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 e PK agradecem apoio financeiro da Fundação de Pesquisa Nacional de Cingapura, atribuído a PK (NRF-NRFF-2011-04 e NRF2012NRF-CRP001-084). Agradecemos também aos abertas de laboratório e de núcleo de microscopia instalações MBI para suporte de infraestrutura.

1. Criação de Imagens Preparação de Amostras <ol><li> Uma vez que os sinais de fluorescência de fundo interferir com fluorescência a partir de fluoróforos etiquetas, limpar os esfregaços por primeiro enxaguar em água desionizada (ddH2O) e, em seguida, ar-secando-os com ar comprimido. Subsequentemente, executar gravação por plasma num dispositivo de limpeza de plasma durante 15 seg, ou mais longo, se necessário. </li><li> Para ativar a correção de deriva e calibração iPALM, utilize # 1.5 roun...

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O sistema óptico de iPALM baseia-se no objectivo de criação de uma dupla-4-oposição π, como mostrado na Figura 1A. A configuração é construído usando peças tanto custom-usinados e comerciais opto-mecânico, como descrito anteriormente 23 e listados na Tabela 1. Além da nossa configuração, o Howard Hughes Medical Institute (HHMI) hospeda um sistema que é acessível à comunidade científica no Imaging Center avançada no Campus Janelia Research....

A visualização das estruturas celulares complexas, desde há muito integrante a insights biológicos e descoberta. Embora a microscopia de fluorescência pode células de imagem com especificidade molecular elevado, o seu poder de resolução é limitada por difracção para ~ 200 nm, no plano de imagem (x, y, ou dimensão lateral) e&gt; 500 nm ao longo do eixo óptico (Z, ou dimensão axial) 1,2. Assim, a observação de características ultra-estruturais historicamente tem sido limitada a microscopia electrónica (ME). Felizmente, o recente desenvolvimento da microscopia de super-resolução tem contornado este limite, permitindo a resolução espacial no 10-100 gama nm 1-6. Em particular, super resolução abordagens baseadas em localização única molécula, conhecida por siglas como PALM (fotoativado Localization Microscopy) 4, FPALM (Fluorescência fotoativado Localization Microscopy) 5 (d) STORM (direto Stochastic Optical Reconstrução Microscopia) 6,7, PINTURA ( PoAcumulação int para imagens em nanoescala Topografia) 8, GSDIM (Estado Chão Depleção Microscopia seguido de retorno moleculares individuais) 9, ou SMACM (Single-molécula activa-Control Microscopia) 10, bem como as suas três dimensões implementações (3D), tal como PALM interferométrico (iPALM) 11 ou 3D-STORM 12, têm sido valiosos em revelar novos insights sobre a organização nanoescala de numerosas estruturas biológicas, incluindo os axônios neuronais e sinapses 13, adesões focais 14,15, junções célula-célula 16, nuclear poros 17 centrossomas, e 18-20, para nomear alguns. Outra característica ultra-estruturais em células para as quais a microscopia de super-resolução é potencialmente útil é o citoesqueleto de actina. O complexo de malha filamentosa (F) -actina no córtex celular desempenha um papel essencial no controlo da forma e propriedades mecânicas celular 21. A organização of f-actina é activa e dinâmica regulada embora numerosas proteínas reguladoras que influenciam fortemente a polimerização, reticulação, volume de negócios, estabilidade e topologia de rede 22. No entanto, embora a caracterização da arquitectura malha de actina F é importante para percepções mecanísticos em uma grande variedade de processos celulares, o pequeno tamanho (~ 8 nm) dos filamentos F-actina dificulta a sua observação por microscopia de luz limitado por difração convencional; Assim, a visualização da estrutura fina actina tem sido até agora exclusivamente realizada por EM. Aqui, nós descrevemos protocolos para a visualização do citoesqueleto de actina F em células de mamífero aderentes, usando a técnica de microscopia de super-resolução iPALM para tirar partido da sua capacidade muito alta precisão em 3D 11,23. Embora o instrumento iPALM é altamente especializada, instruções sobre a criação de um tal instrumento foi descrito recentemente 23, enquanto o acesso ao microscópio iPALM organizada pelo Hoala Hughes Medical Institute, também foi disponibilizado para a comunidade científica com um custo mínimo. Além disso, os métodos de preparação de amostras aqui descritas são directamente aplicáveis aos abordagens alternativas 3D Super resolução, como aquelas baseadas em desfocagem astigmatic da função de propagação do ponto (PSF) de 12 ou bi-plano de detecção 24, que são mais amplamente disponível. Notamos que um ingrediente necessário para a microscopia de super resolução baseada em localização única molécula, em geral, é o fluoróforo foto induzida 25, que permite que os três requisitos essenciais para a microscopia de super-resolução com base em localização única molécula de ser cumprido: i) alta single-molécula brilho e contraste em relação a sinais de fundo; ii) distribuição esparsa de moléculas individuais em um determinado quadro de imagem; e iii) densidade espacial de alta rotulagem suficiente para capturar o perfil da estrutura de base (também conhecida como Nyquist-Shacritério de amostragem nArquivos não) 26. Assim, para obter resultados satisfatórios, a ênfase deve ser colocada igualmente em ambos a preparação adequada das amostras para otimizar fluoróforo photoswitching e preservar a ultra-estrutura subjacente, bem como sobre os aspectos de instrumentação e aquisição dos experimentos.

