準連続シリーズを発するレーザダイオードを励起光源として使用されます。 図において、活性化されたTP53腫瘍抑制遺伝子を持つとlaurdan（8μM、60分）と一緒にインキュベートU251-MGの神経膠芽腫細胞の3 TIRFM画像は、66.0°の入射角69.0°、73.0°の侵入深さに対応するために描かれているそれぞれ約140nm、75 nmおよび60 nmのエバネセント電磁界。侵入深さの蛍光の減少に伴って強度が低下（スケールを参照）、細胞のより表面的な部位（その辺で例えばフォーカルコンタクト）に由来する。細胞-基質間距離（0から600 nmの範囲のカラーコードによって図3dで可視化）に強く間のセルにわたって変化LY数ナノメートル（白黄色）と250から300ナノメートル（暗赤色）、 すなわち焦点連絡先やより大きなセル基板の距離はすぐ近くにあります。細胞-基質距離はU251-MG制御と80から100ナノメートル（黄色の典型的な値を持つ野生型細胞のためではなく、一定である同じことが、活性化抑制遺伝子PTEN（ 図4d）とU251-MGの神経膠芽腫細胞のために保持します。 図4b ）と150から200ナノメートル（橙赤色、 図4a）。 <img alt="図1" src="/files/ftp_upload/4133/4133fig1.jpg" /> 図1：正立顕微鏡のVA-TIRFM用の照明装置。ステップモータにより駆動される調整可能なミラーが±0.25°の角度分解能を可能にします。 <img alt="図2" src="/files/ftp_upload/4133/4133fig2.jpg" /> 1つのPIXについては、 図2。評価プロファイルEL（図）。 LN [I、F / Tは（Θ）] 1 / D（Θ）の関数としてプロットすると、傾きは、細胞-基質距離Δを表しています。このスロープは、一般的に±10％程度の精度で決定されます。つの値が - 不均一な照明に起因する - のマークが付けられ、評価から除外されました。 <img alt="図3" src="/files/ftp_upload/4133/4133fig3.jpg" /> 照明の様々な角度（AC）で記録され、示されて0から600 nmの範囲で細胞-基質間の距離。laurdan（60分8μM）とのインキュベーションにより活性TP53抑制遺伝子とU251-MGの神経膠芽腫細胞の図3 TIRFM画像カラーコードにより、（d）は、励起波長：391 nmで検出：≥420 nmで、画像サイズ：210×210μmのμmの<a href="http://www.jove.com/files/ftp_upload/4133/4133fig3large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 図4に、カラーコードで示さ0から600 nmの範囲でU251-MGのglioblatoma細胞の細胞-基質間距離;（a）は野生型（b）管理;活性化抑制遺伝子TP53と（c）の細胞と、（d）活性化抑制遺伝子PTENと細胞;励起波長：391 nmで検出：≥420 nmで、画像サイズ：210×210μmのμmの<a href="http://www.jove.com/files/ftp_upload/4133/4133fig4large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

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メソッドは、ナノメートルの精度で細胞 - 基質間の距離を測定するために記述されている。現在、 例えば構造化照明7または単一分子検出8-10と同様に誘導放出の枯渇に基づく超解像顕微鏡の方法、（STED）顕微鏡11は、かなりの関心が持たれている。これらの技術はいずれも、しかし、50 nm以下の軸方向の分解能を可能にしていません。また、50100 W / cm 2?...

屈折率n 1の媒質中を伝搬する光ビームが屈折率n 2の第2の媒質へのインターフェースを満たしたときに、&lt;nの1、全反射は、臨界角より大きい発生率Θのすべての角度で発生ΘC =アークサイン（N 2 / N 1）。全く反映されているにもかかわらず、入射光ビームは、I（Z）= I 0 EZ / D（Θ）に従ってインターフェイスから垂直距離zで指数関数的に減衰し、第二の媒体に浸透エバネセント電磁界を彷彿とさせます。で与えられるように私は（z）は、波長λにおける浸透深さに電磁場の強度に対応するとd（Θ） D（Θ）=（λ/4π）（N 1 2罪Θ2 - N 2）-1 / 2 文献1,2に報告されているように、私は0 </sUB&gt;は入射光の強度に対応して、私はeが透過率T（Θ）と比n 2 / nは1で乗算。この光の電場ベクトルが入射面（入射と反射ビームによって張ら平面をIE）に対して垂直に偏光である場合は、この透過率は次式で与えられます。 T（Θ）= 4 cosの2Θ/ [1 - （N 2 / NL 1）2] TIRFM測定で検出された蛍光強度は、光吸収は、サンプルのすべての層の上に統合されており、検出Ωの立体角と同様に、関連する色素の蛍光量子収率ηと乗算する必要があります。以前に3報告したように、この強度は次のように記述することができます I F（Θ）=（Θ）OC（z）での電子のz / D（Θ）DZ もし独立したfはすべての要因<html>蛍光色素の濃度c（z）はほぼ一定であるとみなされた場合ROM入射角Θと座標zは実験定数Aの式（3）内に含まれているが、解析的に解くことができます -いずれかのすべての座標のz≥Δ、界面からの距離Δを有する細胞の細胞質内に例えばで。この場合、積分はz =Δがために、Z =∞の結果から計算しなければならない 私はF = A C T（Θ）はD（Θ）のe-Δ/ D（Θ） -インタフェースから距離Δに位置するT / 2とz =Δ+ T / 2、厚さtの薄い層内すなわち 、 例えば細胞膜-またはz =Δの間。この場合、蛍光強度は次のように計算できます 私はF = A C T（Θ）TE-Δ/ D（Θ）は 、 トンはΔに比べて小さい場合。 ENT &quot;&gt;蛍光強度の画像は私がFを Θ、およびln IF可変角度（I、F / T d）は（式4）のために記録されている場合、式（4）及び（5）細胞-基質距離からΔは、計算することができます。またはLN（I、F / T）（式5）は、これらの角度の1 / dの関数として評価されます。本稿で用いる膜マーカーlaurdan（S.以下）については評価は式5に準じて行った。 画像収集と評価が必要では、以前に報告したように3 - サンプルの上に励起光の均一な分布、 - 2層モデルの妥当性（基質と細胞質の屈折率n 1とn 2と、それぞれ）。原形質膜が非常に薄い場合には、保持しており、細胞外培地（セルと基板との間）の層は、入射光の波長に比べて小さい場合。 加えて、適度な開口数（通常≤0.90）顕微鏡対物レンズのは、誘電体の界面近傍界での放射の異方性に起因する輝度のばらつきを避けるために有利である。

