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RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.
Assuntos
Axônios , Cones de Crescimento , Microtúbulos , Transdução de Sinais , Proteína rhoA de Ligação ao GTP , Microtúbulos/metabolismo , Animais , Proteína rhoA de Ligação ao GTP/metabolismo , Axônios/metabolismo , Cones de Crescimento/metabolismo , Quinases Associadas a rho/metabolismo , Hipocampo/metabolismo , Hipocampo/citologia , Ratos , Forminas/metabolismo , Células Cultivadas , Neurônios/metabolismoRESUMO
The development of efficient nanoscale photon absorbers, such as plasmonic or high-index dielectric nanostructures, allows the remotely controlled release of heat on the nanoscale using light. These photothermal nanomaterials have found applications in various research and technological fields, ranging from materials science to biology. However, measuring the nanoscale thermal fields remains an open challenge, hindering full comprehension and control of nanoscale photothermal phenomena. Here, we review and discuss existent thermometries suitable for single nanoparticles heated under illumination. These methods are classified in four categories according to the region where they assess temperature: (1) the average temperature within a diffraction-limited volume, (2) the average temperature at the immediate vicinity of the nanoparticle surface, (3) the temperature of the nanoparticle itself, and (4) a map of the temperature around the nanoparticle with nanoscale spatial resolution. In the latter, because it is the most challenging and informative type of method, we also envisage new combinations of technologies that could be helpful in retrieving nanoscale temperature maps. Finally, we analyze and provide examples of strategies to validate the results obtained using different thermometry methods.
Assuntos
Nanopartículas , Nanoestruturas , Temperatura Alta , Nanopartículas/química , Nanoestruturas/química , TemperaturaRESUMO
Fluorescence Resonance Energy Transfer (FRET)-based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.
Assuntos
DNA , Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , DNA/química , Microscopia Confocal , Imagem Individual de MoléculaRESUMO
Obtaining arrays of single nanoparticles with three-dimensional complex shapes is still an open challenge. Current nanolithography methods do not allow for the preparation of nanoparticles with complex features like nanostars. In this work, we investigate the optical printing of gold nanostars of different sizes as a function of laser wavelength and power. We found that tuning the laser to the main resonances of the nanostars in the near-infrared makes it possible to avoid nanoparticles reshaping due to plasmonic heating, enabling their deposition at the single particle level and in ordered arrays.
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Localization of single fluorescent molecules is key for physicochemical and biophysical measurements, such as single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Over the last two decades, several methods have been developed in which the position of a single emitter is interrogated with a sequence of spatially modulated patterns of light. Among them, the recent MINFLUX technique outstands for achieving a â¼10-fold improvement compared with wide-field camera-based single-molecule localization, reaching â¼1-2 nm localization precision at moderate photon counts. Here, we present a common framework for this type of measurement. Using the Cramér-Rao bound as a limit for the achievable localization precision, we benchmark reported methods, including recent developments, such as MINFLUX and MINSTED, and long-established methods, such as orbital tracking. In addition, we characterize two new proposed schemes, orbital tracking and raster scanning, with a minimum of intensity. Overall, we found that approaches using an intensity minimum have a similar performance in the central region of the excitation pattern, independent of the geometry of the excitation pattern, and that they outperform methods featuring an intensity maximum.
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Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging. Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Among the numerous localization methods available, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups. Here, we present RASTMIN, a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity. RASTMIN delivers ~1-2 nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications. We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imaging of DNA origami structures.
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Using sequential excitation with a minimum of light to localize single fluorescent molecules represented a breakthrough because it delivers 1-2 nm precision with moderate photon counts, enabling tracking and super-resolution imaging with true molecular resolution. Expanding this concept to multi-photon regimes may be a useful complement to reach even higher localization precision and get deeper into biological specimens.
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While colloidal chemistry provides ways to obtain a great variety of nanoparticles with different shapes, sizes, material compositions, and surface functions, their controlled deposition and combination on arbitrary positions of substrates remain a considerable challenge. Over the last ten years, optical printing arose as a versatile method to achieve this purpose for different kinds of nanoparticles. In this article, we review the state of the art of optical printing of single nanoparticles and discuss its strengths, limitations, and future perspectives by focusing on four main challenges: printing accuracy, resolution, selectivity, and nanoparticle photostability.
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Super-resolution fluorescence microscopy and Förster Resonance Energy Transfer (FRET) form a well-established family of techniques that has provided unique tools to study the dynamic architecture and functionality of biological systems, as well as to investigate nanomaterials. In the last years, the integration of super-resolution methods with FRET measurements has generated advances in two fronts. On the one hand, FRET-based probes have enhanced super-resolution imaging. On the other, the development of super-resolved FRET imaging methods has allowed the visualization of molecular interaction patterns with higher spatial resolution, less averaging and higher dynamic range. Here, we review these advances and discuss future perspectives, including the possible integration of FRET with next generation super-resolution techniques capable of reaching true molecular-scale spatial resolution.