The authors thank Drs. Steven Chu and Joe W. Gray for helpful discussions, Henry Marr for his initial work on the BiFC-PALM project, and Alexis Shoemaker for her technical assistance. This work is supported by startup funds to X.N. from OHSU. Research in the Nan laboratory was also supported by NIH 5U54CA143836-05, the Damon Runyon Cancer Research Foundation, the M. J. Murdock Charitable Trust, and the FEI company.

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The authors have nothing to disclose.

PALMはそのまま生物学的試料のナノスケールイメージングを可能にし、最近の単一分子の超解像顕微鏡技術である間BIFCは、細胞内でのPPIを検出し、可視化するために一般的に使用される技術となっています。手のひらでBIFCの組み合わせは、ナノメートルの空間分解能と単一分子感度で、細胞内のPPIの選択的イメージングを達成しました。 BIFC-PALMは、両方の技術の有用性を拡張し、この作品?...

タンパク質-タンパク質相互作用薬（PPI）は、生物学1の基本である、しっかりと多くの時間と長さスケール全体の時空間的機構を介して調節されています。細胞膜上の細胞シグナル伝達に対する研究は、例えば、特定のPPIおよび細胞プロセス2を容易に動的な、ナノスケール空間区画を明らかにしました。したがって、十分な空間的・時間的解像度、高い特異性、高感度で生物学的システムでのPPIを調査する能力は、生物学のメカニズムの理解を達成するための鍵です。 二分子蛍光相補（BIFC）は、細胞内の分解能でセル内のPPIを可視化するためのいくつかの既存のツールの一つであり、生細胞の互換性3から5 を 。技術は比較的簡単であり、2つの非蛍光フラグメントに蛍光タンパク質の分裂を伴います。遺伝的に2つの相互作用するタンパク質にタグ付けされたときに近接させ、フラグメントは、蛍光シグナルを生じる、完全な蛍光タンパク質を形成するように再構成することができます。適切に設計すると、BIFCプローブは自発的にのPPIの不存在下で再構成してはいけません。このように、BIFCアッセイにおける蛍光シグナルは、高い特異性でのPPIの直接可視化を可能にするのPPIの存在下で生じます。読み出しとして蛍光を使用することのさらなる利点は、とりわけ、高いスループットと高含有量スクリーニングアッセイ、と高感度、細胞内の解像度、および互換性があります。これらの利点のために、異なる親蛍光タンパク質に基づくBIFCプローブの数が開発されています。従来の光学顕微鏡法に基づいて、すべての他の検出手法のように、しかしながら、BIFCの空間分解能は、光の回折による〜250nmのに限定されています。これは、ナノスケールでのPPIの調節を研究するための挑戦になり、先に言及したと脂質ラフト6 aで例示されるようにします、NDラスナノクラスター7は 、そのようなシグナル伝達などの多くの細胞プロセスを理解するための重要な長さスケールです。 BIFCはPPIの10を撮像するための空間分解能でこの制限を克服するために、光活性化ローカライゼーション顕微鏡（PALM）8,9と組み合わされています。 PALMは、確率論的活性化し、単一の蛍光分子のsubdiffractiveローカライズを通じて蛍光イメージングでの回折限界を回避する最近の超解像顕微鏡技術です。