A. Ozcan agradece o apoio do Prémio Carreira presidencial antecipada para Cientistas e Engenheiros (PECASE), Army Research Office (ARO) Young Investigator Award, National Science Foundation (NSF) Prémio Carreira, o Office of Naval Research (ONR) Young Investigator Award e National Institutes of Health (NIH) Prêmio Diretor Innovator Nova DP2OD006427 do Escritório do Diretor, National Institutes of Health.

Nesta seção, apresentamos os protocolos experimentais para o nosso telefone celular baseado microscopia de fluorescência de campo largo 13 e opto-fluídico plataforma de citometria de imagem 14. Nós vamos usar partículas fluorescentes e fluorescente etiquetado células brancas do sangue para testar essas plataformas de imagem. A. Preparação do telemóvel Baseado Largo-campo microscópio de fluorescência e Opto-fluídico citômetro de fluxo de imagem O telemóvel baseado microscópio de campo amplo fluorescente ou citômetro de fluxo consiste de duas partes principais: um celular com câmera e um compacto opto-fluídico add-on acessório. 1. A câmera do telefone Enquanto as técnicas apresentadas são aplicáveis ​​a quase qualquer câmera de telefone, escolhemos Sony Erickson Aino como a base para estes dispositivos. Este celular tem um ~ 8 Megapixels RGB sensor CMOS instalado e uma lente interna que tem uma distância focal (f 1) de ~ 4,65 milímetros. </p&gt; 2. Opto-fluídico anexo para Wide-field Microscopia fluorescente O acessório óptico é projetado pela Autodesk e é impresso por uma elite Dimensão 3-D impressora usando ABSplus material termoplástico. Neste processo de impressão, e materiais de suporte de modelo são aquecidas em uma cabeça de extrusão no interior da impressora e são depositados camada por camada sobre uma base de modelação. Quando este passo é completado, o material de suporte pode ser dissolvida, deixando um modelo robusto 3D do protótipo desejado nossa concepção acessório óptico é composto por LEDs (centro de comprimento de onda de 470 nm, ~, Digikey), um filtro de plástico (# NT54-46, Edmund Optics), um tabuleiro de amostras, e uma lente plano-convexa f 2 = 15 mm (# NT45-302, Edmund Optics). Todos os LEDs e filtros de plástico podem ser facilmente alterados com base em espectros de fluoróforos. Os passos para montar esse apego opto-fluídico incluem: <ol><li> Coloque a lente na sua posição de fixação no interior da lente específica titular. </li><li> Coloque o filtro de plástico sobre a bandeja do filtro e deslize-o anexo; ou fita o filtro de plástico na frente da lente da câmera de telefone celular. </li><li> Insira a bandeja de LED para o anexo. </li><li> Coloque as lâminas de vidro de amostra na bandeja de amostra. Deslize a bandeja de amostra no anexo. Enfrentar os LEDs em direção à amostra. </li><li> Clipe da penhora para o telemóvel, de modo que a lente extra está diretamente em contato com a lente da câmera de telefone celular. </li><li> Use a chave no anexo para ligar os LEDs. </li><li> Imagem da amostra de interesse com a unidade de câmera de telefone celular, usando o seu &quot;modo noite&quot;. </li></ol> 3. Opto-fluídico Attachment para Citometria de imagem fluorescente Quando existe a necessidade de filtrar grandes volumes de amostras líquidas para a detecção de eventos raros, o dispositivo de citometria de fluxo optofluidic podia ser preferido. Nós podemos modificar a nossa grande área de design de microscópio de fluorescência e convertê-lo em um citômetro de fluxo, waqui um PDMS canal microfluídico base é usado para continuamente fornecer a amostra de líquido através do volume de imagem. O acessório óptico também é projetado pela Autodesk e impresso pela Dimensão impressora 3-D Elite. Também é composto por LEDs (centro de