Photoswitching Reagent for Super-Resolution Fluorescence Microscopy.

Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy by exceeding the diffraction limit and revealing previously unattainable nanoscale details of cellular structures and molecular dynamics. This super-resolution imaging capability relies on fluorophore photoswitching, which is crucial for optimizing the imaging conditions and accurately determining the fluorophore positions. To understand the general on and off photoswitching mechanisms of single dye molecules, various photoswitching reagents were evaluated. Systematic measurement of the single-molecule-level fluorescence on and off rates (kon and koff) in the presence of various photoswitching reagents and theoretical calculation of the structure of the photoswitching reagent-fluorophore pair indicated that the switch-off mechanism is mainly determined by the nucleophilicity of the photoswitching reagent, and the switch-on mechanism is a two-photon-induced dissociation process, which is related to the power of the illuminating laser and bond dissociation energy of this pair. This study contributes to a broader understanding of the molecular photoswitching mechanism in SMLM imaging and provides a basis for designing improved photoswitching reagents with potential applications extending to materials science and chemistry.

Visualizing extracellular vesicle biogenesis in gram-positive bacteria using super-resolution microscopy.

BACKGROUND: Recently, bacterial extracellular vesicles (EVs) have been considered to play crucial roles in various biological processes and have great potential for developing cancer therapeutics and biomedicine. However, studies on bacterial EVs have mainly focused on outer membrane vesicles released from gram-negative bacteria since the outermost peptidoglycan layer in gram-positive bacteria is thought to preclude the release of EVs as a physical barrier. RESULTS: Here, we examined the ultrastructural organization of the EV produced by gram-positive bacteria using super-resolution stochastic optical reconstruction microscopy (STORM) at the nanoscale, which has not been resolved using conventional microscopy. Based on the super-resolution images of EVs, we propose three major mechanisms of EV biogenesis, i.e., membrane blebbing (mechanisms 1 and 2) or explosive cell lysis (mechanism 3), which are different from the mechanisms in gram-negative bacteria, despite some similarities. CONCLUSIONS: These findings highlight the significant role of cell wall degradation in regulating various mechanisms of EV biogenesis and call for a reassessment of previously unresolved EV biogenesis in gram-positive bacteria.

Super-Resolution Fluorescence Imaging for Semiconductor Nanoscale Metrology and Inspection.

The increase in the number and complexity of process levels in semiconductor production has driven the need for the development of new measurement methods that can evaluate semiconductor devices at the critical dimensions of fine patterns and simultaneously inspect nanoscale contaminants or defects. However, conventional optical inspection methods often fail to resolve device patterns or defects at the level of tens of nanometers required for device development owing to their diffraction-limited resolutions. In this study, we used the stochastic optical reconstruction microscopy (STORM) technique to image semiconductor nanostructures with feature sizes as small as 30 nm and detect individual 20 nm-diameter contaminants. STORM imaging of semiconductor nanopatterns is based on the development of a selective labeling method of fluorophores for a negative silicon oxide surface using the charge interaction of positive polyethylenimine molecules. This study demonstrates the potential of STORM for nanoscale metrology and in-line defect inspection of semiconductor integrated circuits.

Development of a New Approach for Low-Laser-Power Super-Resolution Fluorescence Imaging.

The development of super-resolution fluorescence microscopy over the past decade has drastically improved the resolution of light microscopy to ∼10 nm. Stochastic optical reconstruction microscopy (STORM) can be used to achieve subdiffraction-limit resolution by sequentially imaging and localizing individual fluorophores. In principle, the super-resolution of STORM can be obtained by high-accuracy localization of photoswitchable fluorophores, which require fast photoswitching and bright fluorescence intensity from a single emitter. It is known that the switching rate of photoswitchable fluorophores depends on the laser power─a high laser power being required for the enhancement of imaging resolution. However, high laser power is usually harmful to biological specimens and limits the imaging time because of its photobleaching effects and high phototoxicity. In this study, we attempted to overcome this problem by improving the STORM resolution at a lower laser power. Through the quantitative analysis of the photoswitching behavior of single fluorophores under different laser power conditions, we developed a new approach to achieve super-resolution fluorescence images at a laser power 10 times lower than had previously been reported. This approach is expected to play an increasingly significant role in super-resolution imaging of power-sensitive samples.