各作動サイクルでは、蛍光分子は、数千光子に数百を放射し、検出器上の単一分子の画像を生じます。画像は、回折限界（幅〜250 nm）であるが、その重心は、典型的には、検出された光子の数に応じて10〜50ナノメートルのオーダで、非常に高い精度で判定することができます。試料中の各蛍光分子を活性化し、局在化することにより、高解像度画像を再構成することができます。 Performe生細胞上のD、単一のセル11からのタンパク質の拡散軌跡の数千の単一分子追跡（smt-）PALMさらに許可取得。重要なのは、Palmは確率的な活性化を達成するために、このような光活性化蛍光タンパク質（PA-のFP）などの特殊な蛍光プローブを使用しています。 BIFCとPalm用の蛍光タンパク質の両方ので、それらは、アミノ酸159と160の間に2つの断片に、Palm用PAmCherry1、一般的に使用されるPA-FPを分割して組み合わせました。 分割PAmCherry1に基づいBIFCシステムは、2つの断片の自発的な再構成からの低バックグラウンドシグナルを示しています。遺伝的に相互作用するタンパク質のペアにタグ付けすると、2つの断片を（RN =は1から159残基; RC =メット+残基160から236）であっても37℃、より低い温度でインキュベートすることなく、完全なPAmCherry1タンパク質を形成するために、効率的に再構成し、これそのような親mCherryを13などの他のBIFCペア12には当てはまりません。 Furthermo再、再構成PAmCherry1タンパク質は、高コントラスト比、正確な単一分子の局在化と高解像度のPALM画像化のために重要である他、間中光子収量、高速光活性化、などの親PAmCherry1の光物理的特性を、保持していました。 このプロトコルでは、分割PAmCherry1（ 図1A）を用いて、U2OS細胞中のRas-Rafの相互作用を撮像するBIFC-PALMの使用が記載されています。最初のステップは、PAmCherry1フラグメント（ すなわち 、RN及びRC）と目的タンパク質との間の融合タンパク質を発現するための構築物を設計することです。理論的には、候補タンパク質（A及びB）の対ごとに、融合タンパク質の8組をテストするために存在する：RN-A / RC-B。 RN-A / B-RC。 RC-A / RN-B; RC-A / B-RN; -RN / RC-B; -RN / B-RC。 -RC / RN-B;そしてA-RC / B-RN。このプロセスは、しばしば考慮候補タンパク質の構造的または生化学的特性を取ることによって簡略化することができます。 Rasの場合には、タンパク質であります翻訳後のC末端CAAXボックスで変更された（C =システイン、A =脂肪族; X =任意の）、その後、AAXモチーフが切断されます。したがって、RNまたはRCは、RasのN末端に融合することができます。これは、4つ（ 図1B）への融合タンパク質のペアの数を減少させます。 Rafは、Rasの結合ドメイン（RBD;残基51から131）が使用され、両端にタグを付けることができます。これら4融合構成が生成されました：RN-KRAS / RC-RafのRBD。 RN-KRAS / RafのRBD-RC。 RC-KRAS / RN-RafのRBD。 RC-KRAS / RafのRBD-RN。 さらに、フラグメントおよび目的のタンパク質との間のリンカーを考慮する必要があります。約10アミノ酸の柔軟なリンカーは、しばしば、それが相補性を生じるのに十分な自由度を提供するように使用されます。マルチクローニングサイト（MCS）14から生成されたランダムな配列を含む、正常に適用されてきた多くの人が、ありますが、そのようなリンカーは、（GGGGS）×2です。リンカーの長さはdependin最適化する必要があるかもしれません目的のタンパク質とその向きの相互作用の大きさにグラム。 PAmCherry1断片はのMCSに隣接すると、小さなクローニングバックボーンに含まれている（材料リストを参照してください）​​。