Cette recherche est parrainée par le Bureau de recherche biologique et environnementale, ministère américain de l&#39;Energie et par l&#39;octroi de financement à partir du Commonwealth de la Virginie Conseil de recherche en santé. Oak Ridge National Laboratory est géré par UT-Battelle, LLC, pour le ministère américain de l&#39;Énergie et du contrat No. DE-AC05-00OR22725.

1. Préparation de Mica: <ol><li> Couper le mica (Sciences Electron Microscopy) avec des ciseaux à la taille nécessaire pour s&#39;adapter au microscope AFM (env. 22 x 30 mm). </li><li> Enchaînement du mica, des deux côtés, généralement avec du ruban pour enlever la couche extérieure, jusqu&#39;à ce que lisse couches ininterrompue demeurent. </li></ol> 2. Préparation de la solution de gélatine: <ol><li> Ajouter 100 ml d&#39;eau distillée à une bouteille de laboratoire. </li><li> Faire chauffer le biberon au micro-ondes jusqu&#39;à ce que l&#39;eau commence à bouillir. (Si un micro-ondes n&#39;est pas disponible, l&#39;eau peut être chauffée dans un bécher sur une plaque chaude.) </li><li> Peser 0,5 g de gélatine (Sigma G-6144, G-2625 ou G-2500) et ajouter à l&#39;eau chaude distillée. </li><li> Agiter doucement le flacon jusqu&#39;à ce que la gélatine soit complètement dissoute. </li><li> Refroidir la solution de gélatine à 60-70 ° C. </li><li> Verser environ 15 ml dans un petit bécher (20 ml) dans lequel le mica clivé coupées peuvent être trempés. </li></ol> 3. Mica revêtement: <ol><li> Plonger les carrés de mica dans la solution chauffée de gélatine et de retirer rapidement de sorte que le mica est couché (fig. 1a). </li><li> Après le revêtement, le soutien du mica immédiatement, sur la pointe, sur une serviette en papier pour sécher à l&#39;air ambiant (Fig. 1b). Le mica recouvert de gélatine peuvent être utilisés pendant au moins 2 semaines. L&#39;excès de solution de gélatine peuvent être conservés au réfrigérateur et utilisé pendant environ un mois par un simple réchauffage de la solution stock à entre 60-70 ° C. Des précautions doivent être prises pour s&#39;assurer que la gélatine n&#39;est pas bouillie pendant le réchauffage. </li></ol> 4. Montage des bactéries sur le mica recouvert de gélatine: <ol><li> Pellet 1 ml d&#39;une culture bactérienne (0,5 - 1,0 DO à 600 nm) de 800 à 4500 fcr. Selon les bactéries, l&#39;utilisation du RCF minimum qui sera un culot cellulaire dans ~ 5 minutes. </li><li> Laver le culot dans le même volume d&#39;eau déminéralisée filtrée ou un tampon. </li><li> Recueillir les cellules à nouveau par centrifugation. </li><li> Reprendre le culot rapidement dans 500 ul d&#39;eau déminéralisée nanopure ou un tampon. (La suspension bactérienne doit être visiblement trouble afin d&#39;avoir une concentration suffisante de cellules pour l&#39;imagerie AFM). </li><li> Appliquer la