The authors have nothing to disclose.

1. Nanostructure Fabrication <ol><li> Escolha de 25 mm de diâmetro, com lamelas de vidro de uma espessura aproximada de 170 fim (N ° 1.5) como substratos para nanofabrication. </li><li> Mergulhar as lamelas em solução de piranha (razão 3: 1 de ácido sulfúrico e peróxido de hidrogénio) durante pelo menos 6 horas. Lavar a piranha lamela embebido com quantidade abundante de ultrapura 18,2 mohms água destilada deionizada (DDW). CUIDADO: Piranha ácido reage violentamente com materiais orgânicos e devem ser manuseados com extremo cuidado. </li><li> Depósito de 10 nm de crómio película fina sobre as lamelas por evaporação e-feixe para evitar os efeitos de carga durante a modelação e de imagem de nanoestruturas. </li><li> Girar a primeira camada da camada dupla de resistir constituído por metacrilato de etil lactato de metilo copolímero (MMA_EL6) a 2.000 rpm durante 45 segundos e, em seguida, cozer a 150 ° C. Girar a segunda camada de poli-metacrilato de metilo (950PMMA_A2) a 3.000 rpm durante 45 segundos, em seguida, cozer a 180 ° C. </li><li> Tamborilarn bicamada resistir usando a litografia por feixe de elétrons (EBL) a 25 kV com uma dose de 300 uC / cm 2 de área. Desenvolver em álcool isopropílico (IPA) / metil-isobutil-cetona (MIBK): 2/1 e enxaguar em IPA. </li><li> Depósito Ti (5 milhas náuticas) / Au (80 nm) filme sobre o substrato usando um evaporador e-feixe. </li><li> Após a deposição de ouro, levante a bicamada copolímero resistir por imersão do substrato em acetona, durante 4 h. </li><li> Inspecione o suporte usando o microscópio eletrônico de varredura (MEV) para confirmar a forma e tamanho nanoestrutura, remover o cromo restante do substrato via etch molhado usando CR-7 etchant durante 60 segundos à temperatura ambiente e depois enxaguar em DDW. </li><li> Projete um espaçamento de centro a centro matriz de 33 mm de deixar espaço para imagens de células entre matrizes. Padrão as nanoestruturas em 20 x 20 padrão para cada matriz com um arremesso de 300 nm usando um escritor e-feixe. Cada chip contém 300 arrays com uma dimensão típica nanoestrutura de altura de 80 ± 2,5 nm e 70 nm ± 2,5 Diameter. </li><li> Inspecione um subconjunto de arrays usando o microscópio de força atômica (AFM) para a verificação do tamanho e uniformidade. </li><li> Anexar um anel de suporte, tipicamente, de silício, para a parte de trás da lamela utilizando um epoxi de cura por UV. </li></ol> 2. Chip Limpeza e Aplicação de auto-montados Monolayer <ol><li> Para a limpeza e regeneração das batatas fritas, as cinzas de plasma a uma potência de 40 W em uma mistura de 300 mTorr de 5% de hidrogénio, 95% de árgon, durante 45 segundos após a limpeza da câmara durante 5 min sob as mesmas condições. </li><li> Funcionalizar o nanoestruturas de ouro imediatamente após a calcinação por plasma pela imersão do chip em uma solução etanólica tiol de dois componentes consistindo de uma proporção 3: 1 de SH-(CH2) 8 EG 3 -OH (SPO) e um componente ou com uma amina carboxilo ou grupo funcional, ou seja, SH- (CH2) 11 EG 3 -NH2 (SPN) ou SH- (CH2) 3-COOH 11 EG (SPC). </li><li> Deixe o chip de in a solução tiol O / N para formar uma monocamada auto-montada (SAM). </li><li> Lavar o chip com etanol e seco com azoto gasoso. </li><li> Se necessário, armazenar o chip funcionalizado por até 2 semanas a 4 ° C. </li><li> Quando pronto para uso reagir o SPN ou componente SPC com o ligando utilizando uma química de acordo com o ligando de escolha (ver abaixo). Nota: Os chips podem ser regenerado e re-funcionalizado dezenas de vezes. Um dado chip pode ser usado por períodos que variam de 6 meses a mais de um ano. O espectro medido em uma determinada matriz são reproduzidas de forma confiável após repetidas regenerações por incineração plasma, seguido de biofunctionalization. 