目的の遺伝子は、制限部位を介して、またはライゲーション非依存の方法で挿入することができます。クローニングおよび配列確認の後、発現カセットは、リコンビナーゼ反応、高忠実度、高効率のクローニング法を使用して発現ベクターに移されます。 次に、得られた発現構築物は、標的細胞株を感染するためにレンチウイルスにパッケージング、安定な細胞株が所望される場合、標的細胞株にトランスフェクトまたはれています。一過性トランスフェクションは、BIFC構成の迅速な検証を可能にしますが、潜在的な問題に注意する必要があります。化学薬品を使用してトランスフェクションは、多くの場合、高い自己蛍光が得られ、細胞を強調。一方、全内部反射蛍光（TIRF）microscoの使用PPIは、細胞膜上で行われるときpyがTIRF理想、照明量を制限することにより、このバックグラウンド信号を軽減することができます。また、一過性トランスフェクションは、多くの場合、遠くのPPIの検出にアーチファクトを引き起こす可能性がある内因性タンパク質のものを超える、高レベルのタンパク質発現を導きます。したがって、それは、適切なBIFC設定が初期テストを通じて決定された後に安定な細胞株を確立することをお勧めします。安定な細胞株はまた、ドキシサイクリン誘導15を介して調節可能な発現の可能性を有します。 感染後、細胞を、抗生物質、典型的にはピューロマイシン、ネオマイシンで各構築物のための1つの選択されています。試料調製、画像収集、およびデータ分析のための後続のステップは、プロトコールに詳細に説明します。 このアプローチでは、ラス-RafのRBDのBIFC-PALM画像内のナノスケールのクラスターの形成が日常観察される（ 図2A &lt;/強いです&gt;）。一貫して、ラス-RafのRBDのSMT-PALM軌道が拡散状態（ 図2B-D）での不均質な分布を示しています。これらの結果は、Ras-Rafの複合体は、潜在的な生物学的な意味で、おそらくモノマー及びクラスターとして、細胞膜上の複数の状態で存在することを示唆しています。この作業は、従来BIFC、蛍光の共局在化、または蛍光共鳴エネルギー移動（FRET）を用いて得ることが困難となり、ナノメートルの空間分解能および単一分子感度を有する細胞でのPPIの選択的なイメージングにBIFC-PALMのパワーを示しています。 BIFC実験を設計し、その結果を解釈する際には、BIFCプロセスが分割PAmCherry1を含め、ほとんどの場合、中に不可逆的であることを心に留めておくことは重要です。二つの断片を組み合わせて、完全なPAmCherry1タンパク質、二つの断片間の結合を形成し、一旦このように、PPIは永続的になります。これはBIFCとBIFC-PALの使用を制限PPIのダイナミクス（ すなわち 、結合反応速度ではなく、それが形成されると、タンパク質複合体の拡散ダイナミクス）を監視すると、時間にMがあっても、PPI複合体の誤局在につながる可能性があります。 BIFC-PALM実験を設計する際に考慮すべき付加的な要因は、蛍光タンパク質に固有の発色団の成熟の遅れです。 2つのタンパク質が相互作用し、FPのフラグメントが一緒に来たら、彼らは一般的に数秒程度（EYFP 3 60秒のハーフタイム）にリフォールディング。しかし、その後の発色団の成熟と蛍光シグナルが分程度で開発しています。蛍光はスプリット金星、高速折りたたみYFP変異体の再構成後10分以内に検出することができるが、一般的には成熟のためのハーフタイムは、多くの場合、約60分で16です。分割PAmCherry1は、同様の速度を有することが観察されました。したがって、BIFCとBIFC-PALMは現在、リアルタイムのPPIの動態を監視するための悪い選択です。他の私このようなFRETと最近開発された二量体化に依存する蛍光タンパク質17としてthodsは、この目的のために、より適しているかもしれません。

