Name: three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)
Uploaded: 2016-12-01T12:30:00
Description: Watch this Scientific Journal Video about Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM at JoVE.com

YWとPKは感謝PK（NRF-NRFF-2011-04とNRF2012NRF-CRP001-084）に授与シンガポール国立研究財団から資金援助を認めます。また、インフラストラクチャのサポートのためのMBIオープンラボと顕微鏡の中核施設に感謝します。

1.イメージング試料の準備<ol><li>ので、バックグラウンド蛍光信号が最初に脱イオン水（のddH 2 O）でそれらをすすぎ、次いで圧縮空気を使用して空気乾燥させてカバーガラスを清掃し、フルオロフォア標識からの蛍光を妨害します。その後、15秒間プラズマクリーナープラズマエッチングを行う、または必要に応じてより長いです。 </li><li>ドリフト補正とiPALMキャリブレーションを?...

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図1（a）に示すようiPALMの光学系は、4-πデュアル対向対物レンズの設計に基づいています。 表 1に先に23を説明し、記載されている設定は、カスタム機械加工や商業の両方の光学機械部品を用いて構成されています。私たちのセットアップに加えて、ハワード・ヒューズ医学研究所（HHMI）がJaneliaリサーチキャンパスでの高度なイメージングセン?...

複雑な細胞構造の可視化は、長い生物学的洞察と発見に不可欠となっています。蛍光顕微鏡は、高分子特異性を有する画像セルは、その分解能は〜像面（X、Y、または横寸法）で200 nmの光軸（Z、または軸方向の寸法）に沿って、&gt; 500nmの回折によって制限されることができるが1,2。したがって、超微細な特徴の観察は、歴史的に、電子顕微鏡（EM）に限られていました。 100 nmの範囲1-6 -幸いなことに、超解像顕微鏡の最近の開発は、10の空間分解能を可能にする、この制限を回避しています。具体的には、超解像は、このようなPALM（光活性化ローカライゼーション顕微鏡）4、FPALM（蛍光光活性化ローカライゼーション顕微鏡）5（d）にSTORM（直接確率的光学再構築顕微鏡）などの頭字語によって知られている単一分子の局在化に基づくアプローチ6,7、PAINT（ポー川int型イメージングナノスケールの地形のための蓄積）8、GSDIM（基底状態枯渇顕微鏡は、個々の分子のリターンが続く）9、またはSMACM（単一分子アクティブ制御顕微鏡）10、ならびに、それらの3次元（3D）の実装、干渉PALM（iPALM）11または3D-STORM 12、神経細胞の軸索とシナプス13を含む多数の生物学的構造のナノスケールの組織に新たな洞察を明らかに貴重なされている、接着斑14,15、細胞間結合16、核膜孔17 、および中心体18-20は 、いくつかの名前を付けます。 超解像顕微鏡は、潜在的に有用であるために、細胞内の別の超微細構造の特徴は、アクチン細胞骨格です。細胞表層における糸状（F）アクチンの複雑な網目構造は、細胞の形状及び機械的特性21の制御において重要な役割を果たしています。組織OF F-アクチンは、積極的かつ動的に強く重合、架橋、売上高、安定性、およびネットワークトポロジー22に影響を与える多数の調節タンパク質も規制されています。 F-アクチン網目構造のキャラクタリゼーションは、細胞プロセスの多様な範囲に機械的な洞察のために重要であるものの、F-アクチンフィラメントの小さいサイズ（〜8 nm）は、従来の回折限界光学顕微鏡によって自分の観察を妨げます。従って、アクチン微細構造の可視化は、これまでもっぱらEMによって実行されています。ここでは、3D 11,23における非常に高精度の能力を活用するiPALM超分解能顕微鏡技術を用いて、接着性哺乳動物細胞におけるF-アクチン細胞骨格を可視化するためのプロトコルを説明します。 iPALM機器は専門性の高いですがiPALM顕微鏡へのアクセスはホーによってホストされている一方で、このような機器の設定に関する指示は、23最近記載されています病棟・ヒューズ医学研究所はまた、最小限のコストとの研究コミュニティに利用可能とされています。さらに、本明細書に記載の試料調製方法は、より広範に利用可能であるような点広がり関数（PSF）の非点収差のデフォーカスに基づくものなどの代替的な3D超解像手法、12又はバイプレーン検出24、に直接適用可能です。 私たちは、一般的には単一分子の局在化ベースの超解像顕微鏡のために必要な成分が満たされなければ、単一分子の局在化ベースの超解像顕微鏡のための3つの重要な要件を可能にするphotoswitchableフォア25、であることに注意した：i）高単一分子背景信号の明るさとコントラストの相対。 ⅱ）所与の画像フレーム内の単一分子のまばらな分布。また、ナイキスト・沙として知られている根本的な構造（のプロファイルをキャプチャするのに十分な標識及びiii）高い空間密度nnonサンプリング基準）26。このように、満足のいく結果を得るために、重点はフルオロフォアphotoswitchingを最適化するために、基礎となる超微細構造を維持するための試験片の適切な準備の両方で、同様の実験の計測および買収側面に均等に配置する必要があります。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>optical table</td>
			<td>Newport, CA&#160;</td>
			<td>RS4000</td>
			<td>iPALM, installed on 4 Newport Stabilizer vibration isolators&#160;</td>
		</tr>
		<tr>
			<td>vibration isolator for optical table</td>
			<td>Newport, CA&#160;</td>
			<td>S-2000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>laser-642</td>
			<td>Newport, CA&#160;</td>
			<td>1185055</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>laser-561</td>
			<td>Newport, CA&#160;</td>
			<td>1168931</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-488</td>
			<td>Newport, CA&#160;</td>
			<td>1137970</td>
