Os autores agradecem o Land Baden-Württemberg e da Europäische União Europäischer Fonds für die Entwicklung regionale - para financiamento ZAFH-PHOTON n, o Bundesministerium für Bildung und Forschung (BMBF) para financiamento da investigação concessão não. 1792C08 bem como a GmbH Baden-Württemberg-Stiftung para o financiamento do projeto &quot;Aurami&quot;.

1. Sementeira e incubação de células <ol><li> Semente de células individuais, por exemplo, U373-MG ou células U-251 MG-glioblastoma a uma densidade típica de 100 células / mm 2 sobre uma lâmina de vidro e crescê-los para 4872 horas em meio de cultura (por exemplo, meio RPMI 1640 suplementado com 10% de soro fetal de vitela e antibióticos), utilizando uma incubadora a 37 ° C e 5% CO 2. </li><li> Incubar as células com um marcador de membrana, por exemplo, 6-dodecanoil-2-dimetilamino-naftaleno (laurdan) aplicado durante 60 minutos a uma concentração de 8 mM (em meio de cultura) 4, ou um marcador de citoplasma, por exemplo, calceína acetomethlyester aplicado durante 40 minutos a uma concentração de 5 uM 3, ou de uma membrana associada fotossensibilizador, por exemplo, protoporfirina IX induzida após 4 horas de incubação com o seu precursor, o ácido 5-aminolevulínico (5-ALA, 1 mM), 4,5. </li><li> Lavar as células com meio de cultura ou solução salina tamponada com fosfato (PBS) antes da microscopia de fluorescência. &lt;/ Li&gt; </ol> 2. VA-TIRFM <ol><li> Usar um microscópio vertical, por exemplo, Zeiss Axioplan, e substituir o condensador por um dispositivo de iluminação, tal como descrito na ref. 3 com um hemi-esférica ou de um prisma de vidro cilíndrico hemi-opticamente acoplado à corrediça objecto (ver Figura 1). </li><li> Usar um feixe laser colimado paralelo que após imagiologia pelo prisma de vidro e outros componentes ópticos (por exemplo, espelho côncavo) novamente resulta num feixe paralelo à superfície da amostra (raio da pupila telecêntrica). Tomar cuidado para que o vector de campo eléctrico é perpendicular polarizada ao plano de incidência. </li><li> Use um espelho ajustável localizado no interior do condensador que é imaginada no plano da amostra e ilumina a amostra sob um ângulo variável de incidência (ray objeto). O espelho ajustável pode ser accionado por um motor de passo, em que cada posição corresponde a um ângulo de incidência bem definida, tal como determinado por medições goniométricas 3 </sup&gt;. </li><li> Calibrar a posição angular do espelho ajustável usando o ângulo crítico Θ c = 61,3 ° para uma transição de vidro-água como um valor fixo. Θ c podem ser visualizados mediante variação do Θ quando um corante fluorescente (por exemplo, 1 mM de mononucleótido de flavina, FMN) é dissolvido em água. A posição do espelho em Θ Θ = c é armazenado no programa de posicionamento do espelho. </li><li> Definir os parâmetros da câmera (iluminação traseira Andor ixon EMCCD, DV887DC, ANDOR Technology, Belfast, Reino Unido 6), incluindo temperatura, tempo de gravação, ganho e velocidade de deslocamento. </li><li> Use uma lente objectiva de ampliação apropriado e preferencialmente moderado abertura numérica (por exemplo, 40 × / 0,75 ou 63 × / 0,90 imersão em água) e localizar o slide objecto no microscópio. Usar óleo de imersão para assegurar um contato óptica da amostra com o prisma de vidro. Gravar imagens de fluorescência das amostras idênticas mediante variação doângulo de iluminação (Θ ≥ Θ Θ c com c correspondendo a 64,5 ° de um interface de célula de vidro). Uma variação angular de 66 ° -74 ° com passos de 0,5 ° ou 1 ° é recomendada. Use um passe longo ou apropriado filtro passa banda para a detecção de fluorescência. </li></ol> 3. Análise de Dados <ol><li> Ler as imagens (por exemplo, em formato ASCII) gravadas em vários ângulos Θ, bem como as configurações da câmara (para a discriminação de fundo), o comprimento de onda de excitação e os índices de refracção do vidro (n 1 = 1,52) e células (n 2 = 1,37) para o programa de avaliação (LabView; NanoCell). Para cada conjunto de parâmetros a profundidade de penetração d (Θ) e de transmissão factor T (Θ) são calculados automaticamente. </li><li> Selecione as imagens utilizadas para avaliação de célula-substrato distâncias Δ acordo com a Equação 5 (marcador de membrana) ou Equação 4 (marcador de