<table><tbody><tr><td>Microscope frame</td><td>Nikon</td><td>Ti-U</td><td></td></tr><tr><td>60X oil immersion TIRF objective with 1.49 NA</td><td>Nikon</td><td>CFI Apo TIRF 60x H</td><td></td></tr><tr><td>EMCCD camera</td><td>Andor</td><td>iXon Ultra 897</td><td></td></tr><tr><td>561 nm laser</td><td>Opto Engine</td><td>MGL-FN-561</td><td></td></tr><tr><td>405 nm laser</td><td>Coherent</td><td>CUBE-405 50mW</td><td></td></tr><tr><td>561 nm dichroic mirror</td><td>Semrock</td><td>&amp;nbsp;Di01-R405/488/561/635-25x36</td><td></td></tr><tr><td>561 nm filter</td><td>Semrock</td><td>FF01-525/45-25</td><td></td></tr><tr><td>405/561 nm notch filter</td><td>Semrock</td><td>NF01-405/488/568-25</td><td></td></tr><tr><td>Temperature and CO&lt;sub&gt;2&lt;/sub&gt; controlled stage</td><td>Tokai Hit</td><td>INUBG2ATW-PLAM</td><td></td></tr><tr><td>pENTR-D-TOPO-PAmCherry1_1-159-MCS</td><td>Addgene</td><td>60545</td><td></td></tr><tr><td>pENTR-D-TOPO-PAmCherry160-236-MCS</td><td>Addgene</td><td>60546</td><td></td></tr><tr><td>pcDNA3.2-DEST</td><td>Life Technologies</td><td>12489-019</td><td></td></tr><tr><td>pLenti-DEST</td><td>Addgene</td><td></td><td>http://www.addgene.org/Eric_Campeau/</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Scientific</td><td>F-531</td><td></td></tr><tr><td>In-Fusion HD Cloning</td><td>Clontech</td><td>639649</td><td></td></tr><tr><td>LR Clonase</td><td>Life Technologies</td><td>11791</td><td></td></tr><tr><td>Vira Power Lentivirus Packaging</td><td>Life Technologies</td><td>K497500</td><td></td></tr><tr><td>X-tremeGENE Transfection Reagent</td><td>Roche</td><td>13873800</td><td></td></tr><tr><td>Lab-Tek II Chambered Coverglass</td><td>Thermo Scientific</td><td>155409</td><td>#1.5 glass bottom dishes</td></tr><tr><td>U2OS cells</td><td>ATCC</td><td>HTB-96</td><td></td></tr><tr><td>293T/17 cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>DMEM with phenol red</td><td>Life Technologies</td><td>11995</td><td></td></tr><tr><td>DMEM no phenol red</td><td>Life Technologies</td><td>21063</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>10082</td><td></td></tr><tr><td>Leibovit&#039;s L-15, no phenol red</td><td>Life Technologies</td><td>21083-027</td><td></td></tr><tr><td>Reduced serum medium</td><td>Life Technologies</td><td>31985</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Life Technologies</td><td>14040</td><td></td></tr><tr><td>Syringe</td><td>BD Biosciences</td><td>309604</td><td></td></tr><tr><td>Syringe filter</td><td>Millipore</td><td>SLHV033RB</td><td></td></tr><tr><td>Lentiviral concentrator</td><td>Clontech</td><td>631231</td><td></td></tr><tr><td>Retroviral concentrator</td><td>Clontech</td><td>631455</td><td></td></tr><tr><td>10 cm culture dish</td><td>BD Biosciences</td><td>353003</td><td></td></tr><tr><td>6-well culture plate</td><td>BD Biosciences</td><td>353046</td><td></td></tr><tr><td>Polybrene</td><td>Sigma</td><td>107689</td><td></td></tr><tr><td>Puromycin</td><td>Life Technologies</td><td>A11138</td><td></td></tr><tr><td>G-418</td><td>Calbiochem</td><td>345812</td><td>Neomycin</td></tr><tr><td>Doxycyline</td><td>Fisher</td><td>BP2653</td><td></td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP152</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>EDS</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>S5881</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>G6257</td><td></td></tr><tr><td>PIPES</td><td>Sigma</td><td>P6757</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>3777</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma</td><td>M2643</td><td></td></tr><tr><td>Potassium Hydroxide</td><td>Sigma</td><td>221473</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>BP358</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>M33</td><td></td></tr><tr><td>100 nm gold particles</td><td>BBI Solutions</td><td>EM.GC100</td><td></td></tr><tr><td>Molecular grade water</td><td>Life Technologies</td><td>10977</td><td></td></tr><tr><td>Dpn1</td><td>New England Biolabs</td><td>R0176</td><td></td></tr><tr><td>PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Miniprep kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>Midiprep kit</td><td>Macherey-Nagel</td><td>740410</td><td></td></tr><tr><td>0.6 ml microcentrifuge tubes</td><td>Fisher</td><td>05-408-120</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Fisher</td><td>05-408-137</td><td></td></tr><tr><td>15 ml tubes</td><td>Fisher</td><td>05-539-12</td><td></td></tr><tr><td>5 ml polypropylene round-bottom tubes</td><td>BD Biosciences</td><td>352063</td><td></td></tr><tr><td>14 ml polypropylene round-bottom tubes</td><td>BD Biosciences</td><td>352059</td><td></td></tr><tr><td>50 ml tube</td><td>BD Biosciences</td><td>352070</td><td></td></tr><tr><td>PCR tube</td><td>GeneMate</td><td>C-3328-1</td><td></td></tr><tr><td>SOC medium</td><td>Life Technologies</td><td>15544</td><td></td></tr><tr><td>LB broth</td><td>BD Biosciences</td><td>244610</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Fisher</td><td>BP906</td><td></td></tr><tr><td>Competent cells</td><td>Life Technologies</td><td>C4040</td><td></td></tr><tr><td>Matlab</td><td>Mathworks</td><td></td><td></td></tr></tbody></table>