comprimento de onda de 470 nm, ~, Digikey), um filtro de plástico (# NT54-46, Edmund Optics), um tabuleiro de amostras, e uma lente asférica (f mm = 4,5) (produto # C230TME-A; Thorlab). Os passos para montar esse apego opto-fluídico incluem: <ol><li> Coloque a lente asférica para o anexo. </li><li> Coloque o filtro de plástico sobre a bandeja do filtro e deslize-o anexo; ou fita o filtro de plástico na frente da lente da câmera de celular. </li><li> Deslize o canal microfluídicos para a fixação opto-fluídico mesmo. </li><li> Clipe da penhora para o telemóvel de modo que a lente extra está diretamente em contato com a lente da câmera de telefone celular. </li><li> Use a chave no anexo para ligar os LEDs. </li><li> Conecte o microfluidic canal para a bomba de seringa e fornecer a amostra de líquido para dentro do dispositivo microfluidico a uma taxa de fluxo constante. </li><li> Capturar um filme das células fluorescentes / partículas que fluem através do canal microfluídicos usando o modo de vídeo da câmera de telefone celular. </li></ol> B. Preparação de Amostras 4. Preparação de Micro-partículas fluorescentes Amostras <ol><li> Esferas fluorescentes com 10 um de diâmetro (contas vermelhas: Produto # F8834 excitação / emissão 580nm/605nm; grânulos verdes: Produto # F8836: excitação / emissão 505nm/515nm) são adquiridos a partir de Invitrogen (Carlsbad, CA). </li><li> Misturar 10 ul de contas fluorescentes verdes, 10 ul de contas fluorescentes vermelhas com 40 ul de água desionizada. </li><li> Place10 ul desta mistura de pérolas sobre uma lâmina de vidro usando uma micropipeta e colocar uma outra lâmina de vidro sobre o topo do mesmo a fazer uma estrutura de sanduíche. </li><li> Insira esta estrutura sanduíche na bandeja de amostra e deslize-o no acessório de celular. </li&gt; </ol> 5. Preparação de fluorescência leucócitos marcados <ol><li> Tome SYTO16 ácido nucleico rotulagem fluorescente kit (# S7578, Tecnologia Vida) e tampão fosfato salino (PBS) para fora do refrigerador e trazê-los à temperatura ambiente. </li><li> Transferir 200 uL da amostra de sangue inteiro a partir de tubo de colheita de sangue EDTA a 1,5 ml tubo de polistireno (# 05-408-129, Fisher Scientific). </li><li> Adicionar 1 ml de tampão de lise de células vermelhas do sangue (# R7757, Sigma-Aldrich) para a amostra de sangue total e 200 ul homogeneizar. </li><li> Após 5 minutos, centrifugar a amostra de sangue lisada e remover a solução sobrenadante. </li><li> Ressuspender o sedimento de células brancas do sangue em 200 ul de tampão PBS e gentilmente misturá-los. </li><li> Adicionam-se 5 ul de solução de 1 mM SYTO16 para a amostra de glóbulos brancos. Enrole a amostra com uma folha de alumínio e incuba-se em ambiente escuro, durante ~ 30 min. </li><li> Centrifugar novamente a amostra. O sobrenadante é removido e as células brancas do sangue marcados pelota é re-Suspenso em tampão de PBS. </li><li> Colocar 5-10 ul rotulado brancas do sangue amostra de líquido de célula para uma lamela de cobertura, e colocar uma lamela segundo sobre o topo da amostra. </li><li> Inserir o slide amostra encaixados na bandeja de amostra e imagem-lo usando o microscópio de telefone celular fluorescente. </li></ol> Alternativamente <ol start="10"><li> Continuamente entregar as fluorescente etiquetado células brancas do sangue através de um canal microfluídicos usando uma bomba de seringa automatizada, além de capturar um filme fluorescente microscópica das células que fluem com a câmara de telemóvel no modo de vídeo. Devemos também enfatizar que uma bateria portátil movido a bomba de seringa ou até mesmo força da gravidade pode ser utilizada para conduzir o fluxo através do canal de microfluídica. </li></ol>

