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.

BiFC tem sido uma técnica vulgarmente utilizada para a detecção e a visualização de IPP em células, enquanto palma é uma técnica de microscopia recente única molécula SuperResolution que permite imagens em nanoescala de amostras biológicas intactas. A combinação de BiFC com PALM alcançado imagem seletiva de PPIs dentro de uma célula com resolução espacial nanométrica e sensibilidade única molécula. BiFC-PALM estende a utilidade de ambas as técnicas, e como demonstrado neste trabalho, mostra uma gran...

Interações proteína-proteína (IBP) são fundamentais para a biologia 1 e sejam estritamente regulado através de mecanismos de espaço-temporais em muitas escalas de tempo e comprimento. Estudos sobre a sinalização celular na membrana celular, por exemplo, revelaram, compartimentos espaciais em nanoescala dinâmicos que facilitam PPIs específicas e processos celulares 2. Assim, a capacidade de sondar os IPP em sistemas biológicos com resoluções suficientes espaciais e temporais, alta especificidade e alta sensibilidade é fundamental para alcançar um entendimento mecanicista da biologia. Bimolecular fluorescência complementação (BiFC) é uma das poucas ferramentas existentes para visualizar PPIs em uma cela com resolução subcelular e compatibilidade de células viver 3-5. A técnica é relativamente simples e envolve a divisão de uma proteína de fluorescência em dois fragmentos não fluorescentes; quando geneticamente marcado para duas proteínas interagem etrazida para a proximidade, os fragmentos podem reconstituir, para formar uma proteína fluorescente completa, obtendo-se o sinal fluorescente. Quando projetado corretamente, as sondas BiFC não deve reconstituir espontaneamente na ausência de PPIs. Como tal, o sinal de fluorescência num ensaio de BiFC só surgem, na presença de IPP, o que permite a visualização directa dos IPP com alta especificidade. Os benefícios adicionais da utilização de fluorescência como a leitura são de alta sensibilidade, resolução subcelular, e compatibilidade com alto rendimento e alta ensaios de rastreio de conteúdo, entre outros. Para estes benefícios, foram desenvolvidos um número de sondas diferentes com base em BiFC proteínas fluorescentes pai. Tal como em todas as outras técnicas de detecção baseadas em microscopia óptica convencional, no entanto, a resolução espacial de BiFC é limitada a ~ 250 nm pela difracção da luz. Isto o torna um desafio para estudar a regulação dos IPP em nanoescala, que, como referi anteriormente e exemplificados por jangadas lipídicas 6 and Ras nanoclusters 7, é uma escala de comprimento crítico para a compreensão de muitos processos celulares, tais como sinalização. BiFC foi combinada com microscopia localização fotoactivado (PALM) 8,9 para superar este limite em resolução espacial para a imagem latente PPIs 10. PALM é uma recente técnica de microscopia SuperResolution que contorna o limite de difração em imagens de fluorescência através da ativação estocástica e localização subdiffractive de moléculas fluorescentes individuais. Em cada ciclo de activação, uma molécula fluorescente emite de algumas centenas a alguns milhares de fotões e dá origem a uma única molécula de imagem no detector. Quando a imagem é limitado por difracção (~ 250 nm de largura), a sua centróide pode ser determinada com muito maior precisão, tipicamente na ordem de 10-50 nm, dependendo do número de fotões detectados. Através da activação e localização de cada molécula fluorescente na amostra, imagem de alta resolução pode ser reconstruído. Performed em células vivas, monitoramento molécula única (smt-) PALM novas autorizações de aquisição de milhares de trajetórias de difusão proteína a partir de uma única célula 11. Importante, PALM utiliza sondas fluorescentes especializados, tais como proteínas fluorescentes fotoactivï¿½eis (PA-FPS) para conseguir a ativação estocástica. Uma vez que tanto BiFC e proteínas fluorescentes uso PALM, eles foram combinados, dividindo PAmCherry1, um comumente usado PA-FP para PALM, em dois fragmentos entre os aminoácidos 159 e 160. O sistema baseado em PAmCherry1 BiFC dividido mostra baixo sinal de fundo a partir de reconstituição espontânea dos dois fragmentos. Quando geneticamente marcado para um par de proteínas que interagem, os dois fragmentos (RN = resíduos 1-159; RC = MET + resíduos 160-236) reconstituído para formar de forma eficiente proteínas PAmCherry1 completa mesmo a 37 ° C e sem incubação a temperaturas mais baixas, o que Não é o caso de outros pares BiFC 12, tais como o pai mCherry 13. Furthermore, a proteína PAmCherry1 reconstituído manteve as propriedades fotofísicas da PAmCherry1 pai, como a alta taxa de contraste, o rendimento médio de fótons, e fotoativação rápido, entre outros, que são críticos para precisas de localização única molécula e de alta resolução de imagem PALM. Neste protocolo, o uso de BiFC-PALM para imagiologia interações Ras-Raf em células U2OS usando divisão PAmCherry1 (Figura 1A) é descrito. O primeiro passo consiste em conceber as construções para expressar proteínas de fusão entre fragmentos PAmCherry1 (isto é, RN e RC) e as proteínas de interesse. Em teoria, para cada par de proteínas candidatas (A e B), existem oito pares de proteínas de fusão a ser testada: RN-A / RC-B; RN-A / B-Rc; RC-A / RN-B; RC-A / B-RN; A-RN / RC-B; A-RN / B-RC; A-RC / RN-B; e A-RC / B-RN. Este processo muitas vezes pode ser simplificada, tendo em conta as propriedades estruturais ou bioquímicas das proteínas candidatas. No caso de Ras, a proteína émodificada após tradução na caixa CAAX C-terminal (C = Cys; A = alifático; X = qualquer), após o que o motivo AAX é clivada. Assim, RN ou RC só pode ser fundido ao N-terminal de Ras; Isto reduz o número de pares de proteínas de fusão para quatro (Figura 1B). Para Raf, domínio de ligação a Ras (RBD; resíduos 51-131) é usado e pode ser marcado em cada extremidade. Esses quatro configurações de fusão foram gerados: RN-Kras / RC-Raf RBD; RN-Kras / Raf RBD-RC; RC-Kras / RN-Raf RBD; e RC-Kras / Raf RBD-RN. Além disso, o ligante entre os fragmentos e as proteínas de interesse terão de ser consideradas. Um ligante flexível de cerca de dez aminoácidos é frequentemente utilizado como proporciona liberdade suficiente para complementação de ocorrer. Um tal ligante é (GGGGS) x2, embora existam muitos outros que foram aplicadas com êxito, incluindo seqüências aleatórias geradas a partir de um local de clonagem múltipla (MCS) 14. O comprimento dos elementos de ligação podem precisar de ser optimizada depending do tamanho das proteínas de interesse e que interagem quando as suas orientações. Os fragmentos PAmCherry1 estão contidos em um pequeno backbone clonagem com flanqueando MCSs (ver Lista de Materiais). Os genes de interesse pode ser inserido através dos locais de restrição ou com um método independente da ligadura. Após a clonagem e sequência de verificação, a cassete de expressão é transferido para um vector de expressão, utilizando uma reacção de recombinase, um processo de clonagem com alta fidelidade e alta eficiência. Em seguida, as construções de expressão resultantes são transfectados para a linha de células alvo ou, se uma linha celular estável é desejada, empacotado em lentivírus para infectar a linha de células alvo. Transfecção transiente permite a validação rápida das configurações BiFC, mas os potenciais problemas devem ser observados. Transfecção utilizando produtos químicos frequentemente sublinha as células, resultando em alta autofluorescência; enquanto que a utilização de um total de fluorescência de reflexão interna (TIRF) microscoaa pode mitigar este sinal de fundo, limitando o volume iluminação, TIRF ideal quando os PPIs ocorrem na membrana da célula. Além disso, transfecções transientes muitas vezes levar a elevados níveis de expressão da proteína, muito superiores às das proteínas endógenas, o que pode causar artefactos na detecção do IPP. Assim, recomenda-se que as linhas celulares estáveis ​​após ser estabelecida uma configuração apropriada BiFC foi determinada através de ensaios iniciais. Linhas de células estáveis ​​também têm o potencial para a expressão sintonizável através da indução de doxiciclina 15. Após a infecção, as células são seleccionadas com antibióticos, tipicamente puromicina e neomicina, um para cada constructo. Os passos subsequentes de preparação das amostras, a aquisição de imagem e análise de dados será descrito em detalhe nos protocolos. Com esta abordagem, a formação de clusters em nanoescala em imagens BiFC-palma da Ras-Raf RBD são rotineiramente observados (Figura 2A &lt;/ strong&gt;). Consistentemente, trajetórias smt-palma da Ras-Raf RBD apresentam uma distribuição heterogênea nos estados de difusão (Figura 2B-D). Estes resultados sugerem que os complexos de Ras-Raf existir em vários estados na membrana celular, presumivelmente como monómeros e aglomerados, com potenciais implicações biológicas. Este trabalho demonstra o poder da BiFC-PALM em imagem seletiva de PPIs em células com resolução espacial nanométrica e sensibilidade única molécula, o que seria difícil de obter com BiFC convencional, fluorescência co-localização, ou a transferência de energia de ressonância de fluorescência (FRET). Ao projetar experimentos BiFC e interpretação dos resultados, é importante ter em mente que o processo é irreversível BiFC na maioria dos casos, incluindo PAmCherry1 divisão. Uma vez que os dois fragmentos se combinar e formar uma proteína PAmCherry1 completo, a ligação entre os dois fragmentos, e, assim, torna-se permanente do PPI. Isto limita a utilização de BiFC e BiFC-PALM para monitorar a dinâmica dos PPIs (ou seja, cinética de ligação e não a dinâmica de difusão do complexo de proteína, uma vez que é formado) e às vezes pode até mesmo levar à mislocalization dos complexos PPI. Um fator adicional a considerar ao projetar experimentos BiFC-palma é o atraso na maturação cromóforo que é inerente a proteínas fluorescentes. Uma vez que duas proteínas interagem e os fragmentos de PF vêm juntos, eles tipicamente redobre na ordem de segundos (tempo de meia 60 seg para EYFP 3). No entanto, a maturação e posterior cromóforo sinal fluorescente desenvolver na ordem de minutos. Embora a fluorescência pode ser detectável dentro de 10 min após a reconstituição de split-Vénus, uma variante rápido dobrar-YFP, a meia-hora para a maturação, em geral, é muitas vezes cerca de 60 min 16. Split-PAmCherry1 foi observada a ter taxas similares. Assim, BiFC e BiFC-PALM são escolhas atualmente pobres para monitorar cinética PPI em tempo real; outro methods, tais como a dimerização de FRET e uma recentemente desenvolvido proteína fluorescente dependente 17, pode ser mais adequado para este fim.