citoplasma), as imagens individuais com heterogêneailuminação (e revelador, g. riscas) podem ser excluídos. </li><li> Calcule imagens de células-substrato topologia e seleccionar um código de cor apropriada para exibição. Durante perfis de avaliação dos pixels individuais podem ser apresentados, como descrito na Figura 2. Estes perfis podem ser usados ​​como um indicador da qualidade do ajuste. </li><li> Armazenar o protocolo de avaliação. </li></ol> 4. Resultados representativos Experiências representativas são realizadas com células geneticamente modificadas U251-MG glioblastoma, gentilmente cedidos pelo Prof Janeiro Mollenhauer, Departamento de Oncologia Molecular, Universidade do Sul da Dinamarca, Odense. Em dois subclones destas células U251 os genes supressores de tumor TP53 ou PTEN estão sobre-expressa utilizando um sistema de Tet-On expressão indutível, de tal modo que as mesmas exibem um potencial tumorigénico reduzida e pode ser considerado como menos maligna. Altamente malignas U251-MG células sem gene supressor são utilizadas como controlos, e U251-Células do tipo MG silvestres servem para uma posterior comparação. Um díodo laser emitindo uma série de impulsos quase contínua picossegundos (LDH400 com condutor PDL 800-B, Picoquant, Berlim, Alemanha; comprimento de onda: 391 nm; pulso de energia: 12 pJ; taxa de repetição: 40 MHz) é usado como uma fonte de excitação. Na Figura 3 imagens TIRFM de U251-MG células de glioblastoma com um gene supressor de tumor TP53 activado e incubado com laurdan (8 uM; 60 min) são representadas por ângulos de incidência de 66,0 °, 69,0 ° e 73,0 ° correspondendo a profundidade de penetração do campo electromagnético evanescente de cerca de 140 nm, 75 nm e 60 nm, respectivamente. Com a diminuição da profundidade de penetração de fluorescência diminui de intensidade (ver tabelas), e origina a partir de locais mais superficiais do que as células (por exemplo, os contactos focais nas suas extremidades). Célula-substrato distâncias (visualizado na Figura 3d por um código de cores que varia de 0-600 nm) variam fortemente sobre as células entre emly alguns nanômetros (branco-amarelo) e 250-300 nm (vermelho escuro), ou seja, contatos focais e maiores de células-substrato distâncias são muito próximos. O mesmo é válido para U251-MG células de glioblastoma com um gene supressor de PTEN activado (Figura 4d), ​​enquanto célula-substrato distâncias são mais ou menos constante para U251-MG e controle de células de tipo selvagem, com os valores típicos de 80-100 nm (amarelo; Figura 4b ) e 150-200 nm (laranja-vermelho; Figura 4a). <img alt="Figura 1" src="/files/ftp_upload/4133/4133fig1.jpg" /> Figura 1. Dispositivo de iluminação para VA-TIRFM em um microscópio vertical. Um espelho ajustável accionado por um motor passo permite uma resolução angular de ± 0,25 °. <img alt="Figura 2" src="/files/ftp_upload/4133/4133fig2.jpg" /> Figura perfil Avaliação 2. Por um pixel (esquemática). Quando ln [I F / T (Θ)] é representada como uma função de 1 / d (Θ), o declive representa a célula-substrato distância Δ. Esta inclinação é normalmente determinada com uma precisão de cerca de ± 10%. Um valor - resultante de iluminação não homogênea - tem sido marcada e excluídas da avaliação. <img alt="Figura 3" src="/files/ftp_upload/4133/4133fig3.jpg" /> Figura 3 imagens TIRFM de U251-MG com células de glioblastoma activado gene supressor TP53 após incubação com laurdan (8 uM; 60 min). Gravada em vários ângulos de iluminação (ac); célula-substrato distâncias na gama de 0-600 nm mostrado por um código de cores (d); comprimento de onda de excitação: 391 nm, detecção: ≥ 420 nm; tamanho da imagem: 210. mM mM × 210 <a href="http://www.jove.com/files/ftp_upload/4133/4133fig3large.jpg" target="_blank">Clique aqui para ver maior figura</a> . Figura 4 Células-substrato distâncias de U251-MG glioblatoma células na gama de 0-600 nm mostrados por um código de cores,. (A) de tipo selvagem (b) Controlos, (c) células activadas com gene supressor TP53, (d) células com PTEN gene supressor ativado; comprimento de onda de excitação: 391 nm, detecção: ≥ 420 nm; tamanho da imagem: 210. mM mM × 210 <a href="http://www.jove.com/files/ftp_upload/4133/4133fig4large.jpg" target="_blank">Clique aqui para ver maior figura</a> .