Development of Deep-Learning-Based Single-Molecule Localization Image Analysis.

Recent developments in super-resolution fluorescence microscopic techniques (SRM) have allowed for nanoscale imaging that greatly facilitates our understanding of nanostructures. However, the performance of single-molecule localization microscopy (SMLM) is significantly restricted by the image analysis method, as the final super-resolution image is reconstructed from identified localizations through computational analysis. With recent advancements in deep learning, many researchers have employed deep learning-based algorithms to analyze SMLM image data. This review discusses recent developments in deep-learning-based SMLM image analysis, including the limitations of existing fitting algorithms and how the quality of SMLM images can be improved through deep learning. Finally, we address possible future applications of deep learning methods for SMLM imaging.

Microtubule-directed transport of purine metabolons drives their cytosolic transit to mitochondria.

To meet their purine demand, cells activate the de novo purine biosynthetic pathway and transiently cluster the pathway enzymes into metabolons called purinosomes. Recently, we have shown that purinosomes were spatially colocalized with mitochondria and microtubules, yet it remained unclear as to what drives these associations and whether a relationship between them exist. Here, we employed superresolution imaging methods to describe purinosome transit in the context of subcellular localization. Time-resolved imaging of purinosomes showed that these assemblies exhibit directed motion as they move along a microtubule toward mitochondria, where upon colocalization, a change in purinosome motion was observed. A majority of purinosomes colocalized with mitochondria were also deemed colocalized with microtubules. Nocodazole-dependent microtubule depolymerization resulted in a loss in the purinosome-mitochondria colocalization, suggesting that the association of purinosomes with mitochondria is facilitated by microtubule-directed transport, and thereby supporting our notion of an interdependency between these subcellular components in maximizing purine production through the de novo purine biosynthetic pathway.

Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

Spectrally Resolved Super-Resolution Microscopy Unveils Multipath Reaction Pathways of Single Spiropyran Molecules.

By recording both the images and emission spectra of thousands of single fluorescent molecules stochastically generated from the ring-opening reaction of a spiropyran, we provide mechanistic insights into its multipath reaction pathways. Through statistics of the measured single-molecule spectra, we identify two spectrally distinct isomers, presumably TTC and TTT cis-trans isomers, for the open-ring merocyanine product, and discover a strong solvent polarity-dependence for the relative population of the two isomers. From single-molecule spectral time traces, we further examine isomerization between the two product merocyanine isomers, as well as their ring-closure reaction back to the spiropyran form.

Site-dependent effects of methylation on the electronic spectra of jet-cooled methylated xanthine compounds.

We obtained the electronic spectra of various methylated xanthine compounds including caffeine in a supersonic jet by resonant two-photon ionization spectroscopy. The methyl group in the tested methylated xanthine compounds has a distinct, site-dependent effect on the electronic spectrum. Methylation at the N3 position causes a significant red shift of the ππ* state, whereas methylation at the N1 position has only minimal effects on the electronic spectrum. The notably broad spectra of theobromine and caffeine result from methyl substitution at the N7 position, which causes a large displacement between the potential energy surfaces of the S0 and S1 states, and a strong vibronic coupling. We also investigated the internal rotation of the methyl group and its effect on the electronic spectrum of the methylated xanthine compounds. We found that the barrier height for the torsional motion in the ground state is significantly affected by a carbonyl or methyl group that lies close to the methyl group of interest. In contrast, the torsional barrier in the excited state is governed by the hyperconjugation interaction in the lowest unoccupied molecular orbital. The agreement between the experimental and simulated spectra of torsional vibronic bands suggested that the low frequency torsional vibrations arising from the tunneling splitting and the coupling between the torsional and molecular motions give theobromine and theophylline the multiplet nature of their origin bands. This study provides a new level of understanding for the methyl substitution effects on the electronically excited states of xanthine compounds, which may very well be applicable to many other methyl substituted biomolecules including DNAs and proteins.