<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)

microscopia de fluorescência permite a visualização direta das biomoléculas específicas dentro das células. No entanto, para a microscopia de fluorescência convencional, a resolução espacial é limitada por difracção para ~ 200 nm, dentro do plano de imagem e&gt; 500 nm ao longo do eixo óptico. Como resultado, a microscopia de fluorescência tem sido severamente limitada na observação das características ultra-estruturais dentro de células. O recente desenvolvimento de métodos de microscopia de super-resolução tem superar essa limitação. Em particular, o advento dos fluoróforos foto induzida permite microscopia de super-resolução à base de localização, que fornece poder de resolução que se aproxima a escala de comprimento molecular. Aqui, descrevemos a aplicação de um método de microscopia de super resolução tridimensional baseado em microscopia de localização única molécula e interferometria multifásico, chamada interferometria fotoativado Localization Microscopy (iPALM). Este método oferece resolução quase isotrópica naordem de 20 nm em todas as três dimensões. Os protocolos para visualizar o citoesqueleto de actina filamentosa, incluindo a preparação da amostra e o funcionamento do instrumento iPALM, são descritos aqui. Estes protocolos são também prontamente adaptável e instrutiva para o estudo de outras características ultra-estruturais nas células.

requisitos críticos para iPALM são o alinhamento, registro e calibração dos sistemas ópticos. Estes são necessários para garantir a interferência adequada dentro do 3-way divisor de feixe requisito para extração coordenada z. Para permitir a monitorização contínua, fontes pontuais constantes de fluorescência são necessárias. Isto pode ser conseguido usando fluorescente Au ou nanopartículas bimetálicas 23 cuja fotoluminescência surgir de ressonância de plasm...

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

Nós apresentamos um protocolo para a aplicação de interferometria fotoativado Localization Microscopy (iPALM), um método de microscopia de super-resolução 3-dimensional única molécula localização, à imagem do citoesqueleto de actina em células de mamíferos aderentes. Esta abordagem permite a visualização à base de luz de características estruturais em nanoescala que, caso contrário permanecem sem solução por microscopia óptica limitado por difração convencional.

Tridimensional Super Resolution Microscopia de filamentos de actina F por Interferometric fotoativado Localization Microscopy (iPALM)

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

Imagens de células vivas de F-actina Dynamics via Microscopia fluorescente Speckle (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 ...

Biologia

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

Nano-Fem: localização de proteínas usando a foto-ativada Microscopia Localização e Microscopia Eletrônica

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

As amostras de teste para otimizar STORM Super-Resolution Microscopy

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

Preparação da amostra para microscopia da força atômica de virion único e imagem de fluorescência de super-resolução

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

Super-resolution Imaging do Z Anel Cytokinetic em bactérias vivas Usando o Fast-estruturados 3D Iluminação Microscopy (F3D-SIM)

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

In vivo Clonal Rastreamento de Stem hematopoiéticas e células progenitoras Marcado por cinco proteínas fluorescentes usando confocal e microscopia multifotônica

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

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

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

Análise de partícula única open-source para Microscopia Super-resolução com VirusMapper

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

Aplicação da microscopia de velocidade de alta velocidade super-resolução no cílio primário ao vivo

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

Preparação de amostras biológicas por congelamento de alta pressão, realce de contraste assistida por microondas e resina mínima incorporação para a imagem latente de Volume

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