Search Results (13)

The resolution of conventional optical microscopy is restricted by the diffraction limit. Light waves containing higher-frequency information about the sample are bound to the sample surface and cannot be collected by far-field optical microscopy. To break the resolution limit, researchers have proposed various far-field super-resolution (SR) microscopy imaging methods using evanescent waves to transfer the high-frequency information of samples to the low-frequency passband of optical microscopy. Optimization algorithms are developed to reconstruct a SR image of the sample by utilizing the high-frequency information. These techniques can be collectively referred to as spatial-frequency-shift (SFS) SR microscopy. This review aims to summarize the basic principle of SR microscopy using evanescent illumination and introduce the advances in this research area. Some current challenges and possible directions are also discussed. Full article

The resolution of an optical microscope is determined by the overall point spread function of the system. When examining structures significantly smaller than the wavelength of light, the contribution of the background or surrounding environment can profoundly affect the point spread function. This research delves into the impact of reflective planar substrate structures on the system’s resolution. We establish a comprehensive forward imaging model for a reflection-type confocal laser scanning optical microscope, incorporating vector field manipulation to image densely packed nanoparticle clusters. Both theoretical and experimental findings indicate that the substrate causes an interference effect between the background field and the scattered field from the nanoparticles, markedly enhancing the overall spatial resolution. The integration of vector field manipulation with an interferometric scattering approach results in superior spatial resolution for imaging isolated particles and densely distributed nanoscale particle clusters even with deep subwavelength gaps as small as 20 nm between them. However, the method still struggles to resolve nanoparticles positioned directly next to each other without any gap, necessitating further work to enhance the resolving ability. This may involve techniques like deconvolution or machine learning-based post-processing methods. Full article

Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the GluRIIA gene have been utilized. Furthermore, mutated and tagged versions of GluRIIA have been examined in the past, but most of these constructs were not expressed under endogenous regulatory control or involved the mentioned imprecise GluRIIA knockouts. We performed CRISPR/Cas9-assisted gene editing at the endogenous locus of GluRIIA. This enabled the investigation of the endogenous expression pattern of GluRIIA using tagged constructs with an EGFP and an ALFA tag for super-resolution immunofluorescence imaging, including structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). All GluRIIA constructs exhibited full functionality and PHP could be induced by philanthotoxin at control levels. By applying hierarchical clustering algorithms to analyze the dSTORM data, we detected postsynaptic receptor cluster areas of ~0.15 µm². Consequently, our constructs are suitable for ultrastructural analyses of GluRIIA. Full article

Optical nanoscopy, also known as super-resolution optical microscopy, has provided scientists with the means to surpass the diffraction limit of light microscopy and attain new insights into nanoscopic structures and processes that were previously inaccessible. In recent decades, numerous studies have endeavored to enhance super-resolution microscopy in terms of its spatial (lateral) resolution, axial resolution, and temporal resolution. In this review, we discuss recent efforts to push the resolution limit of stimulated emission depletion (STED) optical nanoscopy across multiple dimensions, including lateral resolution, axial resolution, temporal resolution, and labeling precision. We introduce promising techniques and methodologies building on the STED concept that have emerged in the field, such as MINSTED, isotropic STED, and event-triggered STED, and evaluate their respective strengths and limitations. Moreover, we discuss trade-off relationships that exist in far-field optical microscopy and how they come about in STED optical nanoscopy. By examining the latest developments addressing these aspects, we aim to provide an updated overview of the current state of STED nanoscopy and its potential for future research. Full article