			<td>output power=200mw</td>
		</tr>
		<tr>
			<td>laser-405</td>
			<td>Newport, CA&#160;</td>
			<td>1142279</td>
			<td>output power=100mw</td>
		</tr>
		<tr>
			<td>broadband dielectric mirrors&#160;</td>
			<td>Thorlabs, NJ</td>
			<td>BB1-E02</td>
			<td>laser combiner</td>
		</tr>
		<tr>
			<td>dichroic beamsplitter&#160;</td>
			<td>Semrock, NY</td>
			<td>LM01-427-25</td>
			<td></td>
		</tr>
		<tr>
			<td>acousto-optic tunable filter&#160;</td>
			<td>AA Opto-Electronic, France</td>
			<td>AOTFnC-VIS-TN</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear polarizer</td>
			<td>Newport, CA&#160;</td>
			<td>05LP-VIS-B</td>
			<td></td>
		</tr>
		<tr>
			<td>baseplate</td>
			<td>local workshop</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr>
			<td>turning mirror (22.5&#176;)</td>
			<td>Reynard Corpporation, CA</td>
			<td>customized</td>
			<td>22.5&#176; mirror</td>
		</tr>
		<tr>
			<td>motorized optic mounts&#160;</td>
			<td>New Focus, CA</td>
			<td>8816</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized XYZ translation stage</td>
			<td>Thorlabs, NJ</td>
			<td>MT3/M-Z6</td>
			<td>sample holder</td>
		</tr>
		<tr>
			<td>T-Cube DC servo motor controller</td>
			<td>Thorlabs, NJ</td>
			<td>TDC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo Phase Shifter</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-303.CD</td>
			<td></td>
		</tr>
		<tr>
			<td>objective lens</td>
			<td>Nikon, Japan</td>
			<td>MRD01691</td>
			<td>objective. Apo TIRF 60X/1.49oil</td>
		</tr>
		<tr>
			<td>translation stage</td>
			<td>New Focus, CA</td>
			<td>9062-COM-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pico Motor Actuator</td>
			<td>New Focus, CA</td>
			<td>8301</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary Solenoid/Shutter</td>
			<td>DACO Instruments, CT</td>
			<td>5423-458</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way beam splitter</td>
			<td>Rocky Mountain Instruments, CO</td>
			<td>customized</td>
			<td>beamsplitter</td>
		</tr>
		<tr>
			<td>Piezo Z/Tip/Tilt scanner</td>
			<td>Physik Instrumente, Germany</td>
			<td>S-316.10</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized five-axis tilt aligner&#160;</td>
			<td>New Focus, CA</td>
			<td>8081</td>
			<td></td>
		</tr>
		<tr>
			<td>Picmotor ethernet controller</td>
			<td>New Focus, CA</td>
			<td>8752</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo controllers/amplifier/digital operation module</td>
			<td>Physik Instrumente, Germany</td>
			<td>E-509/E-503/E-517</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-523/20</td>
			<td>filters</td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-588/21</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-607/30</td>
			<td></td>
		</tr>
		<tr>
			<td>band-pass filter</td>
			<td>Semrock, NY</td>
			<td>FF01-676/37</td>
			<td></td>
		</tr>
		<tr>
			<td>notch filter</td>
			<td>Semrock, NY</td>
			<td>NF01-405/488/561/635</td>
			<td></td>
		</tr>
		<tr>
			<td>motorized filter wheel with controllter</td>
			<td>Thorlabs, NJ</td>
