Esta investigación está patrocinada por la Oficina de Investigación Biológica y Ambiental, Departamento de Energía de EE.UU. y por subvenciones de la Junta de Virginia Commonwealth Investigación en Salud. Oak Ridge National Laboratory es administrado por UT-Battelle, LLC, para el Departamento de Energía de EE.UU. bajo el Contrato No. DE-AC05-00OR22725.

1. Mica de preparación: <ol><li> Cortar la mica (Microscopía Electrónica de Ciencias) con unas tijeras al tamaño necesario para ajustarse al microscopio AFM (aprox. 22 x 30 mm). </li><li> Cleave la mica de ambas partes, generalmente por medio de cinta para quitar la capa externa, hasta que sólo quedan las capas continua suave. </li></ol> 2. Preparación de la solución de gelatina: <ol><li> Añadir 100 ml de agua destilada a una botella de laboratorio. </li><li> Calentar el biberón en el microondas hasta que el agua comience a hervir. (Si el microondas no está disponible, el agua se puede calentar en un vaso sobre un plato caliente.) </li><li> Pesar 0,5 gramos de la gelatina (Sigma G-6144, G 2625-G o 2500) y añadir el agua destilada caliente. </li><li> Haga girar suavemente la botella hasta que la gelatina se disuelva por completo. </li><li> Enfriar la solución de gelatina a 60-70 º C. </li><li> Vierta aproximadamente 15 ml en un vaso pequeño (20 ml) en el que se puede cortar la mica escindida de cruce. </li></ol> 3. Mica de recubrimiento: <ol><li> Sumerja las plazas mica en la solución de gelatina caliente y retirar rápidamente de tal manera que la mica está cubierta (Fig. 1a). </li><li> Después del recubrimiento, el apoyo a la mica de inmediato, en la orilla, sobre una toalla de papel para secarse en el aire ambiente (fig. 1b). La mica recubierta de gelatina se puede utilizar por lo menos 2 semanas. El exceso de solución de gelatina se puede mantener refrigerada y usada por aproximadamente un mes, simplemente calentar la solución de reserva de entre 60-70 º C. Se debe tener cuidado para asegurar que la gelatina no se hierve durante el recalentamiento. </li></ol> 4. Montaje de las bacterias en la mica recubierta de gelatina: <ol><li> Pellet 1 ml de un cultivo bacteriano (0,5 a 1,0 DO a 600 nm) de 800 a 4500 fcr. Dependiendo de la bacteria, el uso de la RCF mínimo que se sedimenten las células de ~ 5 minutos. </li><li> Lavar el sedimento en el mismo volumen de agua filtrada desionizada o tampón. </li><li> Recoger las células de nuevo por centrifugación. </li><li> Resuspender el precipitado con prontitud en 500 L de agua desionizada o tampón Nanopure. (La suspensión bacteriana deberá presentar una apariencia turbia con el fin de tener una concentración adecuada de las células de AFM de imágenes.) </li><li> Solicitar la suspensión de células (10-20 l) de la mica recubiertos de gelatina y se extendió sobre la superficie con una punta de la pipeta (Fig. 1c). Se debe tener cuidado de no contacto físico con la superficie de la gelatina con la punta de la pipeta, mientras que la aplicación o la difusión de la solución bacteriana. </li><li> Permita que la superficie en reposo durante 10 minutos y enjuagar con un chorro de agua o tampón (Fig. 1 cd). A lo largo del procedimiento, tener cuidado de asegurarse que la muestra no se deja secar. </li></ol> 5. Los resultados representativos: La figura 2 muestra imágenes de ejemplo de las células bacterianas que se han montado en gelatina mica recubierta y fotografiada por la AFM. En nuestros laboratorios, las muestras suelen ser fotografiado usando un microscopio de fuerza atómica PicoPlus (Agilent Technologies, Temple, AZ) equipado con un cabezal de escaneado 100 micras. El instrumento es operado a 128-512 píxeles por línea de exploración a una velocidad de escaneo de líneas de 0,5-1,0 / s. Los voladizos que se suelen utilizar son Veeco sondas de nitruro de silicio (MLCT-AUHW, Veeco, Santa Barbara, CA) con las constantes de la primavera nominales que van desde 0,01 hasta 0,1 nN / nm. <img src="/files/ftp_upload/2880/2880fig1.jpg" alt="Figura 1"/> Figura 1. Gelatina a una temperatura de 60-70 º C se coloca en un vaso de precipitados de 20 ml. Con un par de pinzas recién cortado plazas mica están totalmente inmersos en la gelatina (a) ha retirado y colocado en posición vertical sobre una toalla de papel apoyado en un bastidor de microcentrífuga (b). Una gota de la muestra bacteriana se coloca en la mica y se extendió con una punta de pipeta, tanto en el X e Y (c). Después de permitir que la muestra se incuba en la superficie de la mica por lo menos durante 10 minutos, enjuague la muestra en una corriente de agua destilada. Si la inmovilización trabajado usted podrá ver una mancha opaca en la mica, como se muestra en el panel de la derecha de (d). Si la inmovilización no funcionó obtendrá un pedazo de mica clara como en el panel de la izquierda. La mancha opaca en las escalas de mica a la concentración de la bacteria en la gota que se coloca en la mica de gelatina tratada. <img src="/files/ftp_upload/2880/2880fig2.jpg" alt="Figura 2"/> Figura 2. AFM imágenes de E. coli. En el panel (a), las células se montan en gelatina mica recubierta y la imagen en 0.005 M de PBS. Por comparación, una imagen de las células bacterianas montados en gelatina-mica recubierta y la imagen en el aire se muestra en el panel (b). A pesar de los tabiques son claramente visibles en la imagen recogida en el líquido, mayor resolución, como lo indica la presencia de pili (panel de inserción (b)), se puede obtener en el aire.