For decades, terahertz (THz) microscopic imaging has been limited by the resolution of the system due to the larger wavelength, the power of the source, and the equivalent noise power of the detector, so a lot of research has focused on single-point scanning imaging. With the development of hardware, full-field THz imaging based on high-power continuous-wave THz sources have been developed such as the direct intensity imaging method and lensless coherent imaging. In particular, the THz direct intensity imaging method requires no complicated computational reconstruction, while the high resolution, as a key issue, still needs to be improved. In this paper, the rotating coherent scattering microscopy was applied to THz imaging for the first time. Here, we designed and fabricated a hemisphere lens with high-resistance silicon. The tilted hemisphere lens transformed the incident divergent beam into a plane wave, and the total internal reflection occurred in the planar surface within the hemispherical lens, and generated evanescent waves in the rare medium. At the same time, the sample was placed very close to the plane of the hemispherical lens, so that the sample was illuminated by the evanescent waves. The scattered waves carried high frequency information to the far field, and thus through an objective, the super-resolution imaging was achieved along a single direction. Then, the hemispherical lens was rotated to obtain coherent scattering microscopic images under different evanescent wave illumination angles. Finally, the full-field super-resolution imaging results were obtained through incoherent superposition. Full article

Supercritical lens can create a sub-diffraction-limited focal spot in the far field, providing a promising route for the realization of label-free super-resolution imaging through the point scanning mechanism. However, all of the reported supercritical lenses have circular shape configurations, and produce isotropic sub-diffraction-limited focal spots in the focal plane. Here, we propose and experientially demonstrate a sub-diffraction transverse optical needle by using an elliptical supercritical lens. Through breaking the circular symmetry and introducing ellipticity to the lens, a uniform sub-diffractive transverse optical needle with lateral length and width of 6λ/NA and 0.45λ/NA, respectively, was successfully created in the focal plane. Further, elliptical sector-shape cutting with an optimized apex angle of 60 degrees can lead to suppressed subsidiary focusing for improved uniformity and condensed field intensity of the transverse optical needle. The demonstration of sub-diffractive transverse optical needle with a high aspect ratio (length to width ratio) of 13:1 may find potential applications in line-scanning microscopy for video-rate label-free super-resolution imaging, and also enable advances in the fields from laser manufacturing to optical manipulation. Full article

We recently assisted in a revolution in the realm of fluorescence microscopy triggered by the advent of super-resolution techniques that surpass the classic diffraction limit barrier. By providing optical images with nanometer resolution in the far field, super-resolution microscopy (SRM) is currently accelerating our understanding of the molecular organization of bio-specimens, bridging the gap between cellular observations and molecular structural knowledge, which was previously only accessible using electron microscopy. SRM mainly finds its roots in progress made in the control and manipulation of the optical properties of (single) fluorescent molecules. The flourishing development of novel fluorescent nanostructures has recently opened the possibility of associating super-resolution imaging strategies with nanomaterials’ design and applications. In this review article, we discuss some of the recent developments in the field of super-resolution imaging explicitly based on the use of nanomaterials. As an archetypal class of fluorescent nanomaterial, we mainly focus on single-walled carbon nanotubes (SWCNTs), which are photoluminescent emitters at near-infrared (NIR) wavelengths bearing great interest for biological imaging and for information optical transmission. Whether for fundamental applications in nanomaterial science or in biology, we show how super-resolution techniques can be applied to create nanoscale images “in”, “of” and “with” SWCNTs. Full article

To overcome the diffraction limit and resolve target structures in greater detail, far-field super-resolution techniques such as stochastic optical reconstruction microscopy (STORM) have been developed, and different STORM algorithms have been developed to deal with the various problems that arise. In particular, the effect of the local structure is an important issue. For objects with closely correlated distributions, simple Gaussian-based localization algorithms often used in STORM imaging misinterpret overlapping point spread functions (PSFs) as one, which limits the ability of super-resolution imaging to resolve nanoscale local structures and leads to inaccurate length measurements. The STORM super-resolution images of biological specimens from the cluster-forming proteins in the nervous system were reconstructed for localization-based analysis. Generally, the localization of each fluorophore was determined by two-dimensional Gaussian function fitting. Further, the physical shape of the cluster structure information was incorporated into the size parameter of the localization structure analysis in order to generate structure-based fitting algorithms. In the present study, we proposed a novel, structure-based, super-resolution image analysis method: structure-based analysis (SBA), which combines a structural function and a super-resolution localization algorithm. Using SBA, we estimated the size of fluorescent beads, inclusion proteins, and subtle synaptic structures in both wide-field and STORM images. The results show that SBA has a comparable and often superior performance to the commonly used full width at half maximum (FWHM) parameter. We demonstrated that SBA is able to estimate molecular cluster sizes in far-field super-resolution STORM images, and that SBA was comparable and often superior to FWHM. We also certified that SBA provides size estimations that corroborate previously published electron microscopy data. Full article