29 </li></ol> 3. A funcionalização de superfície e Caracterização Cinética Nota: usar o chip funcionalizado no instrumento comercial SPR para caracterizar as constantes cinéticas entre o ligando e o analito, bem como para o estudo da resistência de SAM para não específica binding. Existe uma vasta gama de taxas de fluxo e desenhos de microfluidos que permitem para a funcionalização da superfície eficiente. Como temos um SPR disponível comercialmente padronizamos em torno de suas taxas de fluxo recomendados. Notamos que estas taxas de fluxo são típicos de todos os instrumentos da SPR e por isso não são restritivas. O instrumento SPR não é uma necessidade uma vez que todos funcionalização pode ser feito diretamente no chip LSPR, mas fez reduzir a nossa carga de trabalho porque é um instrumento multiplexado ao passo que a nossa configuração microfluídico LSPR não é. <ol><li> Funcionalizar um chip nua ouro comercial com a SAM, conforme descrito na Seção 2. </li><li> Se for utilizada uma SAM com base SPC, activar o grupo carboxilo, com uma mistura 1: 1 de 133 mM de 1-etil-3- (3-dimetilaminopropil) carbodiimida (EDC) 33 mM e N-hidroxissuccinimida de (NHS) em DDW durante 10 min. </li><li> Conjugar o grupo carboxilo activado com o anticorpo / ligando de interesse para 300 seg usando um caudal de 30 ul / min. Prepare os ligandos empH 6 tampão fosfato, tipicamente, mas este pode variar dependendo da molécula. </li><li> Após a conjugação ligando, fluxo de 0,1 M de etanolamina em solução salina tamponada com fosfato (PBS) como um desactivador de passo para 300 seg a uma taxa de 30 ul / min. Etanolamina ajuda a minimizar a ligação não específica. </li><li> Introduzir o analito de interesse a um caudal de 100 ul / min usando uma gama de concentrações e calcular as constantes de velocidade cinéticas, utilizando um software de análise cinética. </li><li> Se a ligação não específica é problemático, aumentar a proporção de SPO SPC ou SPN. </li></ol> Definições 4. LSPR Gerais <ol><li> Microscópio configurações: <ol><li> Use uma lâmpada de 100 W halogênio para Koehler iluminar a amostra. Usar um filtro de passagem longa (tipicamente 593 nm de corte) no caminho da luz para eliminar comprimentos de onda que não contribuem para o deslocamento de ressonância (Figura 2). </li><li> Para a coleta de dados LSPRi, use um microscópio invertido com um immersio óleo de 40Xobjectivo N (1,4 NA) e uma câmara CCD arrefecida termoelectricamente de 16 bits. </li><li> Coloque um divisor de feixe para a porta de saída do microscópio para obter espectros de aparência e simultaneamente. </li><li> Regule a temperatura controlada palco microscópio para 37 ° C e equilibrar durante 4 h. </li><li> Incorporar uma montagem de incubação adicional sobre o microscópio para regular a concentração de humidade e de CO2 a 5% e 95%, respectivamente. </li></ol></li><li> Chip preparação e montagem: <ol><li> Funcionalizar o chip LSPR como descrito na secção 2, com as relações de SAM de dois componentes óptimos determinados a partir das experiências de SPR. </li><li> Coloque o chip dentro de um titular microfluídico feito sob encomenda da seguinte forma. Coloque o chip em uma parte inferior de alumínio. Sanduíche do chip entre esta parte inferior e uma junta de silicone e um pedaço de plástico transparente superior. Use 4 parafusos para fixar o conjunto. </li><li> Para uma aplicação tiol típico baseado no SPC, queda revestimento 300 ï¿½ de 1: 1 mixtura de 133 mM de EDC e 33 mM de NHS em DDW para activar os grupos carboxilo do componente tiol SPC. </li><li> Esperar durante 10 min e enxaguar manualmente a superfície com PBS 10 mM. </li><li> Conjugar o grupo carboxilo activado com o ligando (tipicamente um anticorpo ou fragmento de anticorpo) de interesse a gota revestimento 300 ul de solução de ligando. </li><li> Aguarde 30 minutos e enxágüe manualmente com PBS 10 mM. </li><li> Largar coat 300 mL de