Los autores agradecen el Estado federado de Baden-Württemberg y la Europäische Union Europäischer-Fonds für die Entwicklung regionale - para la financiación ZAFH-PHOTON n, el Bundesministerium für Bildung und Forschung (BMBF) para la financiación de la investigación subvención no. 1792C08, así como la GmbH Baden-Württemberg-Stiftung para la financiación del proyecto &quot;Aurami&quot;.

1. Siembra y la incubación de células <ol><li> Se siembran las células individuales, por ejemplo U373-MG o U-251-MG células de glioblastoma a una densidad típica de 100 células / mm 2 sobre un portaobjetos de vidrio y crecer para 4872 hr en medio de cultivo (por ejemplo, RPMI 1640 suplementado con 10% suero de ternera fetal y antibióticos), utilizando una incubadora a 37 ° C y 5% CO 2. </li><li> Se incuban las células con un marcador de membrana, por ejemplo, 6-dodecanoil-2-dimetilamino naftaleno (laurdan) aplicada durante 60 min a una concentración de 8 M (en medio de cultivo) 4, o un marcador de citoplasma, por ejemplo calceína acetomethlyester aplicado durante 40 min a una concentración de 5 mM 3, o un fotosensibilizador asociado a la membrana, por ejemplo, la protoporfirina IX inducida tras la incubación de 4 horas con su precursor ácido 5-aminolevulínico (5-ALA; 1 mM) 4,5. </li><li> Lavar las células con medio de cultivo o tampón fosfato salino (PBS) antes de la microscopía de fluorescencia. &lt;/ Li&gt; </ol> 2. VA-TIRFM <ol><li> Usar un microscopio vertical, por ejemplo, Zeiss Axioplan, y sustituir su condensador por un dispositivo de iluminación, como se describe en la ref. 3 con un semiesférica o un prisma de vidrio hemi-cilíndrica acoplada ópticamente a la corredera objeto (véase la Figura 1). </li><li> Utilice un haz colimado láser paralelo que después de la impresión por el prisma de vidrio y otros componentes ópticos (por ejemplo espejo cóncavo) vuelve a producirse en un haz paralelo en la superficie de la muestra (rayo telecéntrico alumno). Tener cuidado de que el vector de campo eléctrico es polarizada perpendicular con el plano de incidencia. </li><li> Use un espejo ajustable situado en el interior del condensador que se forma la imagen en el plano de muestra y se ilumina la muestra bajo un ángulo variable de incidencia (rayos objeto). El espejo ajustable puede ser accionado por un motor paso a paso, donde cada posición corresponde a un ángulo bien definido de incidencia, tal como se determina por medidas goniométricas 3 </sup&gt;. </li><li> Calibrar la posición angular del espejo ajustable mediante el ángulo crítico Θ = 61,3 ° C para una transición vidrio-agua como un valor fijo. Θ c puede ser visualizado en la variación de Θ cuando un colorante fluorescente (por ejemplo, 1 mM mononucleótido de flavina, FMN) se disuelve en agua. La posición del espejo a Θ = Θ C se almacena en el programa de posicionamiento espejo. </li><li> Establezca los parámetros de la cámara (de nuevo iluminado Andor Ixon EMCCD, DV887DC, Andor Technology, Belfast, Reino Unido 6), incluyendo la temperatura, el aumento de tiempo de grabación y la velocidad de desplazamiento. </li><li> Utilice una lente de objetivo de ampliación adecuada y apertura numérica preferentemente moderada (por ejemplo 40 × / 0,75 o 63 × / 0,90 inmersión en agua) y localizar el portaobjetos en el microscopio. Usar aceite de inmersión para asegurar un contacto óptica de la muestra con el prisma de vidrio. Grabar imágenes de fluorescencia de muestras idénticas en la variación de laángulo de iluminación (Θ ≥ Θ Θ c con c correspondiente a 64,5 ° para una interfaz de celda de vidrio). Un intervalo angular de 66 ° -74 ° con pasos de 0,5 º o 1 se recomienda. Utilice un pase largo adecuado o filtro de paso de banda para la detección de fluorescencia. </li></ol> 3. Análisis de Datos <ol><li> Leer las imágenes (por ejemplo, en formato ASCII) registrados en varios ángulos Θ, así como los ajustes de la cámara (para la discriminación de fondo), la longitud de onda de excitación y los índices de refracción de vidrio (n 1 = 1,52) y células (n = 2 1,37) en el programa de evaluación (LabView; nanocélula). Para cada conjunto de parámetros de la profundidad de penetración d (Θ) y el factor de transmisión T (Θ) se calculan automáticamente. </li><li> Seleccione las imágenes utilizadas para la evaluación de las distancias de sustrato de células Δ acuerdo con la ecuación 5 (marcador de membrana) o la ecuación 4 (marcador citoplasma), las