Progress in developing fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, is inseparable from the advancement in optical fluorescence microscopy. Super-resolution microscopy, or optical nanoscopy, overcame the far-field optical resolution limit, known as Abbe’s diffraction limit, by taking advantage of the photophysical properties of fluorescent probes. Therefore, fluorescent probes for super-resolution microscopy should meet the new requirements in the probes’ photophysical and photochemical properties. STED optical nanoscopy achieves super-resolution by depleting excited fluorophores at the periphery of an excitation laser beam using a depletion beam with a hollow core. An ideal fluorescent probe for STED nanoscopy must meet specific photophysical and photochemical properties, including high photostability, depletability at the depletion wavelength, low adverse excitability, and biocompatibility. This review introduces the requirements of fluorescent probes for STED nanoscopy and discusses the recent progress in the development of fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, for the STED nanoscopy. The strengths and the limitations of the fluorescent probes are analyzed in detail. Full article

Super-resolution optical imaging is a consistent research hotspot for promoting studies in nanotechnology and biotechnology due to its capability of overcoming the diffraction limit, which is an intrinsic obstacle in pursuing higher resolution for conventional microscopy techniques. In the past few decades, a great number of techniques in this research domain have been theoretically proposed and experimentally demonstrated. Graphene, a special two-dimensional material, has become the most meritorious candidate and attracted incredible attention in high-resolution imaging domain due to its distinctive properties. In this article, the working principle of graphene-assisted imaging devices is summarized, and recent advances of super-resolution optical imaging based on graphene are reviewed for both near-field and far-field applications. Full article

Through rigorous electromagnetic simulations, the natural coupling of high-spatial-frequency evanescent waves from the near field to the far field by dielectric microspheres is studied in air. The generation of whispering gallery modes inside the microspheres is shown independently of any resonance. In addition, the conversion mechanism of these evanescent waves into propagating waves is analysed. This latter point leads to key information that allows a better physical understanding of the super-resolution phenomenon in microsphere-assisted microscopy where sub-diffraction-limit revolving power is achieved. Full article

Plasmonic nanoparticles have recently emerged as a promising platform for photocatalysis thanks to their ability to efficiently harvest and convert light into highly energetic charge carriers and heat. The catalytic properties of metallic nanoparticles, however, are typically measured in ensemble experiments. These measurements, while providing statistically significant information, often mask the intrinsic heterogeneity of the catalyst particles and their individual dynamic behavior. For this reason, single particle approaches are now emerging as a powerful tool to unveil the structure-function relationship of plasmonic nanocatalysts. In this Perspective, we highlight two such techniques based on far-field optical microscopy: surface-enhanced Raman spectroscopy and super-resolution fluorescence microscopy. We first discuss their working principles and then show how they are applied to the in-situ study of catalysis and photocatalysis on single plasmonic nanoparticles. To conclude, we provide our vision on how these techniques can be further applied to tackle current open questions in the field of plasmonic chemistry. Full article

Tomographic diffractive microscopy (TDM) is a label-free, far-field, super-resolution microscope. The significant difference between TDM and wide-field microscopy is that in TDM the sample is illuminated from various directions with a coherent collimated beam and the complex diffracted field is collected from many scattered angles. By utilizing inversion procedures, the permittivity/refractive index of investigated samples can be retrieved from the measured diffracted field to reconstruct the geometrical parameters of a sample. TDM opens up new opportunities to study biological samples and nano-structures and devices, which require resolution beyond the Rayleigh limit. In this review, we describe the principles and recent advancements of TDM and also give the perspectives of this fantastic microscopy technique. Full article