etanolamina 0,1 M em PBS no chip para minimizar a ligação não específica. Aguarde 10 min. </li><li> Lava-se a etanolamina com PBS contendo 0,005% de Tween 20 (PBS-T20). </li><li> Coloque um pedaço de quartzo acima do chip para reduzir as flutuações nos dados relacionados a um menisco mudança. </li><li> Mantenha o chip molhado com tampão PBS-T20, enquanto a montagem em microscópio. </li><li> Coloque o conjunto de chips LSPR firmemente no suporte de amostras a fase aquecida e prenda o tubo de microfluidos. </li><li> Conecte a tubulação de microfluídica à montagem e de fluxo de buffer (ou meio livre de soro para estudos de células) até um estado de equilíbrio é atingido. </li><li> Permitir que o conjunto de microscópio e a equilibrar durante pelo menos 2 horas. </li><li> Alinhar o chip utilizando joystick de modo que a matriz central é alinhado com a fibra óptica para a espectroscopia. Os dados espectroscópicos são feitas usando um espectrómetro e software de análise espectral. </li><li> Mantenha matrizes em foco durante todo o experimento usando autofocus software, Zeiss Definite Focus ou dispositivo equivalente autofocus. </li></ol></li></ol> 5. LSPR imagem de secreções-c-myc Anti de Células de hibridoma 9E10 Nota: A linha celular de hibridoma utilizado para este estudo expressam o anticorpo anti-c-myc de forma constitutiva e, portanto, não requerem um gatilho química <ol><li> Funcionalizar o nanoestruturas com péptido c-myc. Isto tem um valor de K D de 1,77 nM para os anticorpos anti-c-myc 9E10 secretados pelo clone de hibridoma de células. </li><li> Cultura as células de hibridoma em complete meio de crescimento com 10% de soro fetal de bovino (FBS) e 1% de antibióticos num balão T75 a 37 ° C sob 5% de CO 2. Manter uma densidade celular de 4 x 10 5 células / ml. </li><li> Para os estudos da secreção de células, as células pellet a partir do balão T75 por centrifugação, lava-se duas vezes com RPMI-1640 livre de soro meios (SFM) para remover os anticorpos segregados e ajustar a densidade celular a 4 x 10 6 células / ml. </li><li> Colheita das células e teste de viabilidade antes de introduzir-los para os chips LSPR. </li><li> Introduzir 50 ul da solução de células manualmente sobre os chips LSPR com uma micropipeta. Depois de alguns minutos, de 25 a 50 células aderem à superfície das batatas fritas LSPR. </li><li> Lave as células restantes em solução com fresco SFM usando o sistema de perfusão microfluídico. </li><li> Selecione as matrizes LSPR para imagens que estejam próximas, num raio de 10 mm, mas não se sobrepõem com as células. </li><li> Para assegurar que o sinal é específico para o ANTIB anti-c-myc segregadaodies introduzir o meio de cultura de células com e sem os anticorpos presentes, bem como com os anticorpos, mas com os seus locais de ligação bloqueada pela presença de uma concentração saturante de c-myc péptido na solução. </li><li> Calibrar os sensores no final de cada ensaio com uma solução satura de anticorpos anti-c-myc (250 nm). Isto ajuda na normalização da resposta dos sensores e determinam ocupação fraccionada com base no perfil biofunctionalization de cada prazo. </li><li> Corrigir a deriva na direção X e Y usando software de alinhamento de imagem. </li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+single-cell+IgG+secretion+frequency+and+rates+in+human+memory+B+cells+by+CpG+DNA,+CD40L,+IL-21,+and+cell+division.">Modulation of single-cell IgG secretion frequency and rates in human memory B cells by CpG DNA, CD40L, IL-21, and cell division.</a> Journal of Immunology. 183, 3177-3187 (2009).</li><li>Bramswig, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+Carcinoembryonic+Antigen+Activates+Endothelial+Cells+and+Tumor+Angiogenesis.">Soluble Carcinoembryonic Antigen Activates Endothelial Cells and Tumor Angiogenesis.</a> Cancer Research. 73, 6584-6596 (2013).</li></ol>