suspension de cellules (10-20 uL) pour le mica recouvert de gélatine et la propagation sur la surface à l&#39;aide d&#39;une pipette (fig. 1c). Il faut prendre soin de ne pas toucher physiquement la surface de gélatine à la pointe de la pipette en appliquant ou la diffusion de la solution bactérienne. </li><li> Laisser la surface pour se reposer pendant 10 minutes et rincez avec un jet d&#39;eau ou de tampon (figure 1 cd). Tout au long de la procédure, prendre soin de s&#39;assurer que l&#39;échantillon n&#39;est pas autorisé à sec. </li></ol> 5. Les résultats représentatifs: La figure 2 montre des images par exemple des cellules bactériennes qui ont été montés sur la gélatine-mica et imagée par l&#39;AFM. Dans nos laboratoires, les échantillons sont généralement imagée en utilisant un microscope à force atomique PicoPlus (Agilent Technologies, le Temple, AZ) équipé d&#39;une tête de balayage 100 um. L&#39;instrument fonctionne à 128-512 pixels par ligne de balayage à une vitesse de balayage de 0,5-1,0 lignes / s. Les cantilevers qui sont typiquement utilisés sont des sondes Veeco nitrure de silicium (MLCT-AUHW, Veeco, Santa Barbara, CA) avec des constantes de printemps nominales allant de 0,01 à 0,1 nN / nm. <img src="/files/ftp_upload/2880/2880fig1.jpg" alt="Figure 1"/> Figure 1. Gélatine à une température de 60-70 ° C est placé dans un bécher de 20 ml. En utilisant une paire de pinces de mica fraîchement clivées carrés sont totalement immergés dans la gélatine (a) retirée et placée debout sur une serviette en papier adossé à un support de micro centrifugeuse (b). Une goutte d&#39;échantillon bactérien est mis sur le mica et étaler avec une pipette dans les deux directions X et Y (c). Après avoir laissé l&#39;échantillon à incuber sur la surface du mica pour au moins 10 minutes, rincer l&#39;échantillon dans un courant d&#39;eau distillée. Si l&#39;immobilisation a travaillé, vous serez capable de voir une tache opaque sur le mica comme indiqué dans le panneau de droite de (d). Si l&#39;immobilisation n&#39;a pas fonctionné, vous obtiendrez un morceau de mica clair que dans le panneau gauche. La tache opaque sur les échelles de mica à la concentration de la bactérie dans la gouttelette que vous placez sur le mica de gélatine traitée. <img src="/files/ftp_upload/2880/2880fig2.jpg" alt="Figure 2"/> Figure 2. Images AFM de E. coli. Dans le graphique (a), les cellules sont montées sur la gélatine-mica et imagée de 0,005 M de PBS. A titre de comparaison, une image de cellules bactériennes monté sur la gélatine-mica et imagée dans l&#39;air est montré dans le panneau (b). Bien septa sont clairement visibles dans l&#39;image recueillie dans un liquide, une plus grande résolution, comme indiqué par la présence de pili (panneau en médaillon (b)), peut être obtenue dans l&#39;air.

