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

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

Investigando única molécula de adesão de Força Atômica Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

Engenharia

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

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

Reflexão de Interferência Simultânea e Microscopia de Fluorescência De Reflexão Interna Total para Microtúbulos Dinâmicos de Imagem e Proteínas Associadas

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

Bioquímica

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Visualizando a dinâmica de acoplamento de actina e microtúbula in vitro por microscopia de fluorescência interna total (TIRF)

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

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 ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Controle da adesão celular usando técnicas de padronização de hidrogel para aplicações em microscopia de força de tração

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Dinâmica de oligomerização dos receptores de superfície celular em células vivas pela microscopia de fluorescência de reflexão interna total combinada com análise de número e brilho

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Biologia

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

A adesão molecular de imagem em rolamento celular por ensaio de pegada de adesão

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to&#160;focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of&#160;the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

Sondas de tether medidor de tensão para quantificar fator de crescimento mediado mecânica integrin e adesão

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...

TIRF Microscopy to Visualize Actin and Microtubule Coupling Dynamics

Source: Henty-Ridilla, J.L., <a href="https://app.jove.com/v/64074">Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy.</a> J. Vis. Exp. (2022)
This video describes TIRF, a total internal reflection microscopy-based technique to visualize actin and microtubule polymerization dynamics. The method allows the high-resolution imaging of actin and microtubule coupling dynamics in real-time, which is essential for understanding cellular crosstalks.

Microscopia TIRF para visualizar a dinâmica de acoplamento de actina e microtúbulos

Source: Henty-Ridilla, J.L., Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy. J. ...