Dual function of CD81 in influenza virus uncoating and budding.

As an obligatory pathogen, influenza virus co-opts host cell machinery to harbor infection and to produce progeny viruses. In order to characterize the virus-host cell interactions, several genome-wide siRNA screens and proteomic analyses have been performed recently to identify host factors involved in influenza virus infection. CD81 has emerged as one of the top candidates in two siRNA screens and one proteomic study. The exact role played by CD81 in influenza infection, however, has not been elucidated thus far. In this work, we examined the effect of CD81 depletion on the major steps of the influenza infection. We found that CD81 primarily affected virus infection at two stages: viral uncoating during entry and virus budding. CD81 marked a specific endosomal population and about half of the fused influenza virus particles underwent fusion within the CD81-positive endosomes. Depletion of CD81 resulted in a substantial defect in viral fusion and infection. During virus assembly, CD81 was recruited to virus budding site on the plasma membrane, and in particular, to specific sub-viral locations. For spherical and slightly elongated influenza virus, CD81 was localized at both the growing tip and the budding neck of the progeny viruses. CD81 knockdown led to a budding defect and resulted in elongated budding virions with a higher propensity to remain attached to the plasma membrane. Progeny virus production was markedly reduced in CD81-knockdown cells even when the uncoating defect was compensated. In filamentous virus, CD81 was distributed at multiple sites along the viral filament. Taken together, these results demonstrate important roles of CD81 in both entry and budding stages of the influenza infection cycle.

Recent development of computational cluster analysis methods for single-molecule localization microscopy images.

With the development of super-resolution imaging techniques, it is crucial to understand protein structure at the nanoscale in terms of clustering and organization in a cell. However, cluster analysis from single-molecule localization microscopy (SMLM) images remains challenging because the classical computational cluster analysis methods developed for conventional microscopy images do not apply to pointillism SMLM data, necessitating the development of distinct methods for cluster analysis from SMLM images. In this review, we discuss the development of computational cluster analysis methods for SMLM images by categorizing them into classical and machine-learning-based methods. Finally, we address possible future directions for machine learning-based cluster analysis methods for SMLM data.

In situ silver nanoparticle development for molecular-specific biological imaging via highly accessible microscopies.

In biological studies and diagnoses, brightfield (BF), fluorescence, and electron microscopy (EM) are used to image biomolecules inside cells. When compared, their relative advantages and disadvantages are obvious. BF microscopy is the most accessible of the three, but its resolution is limited to a few microns. EM provides a nanoscale resolution, but sample preparation is time-consuming. In this study, we present a new imaging technique, which we termed decoration microscopy (DecoM), and quantitative investigations to address the aforementioned issues in EM and BF microscopy. For molecular-specific EM imaging, DecoM labels proteins inside cells using antibodies bearing 1.4 nm gold nanoparticles (AuNPs) and grows silver layers on the AuNPs' surfaces. The cells are then dried without buffer exchange and imaged using scanning electron microscopy (SEM). Structures labeled with silver-grown AuNPs are clearly visible on SEM, even they are covered with lipid membranes. Using stochastic optical reconstruction microscopy, we show that the drying process causes negligible distortion of structures and that less structural deformation could be achieved through simple buffer exchange to hexamethyldisilazane. Using DecoM, we visualize the nanoscale alterations in microtubules by microtubule-severing proteins that cannot be observed with diffraction-limited fluorescence microscopy. We then combine DecoM with expansion microscopy to enable sub-micron resolution BF microscopy imaging. We first show that silver-grown AuNPs strongly absorb white light, and the structures labeled with them are clearly visible on BF microscopy. We then show that the application of AuNPs and silver development must follow expansion to visualize the labeled proteins clearly with sub-micron resolution.