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No hay conflictos de interés declarado.

Hay varios factores que pueden afectar a las células microbianas y de montaje de imágenes por AFM. La gelatina que se utiliza para el recubrimiento de la mica es importante. Comerciales de gelatina está aislado de una serie de vertebrados, incluyendo peces, vacas y cerdos. El origen y el método de procesamiento de determinar la idoneidad de la gelatina para la inmovilización de bacterias. Numerosas fuentes y tipos de gelatina fueron evaluados para determinar su eficacia en las bacterias inmovilizar [1]. Los dos gel...

<table border="1"><tbody><tr><td> Nombre </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Gelatina </td><td> Sigma, St. Louis, MO </td><td> G6144, G2625 o G2500 </td></tr><tr><td> PicoPlus Microscopio de Fuerza Atómica </td><td> Agilent Technologies, Tempe, AZ </td><td></td></tr><tr><td> AFM voladizos </td><td> Veeco, Santa Barbara, CA </td><td> MLCT-AUHW </td></tr></tbody></table>

bacterial immobilization for imaging by atomic force microscopy

AFM es una de alta resolución (escala nm) herramienta de imagen que las sondas de una superficie mecánicamente. Tiene la capacidad de las células de la imagen y las biomoléculas, en un medio líquido, sin la necesidad de tratar químicamente la muestra. Con el fin de lograr este objetivo, la muestra suficientemente que se adhieren a la superficie de montaje para evitar la extracción de las fuerzas ejercidas por su punta de exploración AFM. En muchos casos, las imágenes éxito depende de la inmovilización de la muestra a la superficie de montaje. De manera óptima, la inmovilización debe ser mínimamente invasiva para la muestra de tal manera que los procesos metabólicos y los atributos funcionales que no están comprometidos. Por el recubrimiento de superficies mica recién abierto, con porcino (cerdo) de gelatina, las bacterias cargadas negativamente pueden ser inmovilizadas en la superficie y la imagen en un líquido por AFM. La inmovilización de las células bacterianas en gelatina mica recubierta es más probable debido a la interacción electrostática entre las bacterias cargadas negativamente y la gelatina con carga positiva. Hay varios factores que pueden interferir con la inmovilización de bacterias, incluyendo los componentes químicos del líquido en el que las bacterias de suspensión, el tiempo de incubación de la bacteria en las características de gelatina mica recubierta de superficie, de la cepa bacteriana y el medio en el que las bacterias son expuestas. En general, el uso de la mica recubierta de gelatina resulta ser de aplicación general para las células microbianas de imágenes.

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

Viven las bacterias Gram-negativas y Gram-positivas pueden ser inmovilizadas en gelatina mica recubierta y la imagen en el líquido utilizando microscopía de fuerza atómica (AFM).

Inmovilización de bacterias para tratamiento de imágenes de microscopía de fuerza atómica

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

Research

JoVE Journal

Bioengineering

17.2K vistas.  Oak Ridge National Laboratory. Viven las bacterias Gram-negativas y Gram-positivas pueden ser inmovilizadas en gelatina mica recubierta y la imagen en el líquido utilizando microscopía de fuerza atómica (AFM).

Video: 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.

La visualización de ADN recombinante y complejos de proteínas utilizando microscopía de fuerza atómica

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

Investigación

Bioingeniería

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.

La cuantificación de las propiedades mecánicas de la glicocálix endotelial con la Microscopía de Fuerza Atómica

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).

Medición de las propiedades mecánicas de las células vivas mediante microscopía de fuerza atómica

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.

De alta resolución espacio-temporal Análisis de la Dinámica del receptor por Casa molécula de microscopía de fluorescencia

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.

Novela Microscopía de Fuerza Atómica Based biopanning para el aislamiento de Morfología reactivos específicos contra la TDP-43 variantes en la esclerosis lateral amiotrófica

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.

Fuerza Atómica Microscopía Imaging y Fuerza Espectroscopia de admitidos Bilayers lípidos

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.

Visualización de angiogénesis por microscopía multifotón En Vivo en modificados genéticamente 3D-PLGA/nHAp andamio para Calvarial críticos de la reparación del defecto óseo

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.

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

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

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.

Microscopía de fuerza de tracción para estudiar la activación de linfocitos 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).

Aplicación de microscopía de fuerza atómica para detectar la osteoartritis temprana

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