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É descrito um método para a medição de célula-substrato distâncias com uma precisão nanométrica. Presentemente, os métodos de resolução super-microscopia, por exemplo, com base na iluminação estruturada 7 ou em 8-10 de detecção de moléculas isoladas, assim como depleção emissão estimulada (STED) Microscopia 11 são de considerável interesse. Nenhuma destas técnicas, contudo, permite uma resolução axial inferior a 50 nm. Além disso, a irradiância bastante...

Quando um feixe de luz que se propaga através de um meio de índice de refracção n 1 encontra uma interface para um segundo meio de índice de refracção n 2 &lt;n 1, a reflexão interna total ocorre em todos os ângulos de incidência Θ, que são maiores do que o ângulo crítico Θ c = arcsin (n 2 / n 1). Apesar de ter sido totalmente reflectido do feixe de luz incidente evoca um campo electromagnético evanescente que penetra no segundo meio e decai exponencialmente com a distância perpendicular a partir da interface de z de acordo com a I (z) = I 0 ez / d (Θ). I (z) corresponde à intensidade do campo electromagnético e d (Θ) para a profundidade de penetração no comprimento de onda λ, como dada pela d (Θ) = (λ/4π) (n 1 2 2 Θ pecado - n 2 2) -1 / 2 Conforme relatado na literatura 1,2, I 0 </sub&gt; corresponde à intensidade da luz incidente I e multiplicada com o factor de transmissão T (Θ) e a relação de n 2 / n 1. Se o vector do campo eléctrico do feixe de luz é perpendicular ao plano de polarização da incidência (isto é, o plano gerado pelo incidente e do feixe reflectido), este factor de transmissão é dada pela T (Θ) = 4 cos 2 Θ / [1 - (n 2 / nL 1) 2] Para a intensidade de fluorescência detectada no TIRFM medições, a absorção de luz tem de ser integrado ao longo de todas as camadas da amostra e multiplicado com o rendimento quântico de fluorescência do corante η relevantes, bem como com o ângulo sólido de detecção Ω. Como relatado anteriormente 3, esta intensidade pode ser descrita pela Eu F (Θ) = AT (Θ) OC (z) e-z / d (Θ) dz se todos os fatores que são f independente<html> rom o ângulo de incidência e do Θ z coordenadas estão incluídos dentro da Equação A. experimental constante 3 pode ser resolvido analiticamente, se a concentração c (z) de fluoróforos é considerado para ser quase constante - Quer no todo z ≥ Δ coordenadas, por exemplo, dentro do citoplasma das células com uma distância Δ partir da interface. Neste caso, a integral tem de ser calculada a partir de z = Δ para z = ∞ resultando Eu F = A c T (Θ) d (Θ) e-Δ / d (Θ) - Ou entre z = Δ - t / 2 e z = Δ + t / 2, ou seja, dentro de uma fina camada de espessura t, por exemplo, uma membrana celular, localizado a uma distância a partir da interface Δ. Neste caso, a intensidade de fluorescência pode ser calculado como Eu F = A c T (Θ) te-Δ / d (Θ), se t é pequeno em comparação com Δ. &gt; ent &quot;A partir das Equações (4) e (5) da célula-substrato distâncias Δ pode ser calculado, se as imagens de intensidade de fluorescência, F I são registados por ângulos variáveis ​​Θ, e se ln (I F / T d) (Eq. 4) ou ln (I F / T) (Eq. 5) é avaliada como uma função de 1 / d para esses ângulos. Para a membrana laurdan marcador utilizado no presente documento (s. abaixo), a avaliação foi realizada de acordo com a Equação 5.. Como relatado anteriormente 3 de aquisição de imagem, e requer a avaliação - Distribuição homogênea da luz de excitação sobre a amostra, - A validade de um modelo de 2-camadas (com os índices de refracção n 1 e n 2 para o substrato e do citoplasma, respectivamente). Isto aplica-se a membrana do plasma é muito fina e, se a camada do meio extracelular (entre a célula e o substrato) é pequeno em comparação com o comprimento de onda da luz incidente. Além disso, a abertura de uma moderada numérica(Tipicamente 0,90 ≤ A) da lente da objectiva do microscópio é vantajosa para evitar desvios de brilho devido a anisotropia da radiação no domínio próximo da interface dielétrico.

