Super-resolution Stimulated Emission Depletion Nanoscopy Research Articles

Uncovering the impact of UV radiation on mitochondria in dermal cells: a STED nanoscopy study

Mitochondria are essential organelles in cellular energy metabolism and other cellular functions. Mitochondrial dysfunction is closely linked to cellular damage and can potentially contribute to the aging process. The purpose of this study was to investigate the subcellular structure of mitochondria and their activities in various cellular environments using super-resolution stimulated emission depletion (STED) nanoscopy. We examined the morphological dispersion of mitochondria below the diffraction limit in sub-cultured human primary skin fibroblasts and mouse skin tissues. Confocal microscopy provides only the overall morphology of the mitochondrial membrane and an indiscerptible location of nucleoids within the diffraction limit. Conversely, super-resolution STED nanoscopy allowed us to resolve the nanoscale distribution of translocase clusters on the mitochondrial outer membrane and accurately quantify the number of nucleoids per cell in each sample. Comparable results were obtained by analyzing the translocase distribution in the mouse tissues. Furthermore, we precisely and quantitatively analyzed biomolecular distribution in nucleoids, such as the mitochondrial transcription factor A (TFAM), using STED nanoscopy. Our findings highlight the efficacy of super-resolution fluorescence imaging in quantifying aging-related changes on the mitochondrial sub-structure in cells and tissues.

Super-Resolution Visualization of Self-Assembling Helical Fibers Using Aggregation-Induced Emission Luminogens in Stimulated Emission Depletion Nanoscopy.

Organic fluorophores for stimulated emission depletion (STED) nanoscopy usually suffer from quenched emission in the aggregate state and inferior photostability, which largely limit their application in real-time, in situ, and long-term imaging at an ultrahigh resolution. Herein, an aggregation-induced emission (AIE) luminogen of DP-TBT with bright emission in solid state (photoluminescence quantum yields = 25%) and excellent photostability was designed to meet the requirements in STED nanoscopy. In addition to its excellent fluorescence properties, DP-TBT could also easily form self-assembling helixes and finally be well-visualized by super-resolution STED nanoscopy. The observations showed that helical fibers of DP-TBT as dashed lines had a much decreased fiber width with also a full width at half-maximum value of only 178 nm, which is ∼6 times higher than solid lines obtained by confocal microscopy (1154 nm). The STED nanoscopic data were also used to reconstruct 3D images of assembled helixes. Finally, by long-term tracking and dynamic monitoring, the formation and growth of helical fibers by DP-TBT in self-assembly processes were successfully obtained. These findings imply that highly emissive AIEgens with good photostability are highly suitable for real-time, in situ, and dynamic imaging at super-resolution using STED nanoscopy.

Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging.

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Super-resolution Stimulated Emission Depletion-Fluorescence Correlation Spectroscopy Reveals Nanoscale Membrane Reorganization Induced by Pore-Forming Proteins.

Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that the affinity for cholesterol in the membrane binding motifs of the LLO subdomains induce cholesterol and lipid reorganization to a greater extent in the distal (upper) leaflet when compared with the proximal (lower) leaflet. The observed length scale-dependent membrane reorganization that occurs due to invasion by LLO could be generalized to other cholesterol-dependent cytolysins and emphasizes the significant advantage of using super-resolution STED nanoscopy to unravel complex lipid-protein interactions in membrane and cellular biophysics.

Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy

Spermatids must precisely integrate specific molecules into structurally supported domains that develop during spermatogenesis. Once established, the architecture of the acrosome contributes to the acrosome reaction, which occurs prior to gamete interaction in mammals. The present study aims to clarify the morphology associated with the integration of the mouse fertilization-related acrosomal protein equatorin (mEQT) into the developing acrosome. EQT mRNA was first detected by in situ hybridization in round spermatids but disappeared in early elongating spermatids. The molecular size of mEQT was approximately 65 kDa in the testis. Developmentally, EQT protein was first detected on the nascent acrosomal membrane in round spermatids at approximately step 3, was actively integrated into the acrosomal membranes of round spermatids in the following step and then participated in acrosome remodeling in elongating spermatids. This process was clearly visualized by high-resolution fluorescence microscopy and super-resolution stimulated emission depletion nanoscopy by using newly generated C-terminally green-fluorescent-protein-tagged mEQT transgenic mice. Immunogold electron microscopy revealed that mEQT was anchored to the acrosomal membrane, with the epitope region observed as lying 5-70 nm away from the membrane and was associated with the electron-dense acrosomal matrix. This new information about the process of mEQT integration into the acrosome during spermatogenesis should provide a better understanding of the mechanisms underlying not only acrosome biogenesis but also fertilization and male infertility.