Bacteria detection and species identification at the single-cell level using super-resolution fluorescence imaging and AI analysis.

The skin microbiome is thought to play a critical role in maintaining skin health and protecting against infection. While most microorganisms that live on the skin are harmless or even beneficial, some can cause skin infections or other health problems, emphasizing the importance of diagnosis of the composition and diversity of the skin flora. However, conventional diagnostic methods for evaluation of the skin microbiome are not sensitive enough to detect bacteria at low concentrations and suffer from poor specificity, thus limiting early diagnosis of bacterial infections. In this study, we developed novel approaches for bacterial species detection and identification methods with single-cell sensitivity using super-resolution microscopy and AI-based image analysis: a protein quantification-based method and an AI-based bacterial image analysis method. We demonstrate that these methods can differentiate between common bacterial members of the skin flora, including Staphylococcus aureus and Staphylococcus epidermidis, and different ribotypes of Cutibacterium acnes, both in purified bacterial samples and in scaling skin samples. The advantages of these methods, including the lack of time-consuming amplification or purification steps and single-cell level detection sensitivity, allow early diagnosis of bacterial infections, even from bacterial samples at extremely low concentrations, thus showing promise as a next-generation platform for microbiome detection as single-cell diagnostics.

Recent Developments in Correlative Super-Resolution Fluorescence Microscopy and Electron Microscopy.

The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the spatial locations of specific molecules with SRM and the context of the cellular ultrastructure by EM. Although the combination of SRM and EM remains challenging owing to the incompatibility of samples prepared for these techniques, the increasing research attention on these methods has led to drastic improvements in their performances and resulted in wide applications. Here, we review the development of correlative SRM and EM (sCLEM) with a focus on the correlation of EM with different SRM techniques. We discuss the limitations of the integration of these two microscopy techniques and how these challenges can be addressed to improve the quality of correlative images. Finally, we address possible future improvements and advances in the continued development and wide application of sCLEM approaches.

Polarity Nano-Mapping of Polymer Film Using Spectrally Resolved Super-Resolution Imaging.

With the rapid development of the nanofabrication of polymer materials, the local measurement of the chemical properties of polymer nanostructures has become crucial because they can be highly heterogeneous at the nanoscale. We developed a spectroscopic imaging approach to characterize the nanoscale local polarity of polymer films via spectrally resolved super-resolution microscopy. We demonstrate the capability of the recently developed single-molecule sensing and imaging method to probe the polarity of polymers either inside a polymer matrix or on the external surface of a polymer. The nanoscale polarity sensing capability of our method facilitates the differentiation of various polymer surfaces based on chemical polarities, and it can further differentiate the polarity of functional side chain groups. Moreover, we demonstrate that a two-component polymer mixture can be locally distinguished based on the contrasting polarities of the lateral phase separation, further allowing for the investigation of nanoscale phase separation depending on the composition of the polymer blend film. This approach is anticipated to open the door to further characterizations of various nanocomposite materials.

LIN28A enhances regenerative capacity of human somatic tissue stem cells via metabolic and mitochondrial reprogramming.

Developing methods to improve the regenerative capacity of somatic stem cells (SSCs) is a major challenge in regenerative medicine. Here, we propose the forced expression of LIN28A as a method to modulate cellular metabolism, which in turn enhances self-renewal, differentiation capacities, and engraftment after transplantation of various human SSCs. Mechanistically, in undifferentiated/proliferating SSCs, LIN28A induced metabolic reprogramming from oxidative phosphorylation (OxPhos) to glycolysis by activating PDK1-mediated glycolysis-TCA/OxPhos uncoupling. Mitochondria were also reprogrammed into healthy/fused mitochondria with improved functional capacity. The reprogramming allows SSCs to undergo cell proliferation more extensively with low levels of oxidative and mitochondrial stress. When the PDK1-mediated uncoupling was untethered upon differentiation, LIN28A-SSCs differentiated more efficiently with an increase of OxPhos by utilizing the reprogrammed mitochondria. This study provides mechanistic and practical approaches of utilizing LIN28A and metabolic reprogramming in order to improve SSCs utility in regenerative medicine.