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Tipo </td><td> Comentários </td></tr><tr><td> Incubadora </td><td> Nunc </td><td> Nunc Cellstar QM1300SVBA </td><td></td></tr><tr><td> Fluxo laminar </td><td> Holten Seguro </td><td> S2010 1,2 PT GG 1LN </td><td></td></tr><tr><td> DMEM + 10% FCS + 1% de Penicilina / Estreptomicina </td><td> Biochrom </td><td></td><td> Meio de cultura </td></tr><tr><td> Objeto lâminas de vidro </td><td> Marienfeld </td><td> Vidro branco puro </td><td> Procedimento de limpeza especial usado </td></tr><tr><td> 6-dodecanoil-2-dimetilamino-naftaleno (laurdan) </td><td> Molecular Probes </td><td></td><td> Marcador fluorescente (solução de armazém: 2 mM em etanol) </td></tr><tr><td> Microscópio </td><td> Carl Zeiss </td><td> Axioplan 1 </td&gt; <td></td></tr><tr><td> Diodo laser </td><td> PicoQuant </td><td> LDH 400 com motorista PDL 800-B </td><td> Comprimento de onda: 391 nm </td></tr><tr><td> Sistema de fibra monomodo </td><td> Fonte ponto </td><td> kineFlex </td><td> Usado com óptica de colimação </td></tr><tr><td> Câmera EMCCD </td><td> Andor </td><td> DV887DC </td><td> Câmera traseira iluminada </td></tr></tbody></table>

nanotopology of cell adhesion upon variable-angle total internal reflection fluorescence microscopy (va-tirfm)

Topologia de superfície, por exemplo, que as células crescem sobre um substrato, é determinada com uma precisão nanométrica por Variable Angle-Fluorescence Microscopy Internal Reflection total (TIRFM-VA). As células são cultivadas em lâminas transparentes e incubaram-se com um marcador fluorescente homogeneamente distribuídos na sua membrana plasmática. Iluminação ocorre por um feixe de laser paralelo sob um ângulo variável de reflexão interna total (TIR), com diferentes profundidades de penetração do campo electromagnético evanescente. A gravação de imagens de fluorescência após irradiação a cerca de 10 diferentes ângulos permite calcular distâncias célula-substrato, com uma precisão de alguns nanómetros. As diferenças de adesão entre as linhas de células diferentes, por exemplo, células cancerosas e células malignas, menos são assim determinados. Além disso, as variações possíveis de adesão das células mediante tratamento químico ou fotodinâmica pode ser examinado. Em comparação com outros métodos de super-resolução de exposição de microscopia de luz é mantidadanos muito pequena, e não de células vivas é de esperar a ocorrência.

Watch this Scientific Journal Video about Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM at JoVE.com

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Topologia de adesão celular num substrato é medida com uma precisão nanométrica de ângulo variável por microscopia de fluorescência de reflexão interna total (TIRFM-VA).

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

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

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Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

10.1K visualizações.  Institut für Angewandte Forschung. Topologia de adesão celular num substrato é medida com uma precisão nanométrica de ângulo variável por microscopia de fluorescência de reflexão interna total (TIRFM-VA).

Vídeo: Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

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

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

Bioengenharia

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

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

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

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Conta Agregação ensaios para a caracterização dos putativos Moléculas de Adesão Celular

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

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

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

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

De fast imagens de fluorescência com a Lei difusão molecular em membranas de células vivas em um microscópio comercial

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Usando impedância Cell-substrato e imagens de células vivas para medir as mudanças em tempo real no celular Adesão e De-adesão Induzidas por Matrix Modificação

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Bicamadas única molécula de microscopia de fluorescência em Planar Apoiado

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Wet Chemistry and Peptide Imobilização em Polytetrafluoroethylene para Melhoria de adesão celular

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

Divisão celular dos mamíferos em matrizes 3D através de microscopia de reflexão Confocal quantitativa

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

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 ...