RESUMEN
The evolution of hemodialysis membranes (dialyzer, artificial kidney) was remarkable, since Dow Chemical began manufacturing hollow fiber hemodialyzers in 1968, especially because it involved industrial chemistry, including polymer synthesis and membrane manufacturing process. The development of hemodialysis membranes has brought about the field of medical devices as a major industry. In addition to conventional electron microscopy, scanning probe microscopy (SPM), represented by atomic force microscopy (AFM), has been used in membrane science research on porous membranes for hemodialysis, and membrane science contributes greatly to the hemodialyzer industry. Practical studies of membrane porous structure-function relationship have evolved, and methods for analyzing membrane cross-sectional morphology were developed, such as the ion milling method, which was capable of cutting membrane cross sections on the order of molecular size to obtain smooth surface structures. Recently, following the global pandemic of SARS-CoV-2 infection, many studies on new membranes for extracorporeal membrane oxygenator have been promptly reported, which also utilize membrane science researches. Membrane science is playing a prominent role in membrane-based technologies such as separation and fabrication, for hemodialysis, membrane oxygenator, lithium ion battery separators, lithium recycling, and seawater desalination. These practical studies contribute to the global medical devices industry.
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Membranas Artificiales , Diálisis Renal , Humanos , Imagenología Tridimensional , Microscopía Electrónica/métodos , Microscopía de Sonda de Barrido/métodos , Porosidad , Diálisis Renal/instrumentaciónRESUMEN
Scanning probe microscopy techniques, such as atomic force microscopy and scanning tunnelling microscopy, are harnessed to image nanoscale structures with an exquisite resolution, which has been of significant value in a variety of areas of nanotechnology. These scanning probe techniques, however, are not generally suitable for high-throughput imaging, which has, from the outset, been a primary challenge. Traditional approaches to increasing the scalability have involved developing multiple probes for imaging, but complex probe design and electronics are required to carry out the detection method. Here, we report a probe-based imaging method that utilizes scalable cantilever-free elastomeric probe design and hierarchical measurement architecture, which readily reconstructs high-resolution and high-throughput topography images. In a single scan, we demonstrate imaging with a 100-tip array to obtain 100 images over a 1-mm2 area with 106 pixels in less than 10 min. The potential for large-scale tip integration and the advantage of a simple probe array suggest substantial promise for our approach to high-throughput imaging far beyond what is currently possible.
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Microscopía de Sonda de Barrido , Nanotecnología , Microscopía de Fuerza Atómica/métodos , Microscopía de Sonda de Barrido/métodos , Microscopía de Túnel de Rastreo , Nanotecnología/métodos , ProteínasRESUMEN
Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM). However, time-resolved imaging techniques tend to either have a lower resolution, are limited to small areas, or cause damage to the cells, thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of eukaryotic cells. By utilizing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering, we developed a microscopy method that can resolve spatiotemporally diverse three-dimensional (3D) processes on the cell membrane at sub-5-nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of subsecond to days, imaging diverse processes ranging from endocytosis, micropinocytosis, and mitosis to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.
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Microscopía de Sonda de Barrido , Orgánulos , Microscopía de Sonda de Barrido/métodos , Microscopía de Fuerza Atómica , Membrana CelularRESUMEN
Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10-20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.
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Células Cultivadas/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía de Sonda de Barrido/métodos , Microscopía/métodos , Neoplasias Óseas/patología , Línea Celular Tumoral , Marcadores Fiduciales , Colorantes Fluorescentes , Oro , Humanos , Microscopía Electrónica de Rastreo , Mitocondrias/ultraestructura , Nanotubos , Osteosarcoma/patología , Flujo de TrabajoRESUMEN
Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties (i.e., elasticity, viscoelasticity, and viscosity) of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.
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Microscopía de Sonda de Barrido/métodos , Medicina Reproductiva/métodos , Animales , Fenómenos Biomecánicos , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/ultraestructura , Células Germinativas/citología , Células Germinativas/metabolismo , Células Germinativas/ultraestructura , Humanos , Microscopía de Fuerza Atómica/métodos , Microscopía de Sonda de Barrido/normas , Imagen Molecular/métodos , Imagen Molecular/normas , Medicina Reproductiva/normas , Imagen Individual de Molécula/métodosRESUMEN
Cellulose nanocrystals (CNC) are the focus of significant attention in the broad area of sustainable technologies for possessing many desirable properties such as a large surface area, high strength and stiffness, outstanding colloidal stability, excellent biocompatibility and biodegradability, low weight and abundance in nature. Yet, a fundamental understanding of the micro- and nanoscale electrical charge distribution on nanocellulose still remains elusive. Here we present direct quantification and mapping of surface charges on CNCs at ambient condition using advanced surface probe microscopy techniques such as Kelvin probe force microscopy (KPFM), electrostatic force microscopy (EFM) and force-distance (F-D) curve measurements. We show by EFM measurements that the surface charge in the solid-state (as contrasted with liquid dispersions) present at ambient condition on CNCs provided by Innotech Alberta is intrinsically negative and the charge density is estimated to be 13 µC/cm2. These charges also result in CNCs having two times the adhesive force exhibited by SiO2 substrates in adhesion mapping studies. The origin of negative surface charge is likely due to the formation of CNCs through sulfuric acid hydrolysis where sulfate half esters groups remained on the surface (Johnston et al., 2018).