Recent Developments in Lanthanide-Doped Alkaline Earth Aluminate Phosphors with Enhanced and Long-Persistent Luminescence.

Lanthanide-activated alkaline earth aluminate phosphors are excellent luminescent materials that are designed to overcome the limitations of conventional sulfide-based phosphors. The increasing research attention on these phosphors over the past decade has led to a drastic improvement in their phosphorescence efficiencies and resulted in a wide variety of phosphorescence colors, which can facilitate applications in various areas. This review article discusses the development of lanthanide-activated alkaline earth aluminate phosphors with a focus on the various synthesis methods, persistent luminescence mechanisms, activator and coactivator effects, and the effects of compositions. Particular attention has been devoted to alkaline earth aluminate phosphors that are extensively used, such as strontium-, calcium-, and barium-based aluminates. The role of lanthanide ions as activators and coactivators in phosphorescence emissions was also emphasized. Finally, we address recent techniques involving nanomaterial engineering that have also produced lanthanide-activated alkaline earth aluminate phosphors with long-persistent luminescence.

Super-resolution imaging reveals cytoskeleton-dependent organelle rearrangement within platelets at intermediate stages of maturation.

A steady supply of platelets maintains their levels in the blood, and this is achieved by the generation of progeny from platelet intermediates. Using systematic super-resolution microscopy, we examine the ultrastructural organization of various organelles in different platelet intermediates to understand the mechanism of organelle redistribution and sorting in platelet intermediate maturation as the early step of platelet progeny production. We observe the dynamic interconversion between the intermediates and find that microtubules are responsible for controlling the overall shape of platelet intermediates. Super-resolution images show that most of the organelles are located near the cell periphery in oval preplatelets and confined to the bulbous tips in proplatelets. We also find that the distribution of the dense tubular system and α granules is regulated by actin, whereas that of mitochondria and dense granules is governed by microtubules. Altogether, our results call for a reassessment of organelle redistribution in platelet intermediates.

Super-resolution imaging of platelet-activation process and its quantitative analysis.

Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation state and improve the existing therapies for hemostatic disorders. Here, we employed various state-of-the-art super-resolution imaging and quantification methods to characterize the platelet spatiotemporal ultrastructural change during the activation process due to phorbol 12-myristate 13-acetate (PMA) stimuli by observing the cytoskeletal elements and various organelles at nanoscale, which cannot be done using conventional microscopy. Platelets could be spread out with the guidance of actin and microtubules, and most organelles were centralized probably due to the limited space of the peripheral thin regions or the close association with the open canalicular system (OCS). Among the centralized organelles, we provided evidence that granules are fused with the OCS to release their cargo through enlarged OCS. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon activation and call for a reassessment of previously unresolved complex and multi-factorial activation processes.

Direct Visualization of Actin Filaments and Actin-Binding Proteins in Neuronal Cells.

Actin networks and actin-binding proteins (ABPs) are most abundant in the cytoskeleton of neurons. The function of ABPs in neurons is nucleation of actin polymerization, polymerization or depolymerization regulation, bundling of actin through crosslinking or stabilization, cargo movement along actin filaments, and anchoring of actin to other cellular components. In axons, ABP-actin interaction forms a dynamic, deep actin network, which regulates axon extension, guidance, axon branches, and synaptic structures. In dendrites, actin and ABPs are related to filopodia attenuation, spine formation, and synapse plasticity. ABP phosphorylation or mutation changes ABP-actin binding, which regulates axon or dendritic plasticity. In addition, hyperactive ABPs might also be expressed as aggregates of abnormal proteins in neurodegeneration. Those changes cause many neurological disorders. Here, we will review direct visualization of ABP and actin using various electron microscopy (EM) techniques, super resolution microscopy (SRM), and correlative light and electron microscopy (CLEM) with discussion of important ABPs in neuron.