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B., Franze, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+individual+glial+cells+and+neurons+in+the+CNS.">Viscoelastic properties of individual glial cells and neurons in the CNS.</a> Proc Natl Acad Sci U S A. 103, 17759-17764 (2006).</li><li>O'Toole, M., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+model+of+axonal+elongation:+force,+viscosity,+and+adhesions+govern+the+mode+of+outgrowth.">A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.</a> Biophys J. 94, 2610-2620 (2008).</li><li>Pfister, B. J., Bonislawski, D. 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Aucun conflit d&#39;intérêt déclaré.

Différents facteurs peuvent affecter la cellule microbienne de montage et d&#39;imagerie par AFM. La gélatine qui est utilisée pour le revêtement du mica est important. Commerce de gélatine est isolé à partir d&#39;un certain nombre de vertébrés, y compris les poissons, les vaches et les cochons. L&#39;origine et la méthode de traitement de déterminer la pertinence de la gélatine pour immobilisation bactérienne. De nombreuses sources et les types de gélatine ont été évalués pour leur efficacité dans l...

<table border="1"><tbody><tr><td> Nom </td><td> Société </td><td> Numéro de catalogue </td></tr><tr><td> Gélatine </td><td> Sigma, St. Louis, MO </td><td> G6144, G2625 G2500 ou </td></tr><tr><td> Microscope à Force Atomique PicoPlus </td><td> Agilent Technologies, Tempe, AZ </td><td></td></tr><tr><td> Cantilevers AFM </td><td> Veeco, Santa Barbara, Californie </td><td> MLCT-AUHW </td></tr></tbody></table>

bacterial immobilization for imaging by atomic force microscopy

L&#39;AFM est une haute résolution (échelle nm) outil d&#39;imagerie que les sondes mécanique d&#39;une surface. Il a la capacité de cellules d&#39;image et de biomolécules, dans un environnement liquide, sans la nécessité de traiter chimiquement l&#39;échantillon. Afin d&#39;atteindre cet objectif, l&#39;échantillon doit suffisamment adhérer à la surface de montage afin de prévenir l&#39;enlèvement par les forces exercées par la pointe de numérisation AFM. Dans de nombreux cas, l&#39;imagerie succès dépend de l&#39;immobilisation de l&#39;échantillon à la surface de montage. Idéalement, l&#39;immobilisation devrait être minimalement invasive à l&#39;échantillon de telle sorte que les processus métaboliques et attributs fonctionnels ne sont pas compromises. En revêtement de surfaces mica fraîchement clivé par porcine (porc) de gélatine, les bactéries chargées négativement peuvent être immobilisés sur la surface et imagée dans le liquide par l&#39;AFM. Immobilisation des bactéries sur la gélatine mica est très probablement due à l&#39;interaction électrostatique entre les bactéries chargées négativement et la gélatine chargée positivement. Plusieurs facteurs peuvent interférer avec immobilisation bactérienne, y compris les constituants chimiques du liquide dans lequel les bactéries sont suspendues, le temps d&#39;incubation de la bactérie sur les caractéristiques de gélatine mica recouvert de surface, de la souche bactérienne et le milieu dans lequel les bactéries sont imagés. Globalement, l&#39;utilisation de gélatine-mica se trouve être généralement applicables pour l&#39;imagerie des cellules microbiennes.

Watch this Scientific Journal Video about Bacterial Immobilization for Imaging by Atomic Force Microscopy at JoVE.com

Bacterial Immobilization for Imaging by Atomic Force Microscopy

En direct des bactéries Gram-négatives et gram-positif peut être immobilisé sur la gélatine-mica et imagée dans un liquide en utilisant la microscopie à force atomique (AFM).

Immobilisation bactérienne pour l&#39;imagerie par microscopie à force atomique

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

bacterial-immobilization-for-imaging-by-atomic-force-microscopy

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Bioengineering

17.2K vues.  Oak Ridge National Laboratory. En direct des bactéries Gram-négatives et gram-positif peut être immobilisé sur la gélatine-mica et imagée dans un liquide en utilisant la microscopie à force atomique (AFM).

Vidéo: Bacterial Immobilization for Imaging by Atomic Force Microscopy

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Visualisation de l&#39;ADN recombinant et des complexes de protéines par microscopie à force atomique

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

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Bio-ingénierie

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Quantification des propriétés mécaniques de la Glycocalyx endothéliale par microscopie à force atomique

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mesurer les propriétés mécaniques des cellules vivantes par microscopie à force atomique

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Haute résolution spatio-temporelle d&#39;analyse de la dynamique des récepteurs par simple molécule microscopie de fluorescence

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Roman microscopie à force atomique Based biopanning pour l&#39;isolement de la morphologie réactifs spécifiques contre TDP-43 variantes dans la sclérose latérale amyotrophique

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Microscopie à force atomique imagerie et spectroscopie de force de bicouches lipidiques supportées

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Visualisation de l’angiogenèse par microscopie Multiphoton In Vivo en échafaudage de 3D-PLGA/PNVS génétiquement modifiés pour la réparation osseuse défaut critique substance

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

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.

Visualiser la Formation d’adhérences dans les cellules par le biais de pointe tourne disque-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 ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

Microscopie de force de traction pour étudier l’activation de lymphocyte de B

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Application de la microscopie de force atomique pour détecter l’arthrose précoce

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...