			<td>FW103H</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD</td>
			<td>Andor, UK&#160;</td>
			<td>DU-897U-CSO-#BV</td>
			<td>3 sets</td>
		</tr>
		<tr>
			<td>Desktop computers for controlling cameras and synchronization</td>
			<td>Dell</td>
			<td>Precision T3500</td>
			<td>PC, 4 sets</td>
		</tr>
		<tr>
			<td>coverslips with fiducial</td>
			<td>Hestzig, VA</td>
			<td>600-100AuF</td>
			<td>sample preparation. fiducial marks with various density and spectra&#160; available</td>
		</tr>
		<tr>
			<td>fibronectin&#160;</td>
			<td>Millipore, MT</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>15710</td>
			<td>fixation. 16%</td>
		</tr>
		<tr>
			<td>glutaraldehyde&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16220</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>triton X-100</td>
			<td>Sigma aldrich, MO</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC cells</td>
			<td>Life Technologies, CA</td>
			<td>C-015-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 200</td>
			<td>Life Technologies, CA</td>
			<td>M-200-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Vessel Endothelial Factors</td>
			<td>Life Technologies, CA</td>
			<td>A14608-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td></td>
			<td>14190367</td>
			<td></td>
		</tr>
		<tr>
			<td>Pennicillin/Streptomycin</td>
			<td></td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Life Technologies, CA</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma aldrich, MO</td>
			<td>P1851</td>
			<td>PHEM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1st base, Malaysia</td>
			<td>BIO-1825</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma aldrich, MO</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Millipore, MT</td>
			<td>5985</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Alexa Fluor 647 Phalloidin</td>
			<td>Invitrogen, CA</td>
			<td>A22287</td>
			<td>staining</td>
		</tr>
		<tr>
			<td>sodium borohydride (NaBH4)&#160;</td>
			<td>Sigma aldrich, MO</td>
			<td>480886</td>
			<td>quenching</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>1st base, Malaysia</td>
			<td>BIO-1101</td>
			<td>imaging buffer</td>
		</tr>
		<tr>
			<td>glucose oxidase</td>
			<td>Sigma aldrich, MO</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr>
			<td>catalase</td>
			<td>Sigma aldrich, MO</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>cysteamine&#160;</td>
			<td>Sigma aldrich, MO</td>
			<td>30070</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Thorlabs, NJ</td>
			<td>G14250</td>
			<td></td>
		</tr>
		<tr>
			<td>vaseline</td>
			<td>Sigma aldrich, MO</td>
			<td>16415</td>
			<td>sample sealing</td>
		</tr>
		<tr>
			<td>lanolin</td>
			<td>Sigma aldrich, MO</td>
			<td>L7387</td>
			<td></td>
		</tr>
		<tr>
			<td>parafin wax</td>
			<td>Sigma aldrich, MO</td>
			<td>327204</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil&#160;</td>
			<td>Electron Microscopy Sciences, PA</td>
			<td>16915-04</td>
			<td>imaging. Cargille Type HF</td>
		</tr>
	</tbody>
</table>