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Celulosa/química , Microscopía de Fuerza Atómica/métodos , Microscopía de Sonda de Barrido/métodos , Nanopartículas/química , Hidrólisis , Fenómenos Físicos , Dióxido de Silicio , Ácidos Sulfúricos/química , Propiedades de SuperficieRESUMEN
Cellular force microscopy (CFM) is a noninvasive microindentation method used to measure plant cell stiffness in vivo. CFM is a scanning probe microscopy technique similar in operation to atomic force microscopy (AFM); however, the scale of movement and range of forces are much larger, making it suitable for stiffness measurements on turgid plant cells in whole organs. CFM experiments can be performed on living samples over extended time periods, facilitating the exploration of the dynamics of processes involving mechanics. Different sensor technologies can be used, along with a variety of probe shapes and sizes that can be tailored to specific applications. Measurements can be made for specific indentation depths, forces and timing, allowing for very precise mechanical stimulation of cells with known forces. High forces with sharp tips can also be used for mechanical ablation of cells with force feedback.
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Módulo de Elasticidad , Microscopía de Sonda de Barrido/métodos , Cebollas/citología , Epidermis de la Planta/citología , Fenómenos Biomecánicos , Pared Celular/química , Diseño de Equipo , Microscopía de Sonda de Barrido/instrumentación , Cebollas/química , Células Vegetales/química , Epidermis de la Planta/química , Programas InformáticosRESUMEN
Atomic force microscopy (AFM) is a widely used imaging technique in material sciences. After becoming a standard surface-imaging tool, AFM has been proven to be useful in addressing several biological issues such as the characterization of cell organelles, quantification of DNA-protein interactions, cell adhesion forces, and electromechanical properties of living cells. AFM technique has undergone many successful improvements since its invention, including fluidic force microscopy (FluidFM), which combines conventional AFM with microchanneled cantilevers for local liquid dispensing. This technology permitted to overcome challenges linked to single-cell analyses. Indeed, FluidFM allows isolation and injection of single cells, force-controlled patch clamping of beating cardiac cells, serial weighting of micro-objects, and single-cell extraction for molecular analyses. This work aims to review the recent studies of AFM implementation in molecular and cellular biology.
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Técnicas Citológicas/métodos , Microscopía de Sonda de Barrido/métodos , Biología Molecular/métodos , Microscopía de Sonda de Barrido/tendenciasRESUMEN
Scanning probe microscopy has enabled the creation of a variety of methods for the constructive ('additive') top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.
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Microscopía de Sonda de Barrido/métodos , Nanotecnología/métodos , Técnicas de Cultivo de CélulaRESUMEN
Identification of nanoparticles and nanosystems into cells and biological matrices is a hot research topic in nanobiotechnologies. Because of their capability to map physical properties (mechanical, electric, magnetic, chemical, or optical), several scanning probe microscopy based techniques have been proposed for the subsurface detection of nanomaterials in biological systems. In particular, atomic force microscopy (AFM) can be used to reveal stiff nanoparticles in cells and other soft biomaterials by probing the sample mechanical properties through the acquisition of local indentation curves or through the combination of ultrasound-based methods, like contact resonance AFM (CR-AFM) or scanning near field ultrasound holography. Magnetic force microscopy can detect magnetic nanoparticles and other magnetic (bio)materials in nonmagnetic biological samples, while electric force microscopy, conductive AFM, and Kelvin probe force microscopy can reveal buried nanomaterials on the basis of the differences between their electric properties and those of the surrounding matrices. Finally, scanning near field optical microscopy and tip-enhanced Raman spectroscopy can visualize buried nanostructures on the basis of their optical and chemical properties. Despite at a still early stage, these methods are promising for detection of nanomaterials in biological systems as they could be truly noninvasive, would not require destructive and time-consuming specific sample preparation, could be performed in vitro, on alive samples and in water or physiological environment, and by continuously imaging the same sample could be used to dynamically monitor the diffusion paths and interaction mechanisms of nanomaterials into cells and biological systems. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.
