Le développement de qFSM est financé par le NIH U01 GM06230.

<ol>
	<li>Waterman-Storer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy+(FSM)+of+microtubules+and+actin+in+living+cells.">Fluorescent speckle microscopy (FSM) of microtubules and actin in living cells.</a> Curr Protoc Cell Biol. Chapter 4, Unit 4.10-Unit 4.10 (2002).</li><li>Ponti, A., Machacek, M., Gupton, S. L., Waterman-Storer, C. M., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+actin+networks+drive+the+protrusion+of+migrating+cells.">Two distinct actin networks drive the protrusion of migrating cells.</a> Science. 305, 1782-1786 (2004).</li><li>Zhang, X. F., Schaefer, A. W., Burnette, D. T., Schoonderwoert, V. T., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-dependent+contractile+responses+in+the+neuronal+growth+cone+are+independent+of+classical+peripheral+retrograde+actin+flow.">Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow.</a> Neuron. 40, 931-944 (2003).</li><li>Salmon, W. C., Adams, M. C., Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-wavelength+fluorescent+speckle+microscopy+reveals+coupling+of+microtubule+and+actin+movements+in+migrating+cells.">Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells.</a> J Cell Biol. 158, 31-37 (2002).</li><li>Danuser, G., Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fluorescent+speckle+microscopy+of+cytoskeleton+dynamics.">Quantitative fluorescent speckle microscopy of cytoskeleton dynamics.</a> Annu Rev Biophys Biomol Struct. 35, 361-387 (2006).</li></ol>

The authors have nothing to disclose.

Plusieurs facteurs clés sont indispensables pour réussir à obtenir des images de speckle, taches aussi bonne commencent et finissent avec la qualité de votre actine marqués par fluorescence. Un ratio élevé de l&#39;étiquetage de teinture à l&#39;actine monomère, autour de 0,4 à 0,7 veillera à ce que taches apparaissent lumineux et discret, tandis que la protéine marquée elle-même doit être soluble et libre de granulats. Tout aussi importante est la procédure la microinjection. Pour assurer l&#39;homéos...

Injection de tampon (IB) 50 mM de glutamate de potassium 0,5 M de MgCl2 (annexe 2A) Stockez jusqu&#39;à 2 ans à -20 ° C &nbsp; G-buffer 2 mM de Tris Cl ⋅, pH 8,0 (annexe 2A) 0,2 mM CaCl2 (annexe 2A) Ajouter juste avant utilisation: 0,2 mM d&#39;ATP (voir recette de 100 mM) 0,5 mM de 2-mercaptoéthanol Stockez jusqu&#39;à 1 jour à 4 ° C

live cell imaging of f-actin dynamics via fluorescent speckle microscopy (fsm)

Dans ce protocole, nous décrivons l&#39;utilisation de la microscopie fluorescente chatoiement (FSM) pour capturer des images haute résolution de la dynamique de l&#39;actine dans les cellules PtK1. Un avantage unique de la FSM est sa capacité à capturer le mouvement et la cinétique chiffre d&#39;affaires (montage / démontage) du réseau de la F-actine dans les cellules vivantes. Cette technique est particulièrement utile dans découlant des mesures quantitatives de F-actine dynamiques quand il est associé avec le logiciel de vision par ordinateur (qFSM). Nous décrivons la sélection, la micro-injection et la visualisation des sondes fluorescentes actine dans les cellules vivantes. Surtout, des procédures similaires sont applicables aux assemblées macomolecular visualisant les autres. FSM a été démontrée pour les microtubules, filaments intermédiaires, et des complexes d&#39;adhérence.

Watch this Scientific Journal Video about Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM at JoVE.com

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Sélection, la microinjection, et l&#39;imagerie de fluorescence étiqueté F-actine fluorescente via chatoiement de microscopie (FSM).

Imagerie de cellules vivantes de la F-actine fluorescente Dynamics via chatoiement de microscopie (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

live-cell-imaging-f-actin-dynamics-via-fluorescent-speckle-microscopy

Research

JoVE Journal

Biology

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

15.8K vues.  Scripps Institute. Sélection, la microinjection, et l'imagerie de fluorescence étiqueté F-actine fluorescente via chatoiement de microscopie (FSM).

Vidéo: Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Co-actine sédimentation Assay; pour l&#39;analyse des Binding Protein à F-actine

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Recherche

Biologie

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Assemblée, de chargement, et l&#39;alignement d&#39;une cellule échantillon ultracentrifugation analytique

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

Marquage fluorescent du COS-7 exprimant des protéines de fusion SNAP-tag pour imagerie de cellules vivantes

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basées sur la fluorescence de mesure de l&#39;entrée du calcium magasin fonctionnant dans des cellules vivantes: à partir de cellules cancéreuses en culture de fibre musculaire squelettique

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopie intravitale pour l&#39;imagerie Subcellular ouvrages chez des souris vivantes exprimant des protéines fluorescentes

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

Imagerie en temps réel des plantes Dynamics de surface cellulaire avec angle variable épifluorescence Microscopie

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Trois dimensions Super Resolution Microscopie de Filaments F-actine par interférométrique photoactive Localisation Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Examen de la dynamique nucléaire de levure de fission mitotique et méiotique par fluorescence Microscopie à cellules vivantes

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

Imagerie cellulaire en direct pour évaluer la dynamique du moment de la métaphase et le destin cellulaire à la suite de perturbations mitotiques spindle

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

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.

Dynamique d'oligomerisation des récepteurs de surface cellulaire dans les cellules vivantes par microscopie de fluorescence interne totale combinée à l'analyse du nombre et de la luminosité

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