<table>
	<tbody>
		<tr>
			<td>TIRF Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60X oil immersion TIRF objective with 1.49 NA</td>
			<td>Nikon</td>
			<td></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>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm dichroic mirror</td>
			<td>Semrock</td>
			<td>&#160;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 CO2 controlled stage</td>
			<td></td>
			<td></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&#39;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&#160; 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.

O exemplo BiFC-PALM mostrado é KRAS mutante G12D interagindo com os Ras domínio de ligação (RBD) de Craf (Figura 1A). Como discutido, a RN ou fragmentos RC não foram etiquetadas para o C-terminal de Ras porque iria perturbar a localização da membrana e, portanto, a actividade biológica de Ras. Isto reduziu as combinações possíveis de oito para quatro (Figura 1B). Cada um destes quatro combinações foi introduzido em células U2OS utilizando infecção lentiviral. As células...

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.

Fotoactivado Localization Microscopy com Bimolecular Fluorescência de Complementação (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)

9.8K visualizações.  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. 

Vídeo: 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.

Visualização de Cortex organização e dinâmica em microorganismos, utilizando microscopia de fluorescência Total Internal Reflection

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

Bioengenharia

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).

Nanotopology de adesão celular em cima de ângulo variável Microscopia de Fluorescência reflexão interna total (VA-TIRFM)

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

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.

Criando Gradientes adesivas e solúvel para a migração celular Imaging com Microscopia de Fluorescência

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

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

De alta resolução espacial e temporal Análise de Receptor Dynamics por uma única molécula de microscopia de fluorescência

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

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.

3D Orbital Rastreamento em um microscópio de dois fotões Modificação: Uma Aplicação para o rastreamento de vesículas intracelulares

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluorescência-têmpera de um Fluoróforo encapsulado em lipossomas Near-infrared como ferramenta para<em&gt; In Vivo</em&gt; Imageamento óptico

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

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.

Bicamadas única molécula de microscopia de fluorescência em Planar Apoiado

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 ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Microscopia de fluorescência de luz-folha para o estudo do coração murino

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Realização de vários modos de imagem com um microscópio de fluorescência

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...