<table border="1"><tbody><tr><td> 名前 </td><td> 会社 </td><td> タイプ </td><td> 注釈 </td></tr><tr><td>インキュベーター</td><td>ヌンク</td><td>ヌンクCellstar QM1300SVBA </td><td></td></tr><tr><td>層流</td><td>セーフHolten </td><td> S2010 1.2 EN GG 1LN </td><td></td></tr><tr><td> DMEM +10％FCS + 1％ペニシリン/ストレプトマイシン</td><td> BIOCHROM </td><td></td><td>培養媒体</td></tr><tr><td>ガラスのオブジェクトのスライド</td><td> Marienfeld </td><td>純粋な白ガラス</td><td>使用される特殊なクリーニング手順</td></tr><tr><td> 6-ドデカノイル-2 - ジメチルナフタレン（laurdan） </td><td>分子プローブ</td><td></td><td>蛍光マーカー（ストック溶液：エタノールさ2mm） </td></tr><tr><td>顕微鏡</td><td>カールツァイス</td><td> Axioplan 1 </tD&gt; <td></td></tr><tr><td>半導体レーザー</td><td> PicoQuant </td><td>ドライバのPDL 800-BとLDH 400 </td><td>波長：391 nmの</td></tr><tr><td>シングルモード光ファイバシステム</td><td>点光源</td><td> kineFlex </td><td>平行光学系と一緒に使用</td></tr><tr><td> EMCCDカメラ</td><td>アンドール</td><td> DV887DC </td><td>戻る照らさカメラ</td></tr></tbody></table>

nanotopology of cell adhesion upon variable-angle total internal reflection fluorescence microscopy (va-tirfm)

表面トポロジー、基板上に成長する細胞の例は 、可変アングル全内部反射蛍光顕微鏡（VA-TIRFM）によってナノメートルの精度で決定されます。細胞は透明なスライド上で培養し、均一にそれらの形質膜に分布する蛍光マーカーと共にインキュベートする。照明はエバネセント電磁界の様々な侵入深さを持つ内部全反射（TIR）の可変角度の下で平行レーザビームによって発生します。約10異なる角度で照射により蛍光画像の記録は、数ナノメートルの精度で細胞 - 基質の距離を計算することを可能にする。種々の細胞株、 例えばがん細胞と少ない悪性細胞との間の密着性の違いは、このように決定されています。また、化学的又は光線力学的治療の際の細胞接着の変化の可能性を調べることができます。超解像顕微鏡光曝露の他の方法と比較して保持されます生きている細胞の非常に小さい、無被害が発生することが予想されています。

Watch this Scientific Journal Video about Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM at JoVE.com

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

基板上への細胞接着のトポロジーは、可変角度全内部反射蛍光顕微鏡（VA-TIRFM）によってナノメートルの精度で測定されます。

可変角度全内部反射蛍光顕微鏡（VA-TIRFM）の際に、細胞接着のナノトポロジー

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

nanotopology-cell-adhesion-upon-variable-angle-total-internal

Research

JoVE Journal

Bioengineering

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

10.1K ビュー.  Institut für Angewandte Forschung. 基板上への細胞接着のトポロジーは、可変角度全内部反射蛍光顕微鏡（VA-TIRFM）によってナノメートルの精度で測定されます。

ビデオ: Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

全内部反射蛍光顕微鏡を用いた微生物のCortex組織とダイナミクスの可視化、

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

生物工学

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

蛍光顕微鏡を用いたイメージング細胞遊走用接着剤及び可溶性グラデーションを作成する

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

推定細胞接着分子のキャラクタリゼーションのためのビーズ凝集アッセイ

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

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 ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

マトリックス修飾により誘導される細胞接着及び脱接着におけるリアルタイムの変化を測定するために、細胞 - 基質インピーダンスと生細胞イメージングを使用して、

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

平面支持された二分子層​​上の単一分子蛍光顕微鏡

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

改善された細胞接着のためのポリテトラフルオロエチレンにウェットケミストリーおよびペプチドの固定化

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

哺乳類の細胞分裂量的共焦点反射顕微鏡を介して 3 D 行列

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

回転ディスク全内部反射蛍光顕微鏡による細胞の接着の形成の可視化詳細

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...