photoactivated localization microscopy with bimolecular fluorescence complementation (bifc-palm)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

示さBIFC-PALMの例は、CRAFのラス結合ドメイン（RBD）（ 図1A）との相互作用KRAS G12D変異体です。議論したように、それはRasの膜局在化、ひいては生物学的活性を破壊になるので、RNまたはRCフラグメントは、RasのC末端にタグ付けされていませんでした。これは、8から4（ 図1B）に可能な組み合わせを減少させました。これらの4つの組み合わせの各々は、レンチウイルス感...

Watch this Scientific Journal Video about Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM at JoVE.com

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

二分子蛍光相補性を持つ光活性化ローカライゼーション顕微鏡（BIFC-PALM）

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

photoactivated-localization-microscopy-with-bimolecular-fluorescence

Research

JoVE Journal

Bioengineering

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

9.9K ビュー.  Oregon Health and Science University. Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes. 

ビデオ: Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

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

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

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM). Cells are cultivated on transparent slides and incubated with a fluorescent marker homogeneously distributed in their plasma membrane. Illumination occurs by a parallel laser beam under variable angles of total internal reflection (TIR) with different penetration depths of the evanescent electromagnetic field. Recording of fluorescence images upon irradiation at about 10 different angles permits to calculate cell-substrate distances with a precision of a few nanometers. Differences of adhesion between various cell lines, e.g. cancer cells and less malignant cells, are thus determined. In addition, possible changes of cell adhesion upon chemical or photodynamic treatment can be examined. In comparison with other methods of super-resolution microscopy light exposure is kept very small, and no damage of living cells is expected to occur.

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

Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).

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 ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

光活性化単一分子追跡を使用してライブ細菌細胞におけるタンパク質-DNA相互作用を可視化する

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

免疫・感染学

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

ライブセル超解像顕微鏡と単一分子トラッキングのための従来のBODIPYコンジュゲート

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

生化学

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 ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定化生体分子の単一分子蛍光測定のための自動化システム

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

生物学

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

二分子蛍光相補

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

単一の膜タンパク質生きた哺乳類細胞の細胞内小器官のマルチカラー ローカリゼーション顕微鏡

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy

To prepare a sample for imaging, plate about 5.5 x 104 stable expression cells per well of an 8-well glass-bottomed chamber slide, in 350 microliters of phenol red-free DMEM. Use fresh paraformaldehyde with glutaraldehyde to fix the cells, and after replacing the fixative with PBS or imaging buffer, vortex 100-nanometer gold particles, and add 35 microliters per well for tracking stage drift during imaging.
Approach the microscope and power on the 405 and 561-nanometer lasers. Keeping the shutters closed at this point, ensure the 561-nanometer dichroic mirror and notch filter are in place. Open the image acquisition software.
Turn on the EMCCD camera and allow it to cool down, and set the exposure time to 100 milliseconds and the EMCCD gain to an appropriate value. Add immersion oil to the objective, and secure the sample on the microscope stage. Next, with either brightfield or the 561-nanometer laser, bring the sample into focus.
For imaging Ras and other membrane proteins, use a 60X apochromat TIRF objective with a 1.49 numerical aperture, and bring the microscope into TIRF configuration.
Adjust the excitation laser so that it is off-centered when hitting the back aperture of the TIRF objective, which will cause the laser to deflect upon reaching the coverslip buffer interface. Keep adjusting the laser incident position until the critical angle is reached and the laser is being reflected back.
Search for a cell to image with several gold particles in view. Then, set a region of interest that encloses the cell or a region of it and the gold particles.
In case sufficient activation is already occurring because of high expression levels, begin acquisition with the 405-nanometer laser off and the 561-laser on. Otherwise, turn on the 405-nanometer laser at the lowest factory power setting, and increase it gradually until there are tens of molecules per frame or so that single molecules are well-separated.
As data acquisition continues, gradually increase the 405-nanometer laser power to keep the spot density roughly constant. Continue image acquisition until high 405 power does not initiate any more activation events.

二分子蛍光相補性を持つ光活性化ローカライゼーション顕微鏡（BIFC-PALM）

要約

さらに動画を探す

この動画の章

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、

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

光活性化単一分子追跡を使用してライブ細菌細胞におけるタンパク質-DNA相互作用を可視化する

ライブセル超解像顕微鏡と単一分子トラッキングのための従来のBODIPYコンジュゲート

平面支持された二分子層上の単一分子蛍光顕微鏡

表面固定化生体分子の単一分子蛍光測定のための自動化システム

二分子蛍光相補

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

単一の膜タンパク質生きた哺乳類細胞の細胞内小器官のマルチカラーローカリゼーション顕微鏡

二分子蛍光相補結合光活性化局在顕微鏡法

二分子蛍光相補性を持つ光活性化ローカライゼーション顕微鏡（BIFC-PALM）

要約

さらに動画を探す

この動画の章

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、

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

光活性化単一分子追跡を使用してライブ細菌細胞におけるタンパク質-DNA相互作用を可視化する

ライブセル超解像顕微鏡と単一分子トラッキングのための従来のBODIPYコンジュゲート

平面支持された二分子層​​上の単一分子蛍光顕微鏡

表面固定化生体分子の単一分子蛍光測定のための自動化システム

二分子蛍光相補

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

単一の膜タンパク質生きた哺乳類細胞の細胞内小器官のマルチカラー ローカリゼーション顕微鏡

二分子蛍光相補結合光活性化局在顕微鏡法

平面支持された二分子層上の単一分子蛍光顕微鏡

単一の膜タンパク質生きた哺乳類細胞の細胞内小器官のマルチカラーローカリゼーション顕微鏡