Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

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.

Thermometries for Single Nanoparticles Heated with Light.

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.

Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy.

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.

Optical Printing of Single Au Nanostars.

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.

A common framework for single-molecule localization using sequential structured illumination.

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 ∼10-fold improvement compared with wide-field camera-based single-molecule localization, reaching ∼1-2 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.

An alternative to MINFLUX that enables nanometer resolution in a confocal microscope.

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.

Multiphoton single-molecule localization by sequential excitation with light minima.

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.

Challenges on optical printing of colloidal nanoparticles.

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.

Super-resolution FRET measurements.

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.

Super-resolution Imaging of Energy Transfer by Intensity-Based STED-FRET.

Förster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides a more accurate quantification of interaction and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging. The method capabilities were first demonstrated on DNA-origami model systems, verified on uniformly double-labeled microtubules, and then utilized to image biomolecular interactions in the membrane-associated periodic skeleton (MPS) of neurons.

Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules.

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Fluorescence nanoscopy at the sub-10 nm scale.

Fluorescence nanoscopy represented a breakthrough for the life sciences as it delivers 20-30 nm resolution using far-field fluorescence microscopes. This resolution limit is not fundamental but imposed by the limited photostability of fluorophores under ambient conditions. This has motivated the development of a second generation of fluorescence nanoscopy methods that aim to deliver sub-10 nm resolution, reaching the typical size of structural proteins and thus providing true molecular resolution. In this review, we present common fundamental aspects of these nanoscopies, discuss the key experimental factors that are necessary to fully exploit their capabilities, and discuss their current and future challenges.

Pulsed Interleaved MINFLUX.

We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1-2 nm localization precision with 2000-1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes.

Analysis of sparse molecular distributions in fibrous arrangements based on the distance to the first neighbor in single molecule localization microscopy.

Single Molecule Localization Microscopy (SMLM) currently attains a lateral resolution of around 10 nm approaching molecular size. Together with increasingly specific fluorescent labeling, it opens the possibility to quantitatively analyze molecular organization. When the labeling density is high enough, SMLM provides clear images of the molecular organization. However, either due to limited labeling efficiency or due to intrinsically low molecular abundance, SMLM delivers a small set of sparse and highly precise localizations. In this work, we introduce a correlation analysis of molecular locations based on the functional dependence of the complementary cumulative distribution function (CCDF) of the distance to the first neighbor (r1). We demonstrate that the log(-log(CCDF(r1))) vs. log(r1) is characterized by a scaling exponent n that takes extreme values of 2 for a random 2D distribution and 1 for a strictly linear arrangement, and find that n is a robust and sensitive metric to distinguish characteristics of the underlying structure responsible for the molecular distribution, even at a very low labeling density. The method enables the detection of fibrillary organization and the estimation of the diameter of host fibers under conditions where a visual inspection provides no clue.

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy.

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronal extensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limit of conventional fluorescent microscopy. Fluorescent nanoscopy, on the other hand, requires costly equipment and special analysis routines, which remain inaccessible to most research groups. This report aims to resolve this issue by using protein-retention expansion microscopy (pro-ExM) to reveal the MPS of axons. ExM uses reagents and equipment that are readily accessible in most neurobiology laboratories. We first explore means to accurately estimate the expansion factors of protein structures within cells. We then describe the protocol that produces an expanded specimen that can be examined with any fluorescent microscopy allowing quantitative nanoscale characterization of the MPS. We validate ExM results by direct comparison to stimulated emission depletion (STED) nanoscopy. We conclude that ExM facilitates three-dimensional, multicolor and quantitative characterization of the MPS using accessible reagents and conventional fluorescent microscopes.

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in situ affinity constant in cells.

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B corticotropin-releasing hormone type 1 receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.e. depression, anxiety, and addictions), neuroendocrinological alterations, and neurodegenerative diseases. Here, we present a strategy to label GPCRs with a small fluorescent antagonist that permits the observation of the receptor in live cells through stochastic optical reconstruction microscopy (STORM) with 23 nm resolution. The marker, an aza-BODIPY derivative, was designed based on computational docking studies, then synthesized, and finally tested in biological cells. Experiments on hippocampal neurons demonstrate antagonist effects in similar concentrations as the well-established antagonist CP-376395. A quantitative analysis of two color STORM images enabled the determination of the binding affinity of the new marker in the cellular environment.

Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells.

Estrogen receptor α (ERα) is expressed in tissues as diverse as brains and mammary glands. In breast cancer, ERα is a key regulator of tumor progression. Therefore, understanding what activates ERα is critical for cancer treatment in particular and cell biology in general. Using biochemical approaches and superresolution microscopy, we show that estrogen drives membrane ERα into endosomes in breast cancer cells and that its fate is determined by the presence of fibronectin (FN) in the extracellular matrix; it is trafficked to lysosomes in the absence of FN and avoids the lysosomal compartment in its presence. In this context, FN prolongs ERα half-life and strengthens its transcriptional activity. We show that ERα is associated with ß1-integrin at the membrane, and this integrin follows the same endocytosis and subcellular trafficking pathway triggered by estrogen. Moreover, ERα+ vesicles are present within human breast tissues, and colocalization with ß1-integrin is detected primarily in tumors. Our work unravels a key, clinically relevant mechanism of microenvironmental regulation of ERα signaling.

The Actin/Spectrin Membrane-Associated Periodic Skeleton in Neurons.

Neurons are the most asymmetric cell types, with their axons commonly extending over lengths that are thousand times longer than the diameter of the cell soma. Fluorescence nanoscopy has recently unveiled that actin, spectrin and accompanying proteins form a membrane-associated periodic skeleton (MPS) that is ubiquitously present in mature axons from all neuronal types evaluated so far. The MPS is a regular supramolecular protein structure consisting of actin "rings" separated by spectrin tetramer "spacers". Although the MPS is best organized in axons, it is also present in dendrites, dendritic spine necks and thin cellular extensions of non-neuronal cells such as oligodendrocytes and microglia. The unique organization of the actin/spectrin skeleton has raised the hypothesis that it might serve to support the extreme physical and structural conditions that axons must resist during the lifespan of an organism. Another plausible function of the MPS consists of membrane compartmentalization and subsequent organization of protein domains. This review focuses on what we know so far about the structure of the MPS in different neuronal subdomains, its dynamics and the emerging evidence of its impact in axonal biology.

Author Correction: Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.