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Microscopía de Sonda de Barrido/métodos , Nanopartículas/ultraestructura , Animales , Materiales Biocompatibles , Células/ultraestructura , Humanos , Fenómenos Magnéticos , Microscopía de Fuerza Atómica , Microscopía Electroquímica de Rastreo , Microscopía de Sonda de Barrido/instrumentación , Nanotecnología , Espectrometría Raman , Biología de SistemasRESUMEN
Scanning ion conductance microscopy (SICM), one kind of scanning probe microscopy technique, featuring the advantage of non-contact imaging of sample surfaces in three dimensions with high resolution, has been widely applied in characterizations of sample topography, especially for soft materials. However, the time consuming imaging process of SICM restricts its further applications, such as in characterization of dynamic change of sample surface. In this work, a fast control mode of SICM, named as a continuous control mode, has been developed. In this mode, the SICM probe (i.e., pipette) is controlled by speed instructions in the axial direction of pipette (Z axis), and the pipette position is determined by the position sensor. Compared to the conventional piezo control mode of SICM (i.e., the stepwise control mode), in which the pipette is controlled by the position instructions and moves step by step, the continuous control mode can perform the continuous movement of the pipette in Z axis and overcome the time consuming problem caused by the repeated acceleration and deceleration of the pipette during the stepwise mode. Moreover, the imaging resolution in Z axis is not restricted by the pipette movement step and the imaging rate in the continuous control mode can be significantly enhanced without losing imaging quality. The approach speed of pipette in the continuous control mode can reach at 300â¯nm/ms, which is much faster than that in the stepwise mode. The surfaces of the soft polydimethylsiloxane (PDMS) samples with three different patterns, the hard metal grating sample and the cardiac fibroblasts as the biological sample demo were comparably scanned by SICM using the continuous control mode and the stepwise approach mode, respectively. The obtained SICM images of the sample topography prove that the continuous control mode can not only reduce the imaging deviation, but also efficiently improve the scanning rate of SICM. Furthermore, the continuous control mode can reconstruct the sample topography more stably compared to the stepwise control mode. The continuous control mode developed in this work can provide an efficient and reliable control strategy for improving the imaging performance of SICM system, and therefore can be potentially applied in dynamic characterizations of various samples in material science, biology and chemistry fields.
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Iones/química , Microscopía de Sonda de Barrido/métodos , Animales , Dimetilpolisiloxanos/química , Diseño de Equipo/métodos , Fibroblastos/fisiología , Corazón/fisiología , Nylons/química , Ratas , Ratas Sprague-DawleyRESUMEN
Scalability is the major challenge for scanning probe lithography (SPL). Recently developed cantilever-free scanning probe technologies provide a solution to the issue of scalability by incorporating massive arrays of polymer pens, which fundamentally overcome the low-throughput nature of SPL. The further development of cantilever-free SPL brings up a variety of applications in electronics, biology, and chemical synthesis. In this Perspective, we highlight the space-confined synthesis of complex nanostructures enabled by different types of cantilever-free SPL technologies.
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Microscopía de Sonda de Barrido/métodos , Nanoestructuras/química , Nanotecnología/métodos , Polímeros/química , Impresión , Propiedades de SuperficieRESUMEN
Water permeability is a key feature of the cell plasma membranes and it has seminal importance for a number of cell functions such as cell volume regulation, cell proliferation, cell migration, and angiogenesis to name a few. The transport of water occurs mainly through plasma membrane water channels , the aquaporins, who have very important function in physiological and pathophysiological states. Due to the above the experimental assessment of the water permeability of cells and tissues is necessary. The development of new methodologies of measuring water permeability is a vibrant scientific field that constantly develops during the past three decades along with the advances in imaging mainly. In this chapter we describe and critically assess several methods that have been developed for the measurement of water permeability both in living cells as well as in tissues with a focus in the first category.
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Acuaporinas/metabolismo , Microscopía de Fuerza Atómica/métodos , Microscopía Electroquímica de Rastreo/métodos , Microscopía de Sonda de Barrido/métodos , Imagen Molecular/métodos , Agua/metabolismo , Animales , Acuaporinas/genética , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Movimiento Celular , Proliferación Celular , Perros , Impedancia Eléctrica , Expresión Génica , Humanos , Células de Riñón Canino Madin DarbyRESUMEN
Charge trapping properties of electrons and holes in copper-doped zinc oxide (ZnO:Cu) films have been studied by scanning probe microscopy. We investigated the surface potential dependence on the voltage and duration applied to the copper-doped ZnO films by Kelvin probe force microscopy. It is found that the Fermi Level of the 8 at.% Cu-doped ZnO films shifted by 0.53 eV comparing to undoped ZnO films. This shift indicates significant change in the electronic structure and energy balance in Cu-doped ZnO films. The Fermi Level (work function) of zinc oxide films can be tuned by Cu doping, which are important for developing this functional material. In addition, Kelvin probe force microscopy measurements demonstrate that the nature of contact at Pt-coated tip/ZnO:Cu interface is changed from Schottky contact to Ohmic contact by increasing sufficient amount of Cu ions. The charge trapping property of the ZnO films enhance greatly by Cu doping (~10 at.%). The improved stable bipolar charge trapping properties indicate that copper-doped ZnO films are promising for nonvolatile memory applications.