imágenes individuales con inhomogéneailuminación (e reveladora, rayas g.) pueden ser excluidos. </li><li> Cálculo de las imágenes de topología célula-sustrato y seleccione un código de color apropiado para la exhibición. Durante la evaluación de los perfiles de los píxeles individuales se puede mostrar, tal como se representa en la Figura 2. Estos perfiles pueden ser utilizados como un indicador de la calidad del ajuste. </li><li> Guarde el protocolo de evaluación. </li></ol> 4. Los resultados representativos Experimentos representativos se llevó a cabo con ingeniería genética células de glioblastoma U251-MG amablemente proporcionados por el Prof. Jan Mollenhauer, Departamento de Oncología Molecular de la Universidad del Sur de Dinamarca, Odense. En dos de estos subclones células U251 los genes supresores de tumores TP53 o PTEN están sobre-expresa con un Tet-On sistema de expresión inducible, de tal manera que estas células presentan un potencial oncogénico reducido y puede ser considerado como menos maligno. Altamente maligno U251-MG células sin gen supresor se usan como controles, y-U251MG células de tipo salvaje servir para una comparación más. Un diodo láser que emite una serie casi continua de impulsos de picosegundos (LDH400 con controlador PDL 800-B, Picoquant, Berlín, Alemania; longitud de onda: 391 nm; energía de pulso: 12 pJ; tasa de repetición: 40 MHz) se utiliza como una fuente de excitación. En la figura 3 imágenes TIRFM de U251-MG células de glioblastoma con un gen supresor de tumor activado TP53 y se incubaron con laurdan (8 micras; 60 min) se representan para ángulos de incidencia de 66,0 °, 69,0 ° y 73,0 ° correspondientes a profundidades de penetración de la campo electromagnético evanescente de alrededor de 140 nm, 75 nm y 60 nm, respectivamente. Con la disminución de la fluorescencia de profundidad de penetración disminuye en intensidad (véase escalas) y se origina en más sitios superficiales de las células (por ejemplo, contactos focales sobre sus bordes). Sustrato celular distancias (visualizado en la figura 3d por un código de color que va desde 0 hasta 600 nm) varían fuertemente en las células entre elly unos pocos nanómetros (blanco-amarillo) y 250-300 nm (rojo oscuro), es decir, los contactos focales y las grandes distancias sustrato de células se encuentran en las cercanías. Lo mismo vale para U251-MG células de glioblastoma con un gen supresor PTEN activado (Figura 4d), ​​mientras que el sustrato de células distancias son bastante constante para U251-MG de control y las células de tipo salvaje con los valores típicos de 80-100 nm (amarillo; Figura 4b ) y 150-200 nm (color naranja-rojo; Figura 4a). <img alt="Figura 1" src="/files/ftp_upload/4133/4133fig1.jpg" /> Figura 1. Dispositivo de iluminación para VA-TIRFM en un microscopio vertical. Un espejo ajustable accionado por un motor paso a paso permite una resolución angular de ± 0,25 °. <img alt="Figura 2" src="/files/ftp_upload/4133/4133fig2.jpg" /> Figura 2. Evaluación perfil para un pixel (esquemática). Cuando ln [I F / T (Θ)] se representa gráficamente como una función de 1 / d (Θ), la pendiente representa la distancia Δ célula-sustrato. Esta pendiente se determina comúnmente con una precisión de aproximadamente ± 10%. Uno de los valores - como resultado de la iluminación no homogénea - ha estado marcada y excluidos de la evaluación. <img alt="Figura 3" src="/files/ftp_upload/4133/4133fig3.jpg" /> Figura 3 imágenes TIRFM de U251-MG células de glioblastoma con activado gen supresor TP53 tras la incubación con laurdan (8 micras; 60 min). Grabado en varios ángulos de iluminación (CA); sustrato celular distancias en el intervalo de 0-600 nm mostrado por un código de color (d); longitud de onda de excitación: 391 nm, la detección: ≥ 420 nm, el tamaño de la imagen: 210 m. × 210 micras <a href="http://www.jove.com/files/ftp_upload/4133/4133fig3large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . Figura 4-sustrato celular distancias de U251-MG células glioblatoma en el intervalo de 0-600 nm mostradas por un código de color;. (A) de tipo salvaje (b) los controles; (c) las células con el gen supresor TP53 activado; (d) células con PTEN activado gen supresor de longitud de onda; excitación: 391 nm, la detección: ≥ 420 nm, el tamaño de la imagen: 210 m. × 210 micras <a href="http://www.jove.com/files/ftp_upload/4133/4133fig4large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> .