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Dr. Ozcan é o fundador de uma empresa start-up que pretende comercializar imagem computacional e ferramentas de microscopia.

Nós apresentamos nossos resultados recentes sobre celular baseado de campo amplo de microscopia fluorescente e opto-fluídico citometria de fluxo de imagens usando leve e compacto opto-fluídicos anexos para câmeras de telefones celulares. Utilizando esta tecnologia de plataforma que imaged objectos fluorescentes incluindo as micro-partículas e rotulados glóbulos brancos em amostras de sangue total. Portanto, este compacto e de baixo custo de telefone celular conjunto de ferramentas de imagem baseada fluorescente po...

Microscopia e citometria de fluxo são amplamente utilizadas técnicas de 1-12 em pesquisa biomédica e científica, bem como o diagnóstico clínico para a contagem e caracterização de vários tipos celulares. No entanto, os microscópios convencionais e de citometria de fluxo-instrumentos são relativamente complexos e caros, o que limita a sua utilização para principalmente bem estabelecidas laboratórios centrais. Recentemente, temos desenvolvido uma citometria de imagem compacto e leve fluorescente e dispositivo microscopia integrados em um telefone celular, que mostra 13,14 promessa de custo-benefício traduzir microscopia de fluorescência, citometria de fluxo e análise relacionados com técnicas de micro-para ambientes de recursos limitados para várias aplicações de telemedicina que impactam a saúde global. Na configuração de citometria de fluxo optofluidic (ver Figura 1C e 1D por exemplo), um design personalizado polydimethysiloxane (PDMS) canal baseado microfluídico é positioned em frente com o telemóvel da unidade de câmara, em que emitem luz-diodos (LEDs) são acoplados à extremidade das bordas do canal. Este chip de microfluidos, juntamente com a amostra de líquido no interior, forma um guia de ondas planar opto-fluídica (composto de, por exemplo PDMS-líquido-PDMS) de tal modo que a luz de excitação é guiada para bombear uniformemente os espécimes fluorescentes marcados no interior do micro-canal. A emissão de fluorescência de esses objetos marcados, por exemplo, células, é mais trabalhada através de uma lente adicional colocada logo após o telemóvel câmera de unidade e é mapeado para o telemóvel Complementar sensor de imagem Metal-Oxide Semiconductor (CMOS). Uma vez que a emissão fluorescente é recolhido perpendicular ao percurso de luz de excitação, um filtro de absorção de plástico barato é suficiente para eliminar a luz de excitação espalhada e pode proporcionar uma base de campo escuro decente necessárias para imagens de fluorescência. Usando um projeto opto-fluídico semelhante, podemos também a imagem dos objetos fluorescentes em estatísticaic modo (ver Figura 1A e 1B), em que as partículas fluorescentes são prensadas entre duas lâminas de vidro, em vez de fluir através de um canal microfluídico e a emissão fluorescente a partir destas partículas fluorescentes são captadas pelo sensor de imagem CMOS celular para contagem de partículas e caracterização. Com base nos requisitos de aplicação diferentes, citometria de fluxo ou de campo amplo de microscopia fluorescente podem ser escolhidos. Por exemplo, o dispositivo celular por citometria de fluxo pode ser especialmente útil para o rastreio de grandes volumes de amostras de líquidos (por exemplo, alguns mililitros) para a detecção de células raras ou agentes patogénicos. Neste artigo, rever alguns dos nossos resultados recentes sobre a integração de microscopia de fluorescência e ferramentas de imagem de citometria de fluxo em um telefone celular usando compactos e de baixo custo opto-fluídicos anexos. Estas celular baseado micro-análise, citometria de imagens e plataformas de sensoriamento poderia fornecer várias oportunidadespara diagnóstico de telemedicina e de ponto-de-cuidado, especialmente impactando nossa luta contra os problemas globais de saúde em regiões com recursos limitados do mundo.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog Number </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell-phone</td>
			<td>Sony</td>
			<td>Sony Ericsson Aino</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plano-convex lens</td>
			<td>Edmund Optics</td>
			<td># NT45-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspherical lens</td>
			<td>Thorlab</td>
			<td># C230TME-A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Edmund Optics</td>
			<td>#NT54-46</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue LED</td>
			<td>Digikey</td>
			<td>#365-1201-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Battery</td>
			<td>Digikey</td>
			<td>#P032-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene tube</td>
			<td>Fisher Scientific</td>
			<td>#05-408-129</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYLGARD 184 SILICONE ELASTOMER KIT</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8834</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Green fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8836</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYTO16 nucleic acid fluorescent labeling</td>
			<td>Life Technologies</td>
			<td># S7578</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone

Microscopia de fluorescência e citometria de fluxo são amplamente utilizadas ferramentas na pesquisa biomédica e diagnóstico clínico. No entanto, estes dispositivos são, em geral, relativamente volumosos e dispendiosos, tornando-as menos eficazes para os ambientes com recursos limitados. Para potencialmente resolver estas limitações, que recentemente demonstraram a integração do grande campo de microscopia de fluorescência e ferramentas de imagem de citometria de fluxo em telefones celulares usando compacto, leve, e de custo-benefício opto-fluídicos anexos. Na nossa concepção de citometria de fluxo, as células marcadas por fluorescência são lavadas por meio de um canal microfluídico que está posicionado por cima da unidade de câmara existente telemóvel. Alimentados por bateria diodos emissores de luz (LEDs) são butt-acoplada ao lado do chip de microfluidos, que actua efectivamente como um guia de ondas multi-modo de laje, onde a luz de excitação é guiada para excitar uniformemente os alvos fluorescentes. A câmera de celular grava um filme lapso de tempo de as células fluorescentes que fluem atravéso canal microfluídico, onde os quadros digital deste filme são processados ​​para contar o número de células marcadas dentro de solução alvo de interesse. Usando um projeto opto-fluídico semelhante, também podemos imagem dessas células marcadas com fluorescência no modo estático, por exemplo, imprensando as partículas fluorescentes entre duas lâminas de vidro e capturar suas imagens fluorescentes usando a câmera de telefone celular, que podem atingir uma resolução espacial de ex ~ 10 m ao longo de um grande campo de visão de ~ 81 mm 2. Este celular baseado citometria de fluxo fluorescente imagem e plataforma de microscopia pode ser útil especialmente em ambientes com recursos limitados, por exemplo, para a contagem de células T CD4 + em direção acompanhamento de pacientes HIV + ou para a detecção de parasitas transmitidas pela água em água potável.

Com o nosso opto-fluídica bombagem / excitação esquema (Figuras 1C e 1D), as células marcadas com fluorescência pode ser continuamente fornecido para dentro do canal microfluídico utilizando uma bomba de seringa, enquanto a câmara celular grava um lapso de tempo de filme microscópico fluorescente das células de fluxo. Estes filmes fluorescentes pode depois ser rapidamente analisados ​​usando detecção de contorno e de algoritmos de rastreamento 14,15 para determ...

Watch this Scientific Journal Video about Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone at JoVE.com

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Analisamos nossos resultados recentes sobre a integração de microscopia de fluorescência e ferramentas de imagem de citometria de fluxo em um telefone celular usando compactos e de baixo custo opto-fluídicos anexos. Estes celular baseado micro-dispositivos de análise pode ser útil para a análise citométrica, como a realização de tarefas de contagem de células diferentes, bem como para rastreio de alto rendimento de amostras, por exemplo, água em ambientes com recursos limitados.

Largo-campo microscopia de fluorescência e citometria de fluxo fluorescente de imagens em um telefone celular

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

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23.5K visualizações.  University of California, Los Angeles. Analisamos nossos resultados recentes sobre a integração de microscopia de fluorescência e ferramentas de imagem de citometria de fluxo em um telefone celular usando compactos e de baixo custo opto-fluídicos anexos. Estes celular baseado micro-dispositivos de análise pode ser útil para a análise citométrica, como a realização de tarefas de contagem de células diferentes, bem como para rastreio de alto rendimento de amostras, por exemplo, água em ambientes com ...

Vídeo: Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Nanopartículas fluorescentes para a medição da concentração de íons em Sistemas Biológicos

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Bioengenharia

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

Microscopia fluorescente sem lente em um Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Preparação de amostras, Imaging, and Analysis Protocolos para Faca de ponta microscopia de varredura

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Analisando internalização celular de nanopartículas e bactérias multi-espectral por Citometria de Fluxo Imagem

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

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 ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

Um novo método para localizar Repórter fluorescentes Beads perto da superfície de cultivo celular para Tração Microscopia de Força

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizando a formação de adesão em células por meio de avançados girando a reflexão interna Total disco microscopia de fluorescência

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

Um método automatizado para realizar o ensaio de micronúcleos in vitro usando citometria de fluxo de imagem multiespectral

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

Detecção de câncer de ovário usando citometria de fluxo fotoacústico

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

Preparação, Administração e Avaliação da Captação Celular Tecido-Específica In Vivo de Lipossomas Marcados com Corante Fluorescente

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...