Este trabajo fue financiado por el Departamento de Defensa de la concesión era de esperanza Scholar (N º W81XWH-05-0396) y una beca Pew en el Premio de Ciencias Biomédicas de Edward B. Brown III.

1. Alinear la óptica. El equipo esencial de un experimento MP-FRAP son: un modo bloqueado fuente de láser, células Pockels (por haz de modulación), generador de impulsos, dicroicos, las lentes objetivo, la fluorescencia de emisión de filtro, tubo fotomultiplicador cerrada, y un sistema de registro de datos (fotones contra y multicanal escalador). 2. Determinará las competencias segura monitor. <ol><li> Atenuar el láser a un mínimo, pero razonable, para la generación de energía de fluorescencia en la muestra. </li><li> Enfoque dentro de la muestra. </li><li> Establecer un contador de fotones para integrar en una escala de tiempo mucho más largo que la recuperación de fluorescencia se esperaba. Con el volumen focal mantiene estacionario dentro de la muestra (en nuestro software de microscopio esto se logra mediante un análisis de punto), el registro de un número razonable de los datos de recuento de fotones. </li><li> Aumentar el poder y tomar otra serie de datos de conteo de fotones. Repita hasta que el aumento en el recuento de fotones parece disminuir de forma significativa con respecto al aumento de potencia. </li><li> Parcela de registro (recuento de fotones) como una función de registro (el poder). Una desviación de una pendiente de dos indica blanqueo en el monitor. Elegir como su poder de controlar un poder que está por debajo de este corte, pero que permite una señal a ruido razonable. </li></ol> 3. Determinar los parámetros de entrada para el generador de impulsos y multicanal escalador. <ol><li> Determinar el dorso de la de la sobre-estimación del coeficiente de difusión y el tiempo de recuperación basado en la literatura y / o la ecuación de Stokes-Einstein y la ecuación de difusión. </li><li> Establecer los valores de los parámetros necesarios en el generador de impulsos y el escalador de la. <ol class="lalpha"><li> En el generador de impulsos, establecer los tiempos pre-cloro y lejía. Un buen conjunto de reglas de oro es que el cloro pre-debe ser 1-2 veces mayor que el tiempo medio de recuperación de espera y el tiempo de cloro debe ser de una décima parte el tiempo medio de recuperación esperado. </li><li> En el escalador, establecer el ancho de caja de aproximadamente la mitad de la duración de la lejía, y establecer el número de contenedores por cada registro de manera que el producto de su ancho de caja seleccionada y contenedores por registro es mayor o aproximadamente igual a la esperada recuperación completa más pre -cloro y los tiempos de lejía. </li><li> Volver al generador de impulsos y establecer la frecuencia de la secuencia de pre-bleach/bleach/monitor igual a un valor justo inferior a la inversa del producto de la papelera de la anchura del número de contenedores por registro. * </li><li> Por último, establezca el número de registros / digitalización en el escalador sobre la base de la intensidad de la señal de su poder controlar elegido. * Es muy importante que el tiempo de una secuencia completa pre-bleach/bleach/monitor (1/Frecuencia) situado en el generador de impulsos es mayor que el tiempo de recogida de datos (ancho x contenedores bin / registro) establecido en el escalador. Si este criterio no se cumple, dispara múltiples cloro pueden aparecer dentro de un registro único en el escalador. </li></ol></li><li> Ajuste la alimentación del monitor a un nivel aceptable, determinado previamente. Ajustar la potencia de cloro a 200 mW o menos por encima del límite de blanqueo determinado durante la prueba de monitor de blanqueo. Estas facultades están establecidas en diferentes tensiones a través del cristal PC. </li><li> Sello del microscopio, apagar las luces, a su vez en el PMT, realizar un escaneo en el punto en el software de microscopio y luego encender el generador de impulsos y escalador. </li><li> Analizar la curva de recuperación como resultado por el trazado de la curva y marca tres puntos importantes: 1) la fluorescencia pre-cloro, 2) la fluorescencia inmediatamente después de la lejía, y 3) la fluorescencia en el extremo