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Se describe un método para la medición de distancias en el sustrato de células con una precisión nanométrica. En la actualidad, los métodos de microscopia de super-resolución, por ejemplo, sobre la base de iluminación estructurada 7 o en un solo 8-10 moléculas de detección, así como el agotamiento de la emisión estimulada (STED) microscopía 11 son de considerable interés. Ninguna de estas técnicas, sin embargo, permite una resolución axial por debajo de 50 nm. Ad...

Cuando un haz de luz que se propaga a través de un medio de índice de refracción n 1 cumple una interfaz a un segundo medio de índice de refracción n 2 &lt;n 1, la reflexión interna total se produce en todos los ángulos de incidencia Θ, que son mayores que el ángulo crítico c = Θ arcsen (n 2 / n 1). A pesar de estar totalmente reflejada del haz de luz incidente evoca un campo electromagnético evanescente que penetra en el segundo medio y decae exponencialmente con la distancia perpendicular z desde la interfaz de acuerdo a I (z) = I 0 ez / d (Θ). I (z) corresponde a la intensidad del campo electromagnético y d (Θ) a la profundidad de penetración en la longitud de onda λ, como se da d (Θ) = (λ/4π) (n 1 2 sen 2 Θ - n 2 2) -1 / 2 Como se informó en la literatura 1,2, I 0 </sub&gt; corresponde a la intensidad de luz incidente I e multiplica por el factor de transmisión T (Θ) y la relación n 2 / n 1. Si el vector de campo eléctrico de este haz de luz es polarizada perpendicular al plano de incidencia (es decir, el plano generado por el incidente y el rayo reflejado), este factor de transmisión está dada por T (Θ) = 4 cos Θ 2 / [1 - (2 n / nL 1) 2] Por la intensidad de fluorescencia detectada en mediciones TIRFM, absorción de la luz tiene que ser integrado en todas las capas de la muestra y multiplicado con el rendimiento cuántico de fluorescencia del colorante η pertinente, así como con el ángulo sólido de detección Ω. Como se informó previamente 3, esta intensidad puede ser descrito por I F (Θ) = AT (Θ) OC (z) e-z / d (Θ) dz si todos los factores que son independientes f<html> rom el ángulo de incidencia Θ y la coordenada z se incluyen dentro de la ecuación experimental A. constante 3 se pueden resolver analíticamente, si la concentración de c (z) de los fluoróforos se considera que es casi constante - Bien en todas las coordenadas z ≥ Δ, por ejemplo, en el citoplasma de las células que tienen una distancia Δ de la interfaz. En este caso, la integral tiene que ser calculado de z = Δ para z = ∞ resulta en I F = A c T (Θ) d (Θ) e-Δ / d (Θ) - O entre z = Δ - t / 2 y z = Δ + t / 2, es decir, dentro de una capa fina de espesor t, por ejemplo, una membrana de célula que se encuentran a una distancia Δ de la interfaz. En este caso, la intensidad de fluorescencia puede ser calculada como I F = A c T (Θ) te-Δ / d (Θ), si t es pequeño en comparación con Δ. ent &quot;&gt; De las ecuaciones (4) y (5) las distancias sustrato de células-Δ se puede calcular, si las imágenes de la intensidad de fluorescencia I F se registran para ángulos variables Θ, y si ln (I F / T d) (Ec. 4) o ln (I F / T) (Ec. 5) se evalúa como una función de 1 / d para estos ángulos. Para la membrana marcador laurdan utiliza en este documento (s. a continuación) la evaluación se realizó de acuerdo a la ecuación. 5. Como se informó anteriormente 3, adquisición de imágenes y requiere una evaluación - Distribución homogénea de la luz de excitación sobre la muestra, - Validez de un modelo 2-capa (con los índices de refracción N 1 y N 2 para el sustrato y el citoplasma, respectivamente). Esto es, si la membrana plasmática es muy delgada, y si la capa del medio extracelular (entre la célula y el sustrato) es pequeño comparado con la longitud de onda de la luz incidente. Además, una apertura numérica moderada(Típicamente un 0,90 ≤) de la lente objetivo del microscopio es ventajoso para evitar desviaciones de brillo debido a la anisotropía de la radiación en el campo cerca de la interfaz dieléctrico.