three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)

蛍光顕微鏡は、細胞内の特定の生体分子の直接可視化を可能にします。しかし、従来の蛍光顕微鏡法のために、空間分解能は、光軸に沿って像面、&gt; 500nmの範囲内〜200nmの回折により制限されています。結果として、蛍光顕微鏡法は、長い細胞内の超微細構造の特徴の観察に厳しく制限されています。超解像顕微鏡法の最近の開発は、この制限を克服しました。特に、photoswitchable蛍光体の出現は、分子長さスケールに近づいて解像力を提供ローカリゼーションベースの超解像顕微鏡を、可能にします。ここでは、干渉光活性化ローカライゼーション顕微鏡（iPALM）と呼ばれる単一分子の局在顕微鏡法および多相干渉に基づいて3次元超解像顕微鏡法の適用を説明します。この方法は、上のほぼ等方性の解像度を提供しますすべての3つの次元で20nmのオーダー。 iPALM機器の試料調製及び動作を含む、繊維状アクチン細胞骨格を可視化するためのプロトコルは、ここに記載されています。これらのプロトコルはまた、細胞内の他の超微細構造の特徴の研究に容易に適合し、有益です。

iPALMのための重要な要件は、光学系のアライメント、登録、およびキャリブレーションされています。これらは、z座標の抽出に3ウェイビームスプリッタ必要内の適切な干渉を確実にするために必要です。継続的な監視を有効にするには、蛍光の一定の点光源が必要です。これは、蛍光のAu又はそのフォトルミネッセンス局在表面プラズモン共鳴（LSPR）から生じる二?...

Watch this Scientific Journal Video about Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM at JoVE.com

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

私たちは、接着性、哺乳動物細胞におけるアクチン細胞骨格のイメージングに、干渉光活性化ローカライゼーション顕微鏡（iPALM）、3次元の単一分子の局在超解像顕微鏡法の適用のためのプロトコルを提示します。このアプローチは、そうでなければ、従来の回折限界光学顕微鏡で未解決のままであるナノスケールの構造的特徴の光ベースの可視化を可能にします。

干渉光活性化ローカライゼーション顕微鏡（iPALM）によってF-アクチンフィラメントの三次元超解像顕微鏡

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Research

JoVE Journal

Biology

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

蛍光を経由してF -アクチンダイナミクスの生細胞イメージングでは、顕微鏡のスペックル（FSM）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

ナノFEM：写真活性化ローカライゼーション顕微鏡と電子顕微鏡を用いてタンパク質の局在

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

最適STORM超解像顕微鏡のためのテストサンプル

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

単一のビリオン原子間力顕微鏡と超解像蛍光イメージングのサンプル調製

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

高速3D-構造化照明顕微鏡（F3D-SIM）を使用したライブ細菌における細胞質分裂のZリングの超解像イメージング

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

共焦点および多光子顕微鏡を用いて5蛍光タンパク質によってマーク造血幹細胞および前駆細胞の生体内でクローン追跡で

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

VirusMapperと超解像顕微鏡のためのオープンソースの単一粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

ライブの一次繊毛で高速超解像顕微鏡の応用

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

高圧急速凍結、マイクロ波を用いたコントラスト強調、およびボリューム イメージングのために埋め込む最小限の樹脂による生体試料作製

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...