Use Of Super-resolution Imaging Research Articles

Giant nonlinear optical responses from photon-avalanching nanoparticles.

Avalanche phenomena use steeply nonlinear dynamics to generate disproportionately large responses from small perturbations, and are found in a multitude of events and materials1. Photon avalanching enables technologies such as optical phase-conjugate imaging2, infrared quantum counting3 and efficient upconverted lasing4-6. However, the photon-avalanching mechanism underlying these optical applications has been observed only in bulk materials and aggregates6,7, limiting its utility and impact. Here we report the realization of photon avalanching at room temperature in single nanostructures-small, Tm3+-doped upconverting nanocrystals-and demonstrate their use in super-resolution imaging in near-infrared spectral windows of maximal biological transparency. Avalanching nanoparticles (ANPs) can be pumped by continuous-wave lasers, and exhibit all of the defining features of photon avalanching, including clear excitation-power thresholds, exceptionally long rise time at threshold, and a dominant excited-state absorption that is more than 10,000 times larger than ground-state absorption. Beyond the avalanching threshold, ANP emission scales nonlinearly with the 26th power of the pump intensity, owing to induced positive optical feedback in each nanocrystal. This enables the experimental realization of photon-avalanche single-beam super-resolution imaging7 with sub-70-nanometre spatial resolution, achieved by using only simple scanning confocal microscopy and without any computational analysis. Pairing their steep nonlinearity with existing super-resolution techniques and computational methods8-10, ANPs enable imaging with higher resolution and at excitation intensities about 100 times lower than other probes. The low photon-avalanching threshold and excellent photostability of ANPs also suggest their utility in a diverse array of applications, including sub-wavelength imaging7,11,12 and optical and environmental sensing13-15.

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging.

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and nanotechnology. Although near-field scanning microscopy and superlenses have improved the resolution in the near-field region, far-field imaging in real-time remains a significant challenge. Recently, the hyperlens, which magnifies and converts evanescent waves into propagating waves, has emerged as a novel approach to far-field imaging. Here, we report the fabrication of a spherical hyperlens composed of alternating silver (Ag) and titanium oxide (TiO2) thin layers. Unlike a conventional cylindrical hyperlens, the spherical hyperlens allows for two-dimensional magnification. Thus, incorporation into conventional microscopy is straightforward. A new optical system integrated with the hyperlens is proposed, allowing for a sub-wavelength image to be obtained in the far-field region in real time. In this study, the fabrication and imaging setup methods are explained in detail. This work also describes the accessibility and possibility of the hyperlens, as well as practical applications of real-time imaging in living cells, which can lead to a revolution in biology and nanotechnology.

Rupture of Stochastically Occurring Vesicle Clusters Limits Bilayer Formation on Alkane-PEG-Type Supports: Uncoupling Clustering from Surface Coverage.

Polymer-supported bilayers (PSBs) are a recognized tool for drug discovery through function-interaction analysis of membrane proteins. While silica-supported bilayers (SSBs) spontaneously form from surface-adsorbed vesicles, successful PSB formation via a similar method has thus far been limited by an insufficient understanding of the underlying vesicle-remodelling processes. Here, we generated a polymer support through the incubation of poly-L-lysine conjugated to alkyl-chain-terminated poly(ethylene)glycol on silica. This polymer-coated silica substrate yielded efficient vesicle adsorption and spontaneous bilayer formation, thereby providing a rare opportunity to address the mechanism of PSB formation and compare it to that of SSB. The combined use of super-resolution imaging, kinetics, and simulations indicates that the rupture of stochastically formed vesicle clusters is the rate-limiting step, which is an order of magnitude higher for silica than for polymer-coated silica. This was confirmed by directly demonstrating increased rupture rates for surface adsorbed multivesicle assemblies formed by vesicle cross-linking in solution. On the basis of this key insight we surmised that a low propensity of cluster rupture can be compensated for by an increase in the number density of clusters: the deposition of a mixture of oppositely charged vesicles resulted in bilayer formation on another alkane-PEG type of interface, which despite efficient vesicle adsorption otherwise fails to support spontaneous bilayer formation. This potentially provides a universal strategy for promoting bilayer formation on resistant surfaces without resorting to modifying the surface itself. Therefore, multivesicle assemblies with tailored geometries not only could facilitate bilayer formation on polymers with interesting functional properties but also could instigate the exploration of vesicle architecture for other processes involving vesicle remodelling such as drug delivery.