We thank George Anderson for helpful comments and discussions. This work was supported by the Naval Research Laboratory&#8217;s Institute for Nanoscience and the National Research Council Research Associateship Award.

The LSPR imaging technique described in this work has numerous advantages over more traditional methodologies for detecting cell secretions. First, the time resolution of our technique is on the order of seconds whereas the commercial alternative, an immunosandwhich assay known as EliSpot, has a typical time resolution of 2 to 3 days.7,32 As a result we were able to resolve sudden changes in the rate of protein secretion, such as that shown in Figure 6. Second, having arrays distributed over the chip allows for the secreted signal to be tracked in space and time which enables more rigorous comparisons to diffusion-based models of cell secretion. In addition, arrays like the control array shown in Figure 6 can be used to subtract out global changes in the image that typically arise from instrumental factors such as focus drift. Third, our technique requires no modification of the cells. If desired, the experiment can incorporate commonly used tags such as fluorescent proteins, but if there is concern that such tags may negatively affect cell viability or homeostasis the label-free nature of our approach does not require them. Fourth, using the spectroscopic data we have demonstrated that quantitative information regarding the fractional occupancy of surface bound ligands can be calculated.
There are numerous alternative methods to EBL for fabricating metallic nanoparticles. However, we have found that the EBL provides considerable flexibility for optimizing nanostructure and array dimensions to best suit the optics and the cells under investigation. Also critical is the fact that the chips can be readily regenerated by plasma ashing. In this way, a typical chip can be used dozens of times. Biofunctionalization details must be modified for the specific application. The protocol presented here conjugated the surface with relatively small c-myc peptide ligands. Larger ligands such as whole antibodies typically require more spacing and thus a higher SPO to SPN/SPC ratio. Regardless, a well formed SAM layer is essential for preventing non-specific binding in live-cell experiments. In general, larger molecular weight analytes are more readily detected by LSPR. Thus, in its single-cell manifestation, this technique may not be appropriate for detecting the secretion of small proteins, such as cytokines. 
The current setup has been used for studying individual non-adherent cells. There are significant number of secreted signaling proteins and vesicles to which the results reported in this work are directly applicable. For example carcinoembryonic antigen (CEA) which for decades now has been a diagnostic marker for cancer. Colon cancer cells are known to secrete CEA at the rates of thousands of molecules/cell/hr and the molecular weight is 180 kDa which exceeds that of IgG antibodies. CEA is believed to be involved in autocrine and paracrine signaling pathways but the spatio-temporal nature of these secretions have never been measured. Our technique can directly address these signaling questions. An extension of this work will be to measure the spatio-temporal nature of CEA secretion from single cells.33 Future work will also focus on integrating LSPRi with two and three dimensional cell cultures of adherent cells. By incorporating multiplexed arrays capable of detecting a number of secreted proteins in parallel, this technique has the potential to open a new window into cell secretions and how they influence neighboring cells.

