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.

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.

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.

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.