del conjunto de datos. Use los valores de fluorescencia pre-e inmediatamente después de cloro para calcular mejor el tiempo medio de recuperación. </li><li> Con esta estimación del tiempo medio de recuperación, utilice las reglas generales señalados anteriormente para determinar los nuevos valores de la pre-lejía, cloro, y la duración de bin ancho. Además, el uso de la fluorescencia pre-cloro y de fluorescencia en el extremo del conjunto de datos para calcular mejor el tiempo necesario para lograr la plena recuperación. Como regla general, los datos deben ser recogidos por un período de tiempo que permite la recuperación total que debe presentarse en la segunda mitad de los datos. </li><li> Ajustar los parámetros en el generador de impulsos y escalador, tomar una nueva curva, y continuar ajustando los parámetros hasta que la curva presenta un blanqueador fuerte y pronta recuperación. Tenga en cuenta que la duración de cloro puede, y debe, ser reducido por debajo de la regla de oro, si es posible. El poder de cloro también se puede ajustar, si es demasiado bajo, o demasiado profundo, un blanqueador se logra. </li></ol> 4. Prueba de la saturación de la excitación. <ol><li> Utilizando el monitor y el poder adecuados cloro, determinado previamente, tomar una serie de MP-FRAP curvas. Analizar cada uno de recuperación, normalización de la fluorescencia pre-lejía, utilizando la forma matemática adecuada y una de mínimos cuadrados no lineal algoritmo de ajuste (ver &quot;Análisis de Datos&quot;). Registrar el valor ajustado del parámetro de profundidad lejía.</li><li> Siga tomando y análisis de series sucesivas de MP-FRAP curvas a aumentar los valores del poder blanqueador. </li><li> Gráfico log (parámetro blanqueo de profundidad) en función de log (el poder). Cualquier desviación de una pendiente de dos indica la saturación de excitación. Elija un poder blanqueador de cloro que produce un fuerte, pero no causa la saturación. </li></ol> 5. Siga tomando MP-FRAP curvas. <ol><li> Seguir tomando las curvas, como el anterior, con todos los parámetros configurados correctamente. </li></ol> 6. Análisis de datos. <ol><li> Eliminar los datos pre-cloro y lejía de los datos de fluorescencia de fijar y ajustar los datos de tiempo de tal manera que t = 0 se corresponde con el valor de fluorescencia inmediatamente después de la lejía. </li><li> Normalizar la recuperación de fluorescencia resultante con respecto a la media pre-cloro fluorescencia. </li><li> Ajustar la curva normalizada utilizando el modelo matemático adecuado y de un algoritmo no lineal, al menos, las plazas ajuste, como por ejemplo la función lsqcurvefit en MATLAB. El modelo aquí presentado representa la recuperación de la fluorescencia influenciada tanto por la difusión y el flujo de convección: <img src="/files/ftp_upload/1636/1636eq.jpg" alt="Ecuación" /> donde F es la fluorescencia o media antes de la lejía, β es el parámetro de profundidad de cloro, τ D es la característica de tiempo de recuperación de difusión, R es la relación entre el cuadrado de la axial (w z) para radial (w r) de ancho de la volumen focal de dos fotones, τ vx = w r / v x, τ vy = w R / V y, τ vz = w z / z v, y v 2 = v x 2 + y 2 + v v z 2 es el velocidad de la corriente de convección. De flujo confinado al plano de imagen, τ vz → ∞ y 1 / τ vx + 1 / τ vy se puede sustituir por una vr / τ. En ausencia de flujo convectivo, tanto en el numerador exponenciales ir a 1 y la expresión se reduce, a sólo dos parámetros de ajuste, β y D τ. El coeficiente de difusión se puede calcular fácilmente como D = w r 2 / D 8τ. </li></ol> 7. Los resultados representativos. <img src="/files/ftp_upload/1636/1636fig1.jpg" alt="Figura 1" /> Figura 1. Lejía Representante y la curva de recuperación de FITC-BSA. Una curva de una buena recuperación exhibe una lejía fuerte y suave recuperación, con la fluorescencia a la recuperación completa se informa de la segunda mitad de los datos. <img src="/files/ftp_upload/1636/1636fig2.jpg" alt="Figura 2" /> Figura 2. Curva de recuperación normalizada de FITC-BSA (rojo) y el correspondiente ajuste de mínimos cuadrados (negro). Este ajuste se obtiene un coeficiente de difusión del 52,9 UM2 / s, de acuerdo con la literatura.