Comunicação inter-celular é crucial para a regulação de muitas atividades fisiológicas dentro e fora da célula. Uma variedade de proteínas e vesículas pode ser secretado que posteriormente desencadear processos celulares complexos, tais como a diferenciação, a cicatrização de feridas, a resposta imune, migração, proliferação e. 1-5 O mau funcionamento de vias de sinalização inter-celulares têm sido implicados em numerosas doenças, incluindo o cancro, aterosclerose , e diabetes, para citar alguns. O ensaio de secreção celular óptima deve ser capaz de espacialmente e temporalmente o mapeamento da proteína segregada de interesse sem interromper as vias de sinalização relevantes. Desta forma relações causais entre os perfis de concentração e a resposta das células receptoras pode ser inferida. Infelizmente, muitas das técnicas baseadas em fluorescência mais geralmente utilizados não satisfazem estes critérios. Proteínas de fusão fluorescentes podem ser utilizadas para etiquetar o analito Within da célula, mas podem interromper a via secretora, ou, se segregado, resulta em uma luz difusa no exterior da célula que é difícil de quantificar. Ensaios baseados em immunosandwich fluorescentes são as técnicas mais vulgarmente utilizadas para a detecção de secreções celulares, mas tipicamente requerem o isolamento de células individuais. 6-11 Além disso, a introdução do anticorpo de detecção, tipicamente interrompe ou termina a experiência e o tamanho das etiquetas de anticorpos, 150 kDa para IgG, é um impedimento para sinalização a jusante. Devido a estas barreiras é preferível que uma técnica livre rótulo ser utilizado para secreção de proteínas e entre tecnologias de imagem livre de etiquetas existentes, a superfície de ressonância de plasmão (SPR) e sensores localizados de ressonância de plasmon de superfície (LSPR) são excelentes candidatos. 12-17 Estes sensores têm sido amplamente utilizados para estudos de ligação de analisável, exossomas de proteínas e outros biomarcadores. 18-24 Em caso de LSPR, o nanostr plasmonicuctures pode ser modelado lithographically em lamelas de vidro e animado usando luz visível através de configurações padrão de microscopia de campo amplo. Devido à sua presença em nanoescala, a maioria do substrato de vidro está disponível para imagens técnicas comuns, tais como de campo brilhante e microscopia de fluorescência tornando assim estas sondas adequados para integração com microscopia de células vivas. 25-28 Demonstrámos a medição em tempo real de secreção de anticorpos a partir de células de hibridoma utilizando nanoestruturas plasmonic ouro funcionalizado com resoluções espaciais e temporais de 225 mseg e 10 uM, respectivamente. A configuração básica do chip é ilustrado na Figura 1. 28 O percurso de luz de saída do microscópio é dividido entre uma câmara CCD usada para aparência e um espectrómetro de fibra-opticamente acoplada para a determinação quantitativa de ocupação fraccionada de uma dada matriz de nanoestruturas (Figura 2 ). O PROTOCol apresentada neste documento descreve a concepção experimental para a medição em tempo real das secreções celulares individuais enquanto monitorando simultaneamente a resposta das células utilizando a microscopia de campo brilhante padrão. A abordagem multidisciplinar inclui a fabricação de nanoestruturas, funcionalização das nanoestruturas para a ligação de analitos de elevada afinidade, a optimização da superfície tanto para minimizar a ligação não específica e caracterização das constantes cinéticas usando uma superfície comercial Plasmon Resonance (SPR) instrumento, a integração de linhas celulares sobre o substrato, e a análise de imagens e de dados espectrais. Prevemos que esta técnica seja uma tecnologia capacitadora para o mapeamento espaço-temporal das secreções celulares e suas relações causais com células receptoras.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>25mm diameter glass coverslips</td>
			<td>Bioscience Tools&#160;</td>
			<td>CSHP-No1.5-25</td>
			<td>&#160; 170&#177;5 &#181;m is optimal</td>
		</tr>
		<tr>
			<td>Poly-methyl methacrylate</td>
			<td>Microchem</td>
			<td>PMMA 950 A4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl lactate methyl metacrylate</td>
			<td>Microchem</td>
			<td>MMA EL6</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron beam evaporator</td>
			<td>Temescal</td>
			<td>FC-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron beam lithography</td>
			<td>Raith</td>
			<td>Series 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>320110</td>
			<td></td>
		</tr>
		<tr>
			<td>CR-7 chromium etchant</td>
			<td>Cyantek</td>
			<td>CR-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Zeiss</td>
			<td>Ultra 55</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic force microscope</td>
			<td>Veeco</td>
			<td>Nanoscope III</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma ashing system</td>
			<td>Technics</td>
			<td>Series 85 RIE</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-(CH2)8-EG3-OH (SPO)&#160;</td>
			<td>Prochimia</td>
			<td>TH 001-m8.n3-0.2</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-(CH2)11-EG3-COOH (SPC)</td>
			<td>Prochimia</td>
			<td>TH 003m11n3-0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-(CH2)11-EG3-NH2 (SPN)</td>
			<td>Prochimia</td>
			<td>TH 002-m11.n3-0.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Surface plasmon resonance system</td>
			<td>Biorad</td>
			<td>XPR36</td>
			<td></td>
		</tr>
		<tr>
			<td>Bare gold chip</td>
			<td>Biorad</td>
			<td>GLC chip</td>
			<td>Plasma ashed to remove the monolayer</td>
		</tr>
		<tr>
			<td>1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide</td>
			<td>Thermo</td>
			<td>22980</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Thermo</td>
			<td>24510</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentylamine-Biotin&#160;&#160;&#160;&#160;&#160;</td>
			<td>Thermo</td>
			<td>21345</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>E9508</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutraavidin</td>
			<td>Thermo</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermo</td>
			<td>28374</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer</td>
			<td>Microscope is equipped with 40X oil immersion objective; CO2 and humidity incubation from Pecon GmbH</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Hamamatsu</td>
			<td>Orca R2</td>
			<td>Thermoelectrically cooled (16 bit)</td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics</td>
			<td>QE65Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrasuite</td>
			<td>Ocean Optics</td>
			<td>version1.4</td>
			<td></td>
		</tr>
		<tr>
			<td>c-myc peptide HyNic Tag</td>
			<td>Solulink</td>
			<td>SP-E003</td>
			<td></td>
		</tr>
		<tr>
			<td>monoclonal anti-c-myc antibody</td>
			<td>Sigma-Aldrich</td>
			<td>M4439</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridoma cell line</td>
			<td>ATCC</td>
			<td>CRL-1729</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum free media RPMI 1640&#160;</td>
			<td>Invitrogen</td>
			<td>&#160;11835-030&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine DHPE</td>
			<td>Life Technologies</td>
			<td>L-1392</td>
			<td></td>
		</tr>
	</tbody>
</table>