<table border="1"><tbody><tr><td> Nombre </td><td> Empresa </td><td> Tipo </td><td> Comentarios </td></tr><tr><td> Incubadora </td><td> Nunc </td><td> Nunc Cellstar QM1300SVBA </td><td></td></tr><tr><td> Flujo laminar </td><td> Holten Safe </td><td> S2010 1.2 EN GG 1LN </td><td></td></tr><tr><td> DMEM + 10% FCS + 1% de penicilina / estreptomicina </td><td> Biochrom </td><td></td><td> Cultivo medio </td></tr><tr><td> Diapositivas de vidrio objeto </td><td> Marienfeld </td><td> Vidrio blanco puro </td><td> Procedimiento de limpieza especial utilizado </td></tr><tr><td> 6-dodecanoil-2-dimetilamino naftaleno (laurdan) </td><td> Molecular Probes </td><td></td><td> Marcador fluorescente (solución madre: 2 mM en etanol) </td></tr><tr><td> Microscopio </td><td> Carl Zeiss </td><td> Axioplan 1 </td&gt; <td></td></tr><tr><td> Láser diodo </td><td> PicoQuant </td><td> LDH 400 con controlador PDL 800-B </td><td> Longitud de onda: 391 nm </td></tr><tr><td> Modo simple sistema de fibra </td><td> Punto de Origen </td><td> kineFlex </td><td> Se utiliza con la óptica de colimación </td></tr><tr><td> EMCCD cámara </td><td> Andor </td><td> DV887DC </td><td> Cámara trasera iluminada </td></tr></tbody></table>

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

Topología de la superficie, por ejemplo de células que crecen sobre un sustrato, se determina con una precisión nanométrica de ángulo variable total Microscopía de Fluorescencia de Reflexión Interna (VA-TIRFM). Las células se cultivan en portaobjetos transparentes y se incubaron con un marcador fluorescente distribuido homogéneamente en su membrana plasmática. Iluminación se produce mediante un rayo láser paralelo bajo ángulos variables de reflexión interna total (TIR) ​​con diferentes profundidades de penetración del campo electromagnético evanescente. Grabación de imágenes de fluorescencia tras la irradiación en alrededor de 10 diferentes ángulos permite calcular distancias sustrato celular con una precisión de unos pocos nanómetros. Las diferencias de adhesión entre las distintas líneas celulares, por ejemplo células cancerosas y las células malignas, son menos así determinada. Además, los posibles cambios de la adhesión celular a un tratamiento químico o fotodinámica puede ser examinado. En comparación con otros métodos de super-resolución exposición microscopía de luz se mantienedaño muy pequeño, y no de las células vivas se espera que ocurra.

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

Topología de la adhesión celular sobre un sustrato se mide con una precisión nanométrica de ángulo variable total de microscopía de fluorescencia de reflexión interna (VA-TIRFM).

Nanotopology de adhesión celular en ángulo variable total microscopía de fluorescencia de reflexión interna (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 vistas.  Institut für Angewandte Forschung. Topología de la adhesión celular sobre un sustrato se mide con una precisión nanométrica de ángulo variable total de microscopía de fluorescencia de reflexión interna (VA-TIRFM).

Video: 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.

La visualización de la corteza organización y dinámica de los microorganismos, con total Microscopía de fluorescencia de reflexión interna

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

Investigación

Bioingeniería

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.

Creación de degradados adhesivas y soluble para la migración celular con imágenes de microscopía de fluorescencia

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.

Grano de agregación Los ensayos para la caracterización de Putativos Moléculas de Adhesión 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.

Rastreo Orbital 3D en un microscopio de dos fotones Modificado: una aplicación para el Seguimiento 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 Fluorescencia Fast Imaging a la Ley de Difusión Molecular en membranas de células vivas en un microscopio 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.

Utilizando Impedancia Cell-sustrato y imágenes de células vivas para medir los cambios en tiempo real en la adhesión celular y De-adhesión inducidas por Matrix Modificación

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.

Bilayers Individual Molécula microscopía de fluorescencia en planar compatibles

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.

Química húmeda y el péptido Inmovilización de politetrafluoroetileno para la adhesión celular mejorada

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.

División de célula mamífera en Matrices 3D mediante microscopia de reflexión Confocal cuantitativa

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.

Visualizar la formación de adherencias en las células por medio de avanzados Spinning microscopía de fluorescencia de reflexión interna Total de disco

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