<ol>
	<li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Biophys J. 60, 73-76 (1990).</li><li>Brown, E. B., Wu, E. S., Zipfel, W., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+molecular+diffusion+in+solution+by+multiphoton+fluorescence+photobleaching+recovery.">Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.</a> Biophys J. 77, 2837-2849 (1999).</li><li>Sullivan, K. D., Sipprell, W. H. B. r. o. w. n., Jr, E. B., Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+model+of+fluorescence+recovery+expands+the+application+of+multiphoton+fluorescence+recover+after+photobleaching+in+vivo.">Improved model of fluorescence recovery expands the application of multiphoton fluorescence recover after photobleaching in vivo.</a> Biophys J. 96, 5082-5094 (2009).</li></ol>

El poder de la multi-fotón recuperación de fluorescencia después de photobleaching radica en su capacidad de conseguir que las muestras de espesor con una resolución en 3D. Desde su desarrollo en la década de 1990, MP-FRAP se ha utilizado para determinar el coeficiente de difusión (o parámetros análogos de transporte) en los cuerpos celulares, ex rebanadas gruesas de tejido vivo, y en el tejido vivo y el intersticio. En este artículo, presentamos el equipo necesario para real...

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<td>Ti:sapphire laser</td>
<td>&nbsp;</td>
<td>Spectra Physics</td>
<td>Mai Tai</td>
<td>&nbsp;</td>
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<td>Pockels Cell</td>
<td>&nbsp;</td>
<td>Conoptics</td>
<td>350-80</td>
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<td>Laser Scanning Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>Fluoview</td>
<td>&nbsp;</td>
<tr>
<td>Short pass dichroic mirror</td>
<td>&nbsp;</td>
<td>Chroma Technologies</td>
<td>670 DCSX-2P</td>
<td>&nbsp;</td>
<tr>
<td>Photomultiplier tube</td>
<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>HC125-02</td>
<td>&nbsp;</td>
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<td>Photon counter</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>SR 400</td>
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<td>Multi-channel scaler/averager</td>
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<td>Stanford Research Systems</td>
<td>SR 430</td>
<td>&nbsp;</td>
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<td>Pulse Generator</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>DG 535</td>
<td>&nbsp;</td>
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<td>FITC-BSA</td>
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<td>Invitrogen</td>
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measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching

Multi-fluorescencia de recuperación después de photobleaching es una técnica de microscopía para medir el coeficiente de difusión (o parámetros análogos de transporte) de las macromoléculas, y se puede aplicar tanto a<em&gt; In vitro</em&gt; Y<em&gt; In vivo</em&gt; Los sistemas biológicos. Multi-fluorescencia de recuperación después de photobleaching se lleva a cabo por photobleaching una región de interés dentro de una muestra fluorescente se utiliza un flash láser intensa, atenuar el haz y el control de la fluorescencia de las moléculas fluorescentes que aún fuera de la región de difusión de interés para reemplazar a las moléculas photobleached . Vamos a empezar nuestra demostración, alineando el haz de láser a través de la célula Pockels (modulador láser) ya lo largo del camino óptico a través del cuadro de escaneo láser y la lente de objetivo de la muestra. Para simplificar, vamos a utilizar una muestra de tinte fluorescente acuoso. A continuación, se determinan los parámetros adecuados para nuestra muestra experimental, incluyendo el seguimiento y los poderes de blanqueo, la duración de la lejía, anchos de caja (de conteo de fotones), y el tiempo de recuperación de fluorescencia. A continuación, se describe el procedimiento para la toma de curvas de recuperación, un proceso que se puede automatizar en gran medida a través de LabVIEW (National Instruments, Austin, TX) para un rendimiento mejorado. Finalmente, el coeficiente de difusión es determinado por la instalación de recuperación de datos para el modelo matemático adecuado utilizar un algoritmo de ajuste por mínimos cuadrados, fácilmente programable utilizando software como MATLAB (The Mathworks, Natick, MA).

Watch this Scientific Journal Video about Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching at JoVE.com

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

En este artículo vamos a describir el procedimiento para medir coeficientes de difusión con multi-fotón recuperación de fluorescencia después de photobleaching. Vamos a empezar por alinear el láser a lo largo del camino óptico de la muestra y la determinación de los parámetros experimentales adecuadas, y luego continuar la generación de curvas de fluorescencia y por último montaje de recuperación.

Medición de los coeficientes de difusión a través de dos fotones de fluorescencia de recuperación después de photobleaching

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) ...

measuring-diffusion-coefficients-via-two-photon-fluorescence-recovery

Research

JoVE Journal

Biology

11.1K vistas.  University of Rochester. En este artículo vamos a describir el procedimiento para medir coeficientes de difusión con multi-fotón recuperación de fluorescencia después de photobleaching. Vamos a empezar por alinear el láser a lo largo del camino óptico de la muestra y la determinación de los parámetros experimentales adecuadas, y luego continuar la generación de curvas de fluorescencia y por último montaje de recuperación.

Video: Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

De dos fotones axotomía y de lapso de tiempo de imagen confocal en embriones de pez cebra en vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Investigación

Biología

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse de imágenes de mitosis después de la transfección de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

De dos fotones basado en fotoactivación en vivo embriones de pez cebra

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

En la cuantificación in vivo de interacciones de proteínas G del receptor acoplado con espectralmente resuelve de dos fotones de Microscopía

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Photobleaching ensayos (FRAP y FLIP) para medir la dinámica de la cromatina de proteínas en células vivas madre embrionarias

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

Cartografía de la difusión molecular en la membrana plasmática de objetivos múltiples de Búsquedas (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Criopreservación de espermatozoides de ratón y de recuperación con el I · Kit de Cryo

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

Fácil Medición de Difusión Coeficientes de etiquetado-EGFP Plasma Proteínas de la Membrana Usando k-espacio Correlación Imagen Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperación de adultos de pez cebra Corazones para aplicaciones de alto rendimiento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

De dos fotones de imágenes de Ca intracelular<sup&gt; 2 +</sup&gt; Manejo y producción de óxido nítrico en el endotelio y las células del músculo liso de la aorta de rata aislada

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...