a label-free technique for the spatio-temporal imaging of single cell secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Em um estudo típico secreção de células vivas existem vários modos de recolha de dados em curso. A Figura 3 mostra uma sobreposição de uma imagem LSPRi, que destaca as matrizes quadradas, e uma imagem iluminada da luz transmitida que destaca a célula no canto inferior esquerdo. Os dados são tipicamente recolhida durante um período de 3 horas seguido pela introdução de uma solução satura da substância a analisar para o cálculo de normalização descrito abaixo. Imagens de fluorescência também pode ser integrado na rotina de recolha de dados pela comutação automática de um cubo de filtro. Na Figura 4 uma célula corada com o corante fluorescente rodamina DHPE membrana apresenta extensões lamellipodia-like (setas). Se essas extensões foram a sobrepor-se com as matrizes que iriam dar um falso-positivo para a secreção de proteínas. Tendo vários modos de imagem podem ajudar a identificar essas ocorrências. A Figura 5 mostra dados de espectrometria de antes e réer a introdução de uma solução de saturação (400 nM) de comercialmente adquirido anticorpos anti-c-myc para as matrizes funcionalizados c-myc. Não células estavam presentes nesta experiência. O espectro mostra tanto um desvio para o vermelho e um aumento na intensidade. A diferença entre as áreas sob as curvas de duas resulta em um aumento na intensidade de imagem na matriz de modo LSPRi na câmara CCD. Uma abordagem menos análise de dados quadrados não-linear foi desenvolvido para inferir ocupação fraccionada ligada à superfície de ligandos a partir dos espectros 30,31. No final da experiência, os valores de intensidade saturado (isto é, ocupação fraccionada ≈ 1) são utilizados para calcular uma resposta normalizada para cada matriz com a seguinte fórmula: Onde são a intensidade normalizada no ponto do tempo t, a intensidade inicial no início da experiência, intensidade final saturado, e a intensidade medida da matriz no ponto de tempo t </em&gt;, respectivamente. Os valores normalizados a partir de duas matrizes são mostrados na Figura 6. Uma matriz estava dentro de 10 um da célula sob investigação enquanto a outra, utilizada como controlo, era uma distância de 130 um a partir da célula. O aumento repentino na resposta normalizados da matriz mais próxima à célula, relativamente à resposta plana da matriz de controlo é indicativo de uma explosão localizada de anticorpos segregados. <img alt="figura 1" src="/files/ftp_upload/53120/53120fig1.jpg" /> Figura 1. Sensor de design. Um desenho que representa a geometria de uma experiência típica a secreção de células vivas. A célula (esferóide azul) é depositada sobre o chip para LSPR que contém as matrizes de nanoestruturas bioativa de ouro. Na vista ampliada em, a secreção de células de interesse, neste caso mostrado anticorpos como moléculas em forma de Y, é medida à medida que se ligam à superfíciedas nanoestruturas funcionalizadas. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53120/53120fig2.jpg" /> Figura 2. Configuração óptica. A luz iluminada por uma lâmpada halógena é filtrado primeiro por um filtro de passagem longa (LP). A luz é polarizada linearmente (P1) e ilumina a amostra através de um NA objetiva de 40X / 1.4. A luz dispersa é recolhido por o objectivo e passados ​​através de um polarizador cruzado (P2). Um divisor de feixe 50/50 (BS) é inserido no caminho de luz coletada para espectroscópica simultânea e análise de imagens. Top Right:. Uma imagem microscopia de força atômica de 9 nanoestruturas individuais separados por um campo de 300 nm <a href="https://www.jove.com/files/ftp_upload/53120/53120fig2large.jpg" target="_blank">por favor clique aqui</a>para ver uma versão maior desta figura. <img alt="Figura 3" src="/files/ftp_upload/53120/53120fig3.jpg" /> LSPRi Figura 3. Estudo de células vivas. Um fundiu imagem LSPRi luz transmitida e que mostra uma única célula de hibridoma (inferior esquerdo), circundado por 12 matrizes. Esta é uma imagem de contraste melhorado. Barra de escala é de 10 mm. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53120/53120fig4.jpg" /> Figura 4. Estudo de fluorescência de células vivas. Uma imagem em cores falsas fluorescente de uma única célula de hibridoma DHPE coradas com rodamina, o qual é um corante de membrana. No modo de imagiologia fluorescente as matrizes não são geralmente visíveis, no entanto, um conjunto próxima é observável como aqui abfalta quadrado no canto inferior direito. A célula pode ser visto para ser separada da matriz embora as extensões tentáculo-like (possivelmente filopodia ou lamellipodia) são estende para fora a partir das células (setas). Barra de escala é de 10 mm. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 5" src="/files/ftp_upload/53120/53120fig5.jpg" /> Figura 5. Modalidade espectral. O espectro obtido a partir de uma matriz funcionalizada C-myc antes e após a introdução da solução de 400 nM de anticorpos anti-c-myc. Não células estavam presentes neste estudo. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig5large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 6" src= &quot;/ files / ftp_upload / 53120 / 53120fig6.jpg&quot; /&gt; Figura 6. A secreção única célula. A resposta de uma matriz de 15 um localizado dentro de uma única célula e um localizado 130 um de distância (de controlo). Barra de escala é de 10 mm. <a href="https://www.jove.com/files/ftp_upload/53120/53120fig6large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions at JoVE.com