Superresolution imaging of viral protein trafficking.

The endoplasmic reticulum (ER) membrane is closely apposed to the outer mitochondrial membrane (OMM), which facilitates communication between these organelles. These contacts, known as mitochondria-associated membranes (MAM), facilitate calcium signaling, lipid transfer, as well as antiviral and stress responses. How cellular proteins traffic to the MAM, are distributed therein, and interact with ER and mitochondrial proteins are subject of great interest. The human cytomegalovirus UL37 exon 1 protein or viral mitochondria-localized inhibitor of apoptosis (vMIA) is crucial for viral growth. Upon synthesis at the ER, vMIA traffics to the MAM and OMM, where it reprograms the organization and function of these compartments. vMIA significantly changes the abundance of cellular proteins at the MAM and OMM, including proteins that regulate calcium homeostasis and cell death. Through the use of superresolution imaging, we have shown that vMIA is distributed at the OMM in nanometer scale clusters. This is similar to the clusters reported for the mitochondrial calcium channel, VDAC, as well as electron transport chain, translocase of the OMM complex, and mitochondrial inner membrane organizing system components. Thus, aside from addressing how vMIA targets the MAM and regulates survival of infected cells, biochemical studies and superresolution imaging of vMIA offer insights into the formation, organization, and functioning of MAM. Here, we discuss these insights into trafficking, function, and organization of vMIA at the MAM and OMM and discuss how the use of superresolution imaging is contributing to the study of the formation and trafficking of viruses.

Visualizing the HoxD Gene Cluster at the Nanoscale Level.

Transcription of HoxD cluster genes in limbs is coordinated by two topologically associating domains (TADs), neighboring the cluster and containing various enhancers. Here, we use a combination of microscopy approaches and chromosome conformation capture to assess the structural changes occurring in this global architecture in various functional states. We observed that despite their spatial juxtaposition, the TADs are consistently kept as distinct three-dimensional units. Hox genes located at their boundary can show significant spatial segregation over long distances, suggesting that physical elongation of the HoxD cluster occurs. The use of superresolution imaging (STORM [stochastic optical reconstruction microscopy]) revealed that the gene cluster can be in an either compact or elongated shape. The latter configuration is observed in transcriptionally active tissue and in embryonic stem cells, consistent with chromosome conformation capture results. Such morphological changes at HoxD in developing digits seem to be associated with its position at the boundary between two TADs and support the idea that chromatin dynamics is important in the establishment of transcriptional activity.

Fast and precise Laguerre–Gaussian beam steering with acousto-optic deflectors

Novel fluorescence microscopy techniques and two-color laser direct imaging photolithography methods that enable resolution an order of magnitude beyond the diffraction limit require Laguerre-Gaussian beams and a fast and precise laser beam steering device to obtain images and produce microstructures. An acousto-optic deflector (AOD) is a suitable choice and provides high-speed random access beam positioning with subnanometer precision as well as beam intensity control in a single element. In high-resolution applications, the impact of an AOD on beam quality plays a major role. We study the transfer function of an AOD for a fundamental Gaussian and a doughnut-shaped Laguerre-Gaussian beam by measuring the beam quality as a function of the diffraction angle after passing through the device. It is demonstrated that an AOD introduces negligible distortion and degradation to the beam profile and is therefore highly suitable for use in super-resolution imaging and photolithography techniques where manipulation of Laguerre-Gaussian doughnut-shaped beams is required.