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Cobre/química , Electricidad , Microscopía de Sonda de Barrido/métodos , Óxido de Zinc/química , Microscopía de Fuerza Atómica , Propiedades de Superficie , VolatilizaciónRESUMEN
We present a scanning Hall probe microscope operating in ambient conditions. One of the unique features of this microscope is the use of the same stepper motors for both sample positioning as well as scanning, which makes it possible to have a large scan range (few mm) in the x and y directions, with a scan resolution of 0.1 µm. Protocols have been implemented to enable scanning at different heights from the sample surface. The z range is 35 mm. Microstructured Hall probes of size 1-5 µm have been developed. A minimum probe-sample distance <2 µm has been obtained by the combination of new Hall probes and probe-sample distance regulation using a tuning fork based force detection technique. The system is also capable of recording local B(z) profiles. We discuss the application of the microscope for the study of micro-magnet arrays being developed for applications in micro-systems.
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Campos Magnéticos , Microscopía de Sonda de Barrido/métodos , Modelos Teóricos , Microscopía de Sonda de Barrido/instrumentaciónRESUMEN
This paper reports an array-designed C84-embedded Si substrate fabricated using a controlled self-assembly method in an ultra-high vacuum chamber. The characteristics of the C84-embedded Si surface, such as atomic resolution topography, local electronic density of states, band gap energy, field emission properties, nanomechanical stiffness, and surface magnetism, were examined using a variety of surface analysis techniques under ultra, high vacuum (UHV) conditions as well as in an atmospheric system. Experimental results demonstrate the high uniformity of the C84-embedded Si surface fabricated using a controlled self-assembly nanotechnology mechanism, represents an important development in the application of field emission display (FED), optoelectronic device fabrication, MEMS cutting tools, and in efforts to find a suitable replacement for carbide semiconductors. Molecular dynamics (MD) method with semi-empirical potential can be used to study the nanoindentation of C84-embedded Si substrate. A detailed description for performing MD simulation is presented here. Details for a comprehensive study on mechanical analysis of MD simulation such as indentation force, Young's modulus, surface stiffness, atomic stress, and atomic strain are included. The atomic stress and von-Mises strain distributions of the indentation model can be calculated to monitor deformation mechanism with time evaluation in atomistic level.
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Fulerenos/química , Microscopía de Sonda de Barrido/métodos , Silicio/química , Módulo de Elasticidad , Simulación de Dinámica Molecular , Nanotecnología/métodosRESUMEN
The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette-which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier-is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface.Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations.
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Cóclea/ultraestructura , Microscopía de Sonda de Barrido/instrumentación , Estereocilios/ultraestructura , Animales , Conductividad Eléctrica , Ratones , Microscopía de Sonda de Barrido/métodos , Nanotecnología/instrumentación , RatasRESUMEN
Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology.
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Imagenología Tridimensional/métodos , Microscopía de Sonda de Barrido/métodos , Adulto , Animales , Células Cultivadas , Medios de Cultivo , Diseño de Equipo , Femenino , Células HeLa , Humanos , Imagenología Tridimensional/instrumentación , Masculino , Ratones , Micromanipulación/instrumentación , Micromanipulación/métodos , Microscopía Electrónica de Rastreo , Microscopía de Sonda de Barrido/instrumentación , Nanotecnología , Técnicas de Placa-Clamp/instrumentación , Técnicas de Placa-Clamp/métodos , Ratas Sprague-DawleyRESUMEN
Skeletal muscles are composed of heterogeneous muscle fibers with various fiber types. These fibers can be classified into different classes based on their different characteristics. MALDI mass spectrometric imaging (MSI) has been applied to study and visualize different metabolomics profiles of different fiber types. Here, skeletal muscles were analyzed by atmospheric pressure scanning microprobe MALDI-MSI at high spatial and high mass resolution.
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Metabolómica/métodos , Fibras Musculares Esqueléticas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Presión Atmosférica , Microscopía de Sonda de Barrido/métodos , RatasRESUMEN
Measuring small forces is a major challenge in cell biology. Here we improve the spatial resolution and accuracy of force reconstruction of the well-established technique of traction force microscopy (TFM) using STED microscopy. The increased spatial resolution of STED-TFM (STFM) allows a greater than 5-fold higher sampling of the forces generated by the cell than conventional TFM, accessing the nano instead of the micron scale. This improvement is highlighted by computer simulations and an activating RBL cell model system.