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

A comunicação inter-celular é crítico para controlar várias actividades fisiológicas dentro e fora da célula. Este documento descreve um protocolo para avaliar a natureza espaço-temporal de secreção das células individuais. Para conseguir isso, uma abordagem multidisciplinar é usado que integra livre-label nanoplasmonic sentindo com imagens de células vivas.

A técnica de livre-Label para a imagem espaço-temporal do Secreções única célula

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

a-label-free-technique-for-spatio-temporal-imaging-single-cell

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Bioengineering

8.4K visualizações.  Naval Research Laboratory.  A comunicação inter-celular é crítico para controlar várias actividades fisiológicas dentro e fora da célula. Este documento descreve um protocolo para avaliar a natureza espaço-temporal de secreção das células individuais. Para conseguir isso, uma abordagem multidisciplinar é usado que integra livre-label nanoplasmonic sentindo com imagens de células vivas. 

Vídeo: A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Espaço-Temporal Manipulação de Atividade GTPase Pequeno em nível subcelular e em escala de tempo de segundos em células vivas

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Bioengenharia

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sem contato, Monitoramento Label livre de células e matriz extracelular utilizando Espectroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

O uso de biossensores ópticos sem etiqueta para detectar modulação dos canais de potássio por G receptores acoplados à proteína

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

A técnica de microfluídica para sondar Deformabilidade celular

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

A Biochip eletroquímica microfluídica à base de Análise de hibridação DNA livre-Label

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Único Detecção de Moléculas livre-Label Usando Microtoroid ópticos Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Eliminação celular seletiva de Cultura 3D Mixed usando uma técnica de infravermelho próximo Photoimmunotherapy

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip: um estudo livre de rótulos de Interações Proteína-fosfoinositide

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain large field-of-view, high-contrast images in thick cells and tissues. Here, we introduce highly inclined swept tile (HIST) microscopy that overcomes this problem. A pair of cylindrical lenses was implemented to generate an elongated excitation beam that was scanned over a large imaging area via a fast galvo mirror. A 4f configuration was used to position optical components. A scientific complementary metal-oxide semiconductor camera detected the fluorescence signal and blocked the out-of-focus background with a dynamic confocal slit synchronized with the beam sweeping. We present a step-by-step instruction on building the HIST microscope with all basic components.

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

Um guia para construir um altamente inclinado varreu o microscópio de telha para a imagem latente de único-molécula campo de visão alargado

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

Implementação de microscopia de reflexão de interferência para imagens sem rótulo e alta velocidade de microtúbulos

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...