Stimulated Emission Depletion Nanoscopy Research Articles

Phenazine Cations as Anticancer Theranostics†.

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.

Combining sophisticated fast FLIM, confocal microscopy, and STED nanoscopy for live-cell imaging of tunneling nanotubes.

Cell-to-cell communication via tunneling nanotubes (TNTs) is a challenging topic with a growing interest. In this work, we proposed several innovative tools that use red/near-infrared dye labeling and employ lifetime-based imaging strategies to investigate the dynamics of TNTs in a living mesothelial H28 cell line that exhibits spontaneously TNT1 and TNT2 subtypes. Thanks to a fluorescence lifetime imaging microscopy module being integrated into confocal microscopy and stimulated emission depletion nanoscopy, we applied lifetime imaging, lifetime dye unmixing, and lifetime denoising techniques to perform multiplexing experiments and time-lapses of tens of minutes, revealing therefore structural and functional characteristics of living TNTs that were preserved from light exposure. In these conditions, vesicle-like structures, and tubular- and round-shaped mitochondria were identified within living TNT1. In addition, mitochondrial dynamic studies revealed linear and stepwise mitochondrial migrations, bidirectional movements, transient backtracking, and fission events in TNT1. Transfer of Nile Red-positive puncta via both TNT1 and TNT2 was also detected between living H28 cells.

Nanoscale myelinogenesis image in developing brain via super-resolution nanoscopy by near-infrared emissive curcumin-BODIPY derivatives

Understanding the intricate nanoscale architecture of neuronal myelin during central nervous system development is of utmost importance. However, current visualization methods heavily rely on electron microscopy or indirect fluorescent method, lacking direct and real-time imaging capabilities. Here, we introduce a breakthrough near-infrared emissive curcumin-BODIPY derivative (MyL-1) that enables direct visualization of myelin structure in brain tissues. The remarkable compatibility of MyL-1 with stimulated emission depletion nanoscopy allows for unprecedented super-resolution imaging of myelin ultrastructure. Through this innovative approach, we comprehensively characterize the nanoscale myelinogenesis in three dimensions over the course of brain development, spanning from infancy to adulthood in mouse models. Moreover, we investigate the correlation between myelin substances and Myelin Basic Protein (MBP), shedding light on the essential role of MBP in facilitating myelinogenesis during vertebral development. This novel material, MyL-1, opens up new avenues for studying and understanding the intricate process of myelinogenesis in a direct and non-invasive manner, paving the way for further advancements in the field of nanoscale neuroimaging.Graphical

One Stone, Two Birds: High-Brightness Aggregation-Induced Emission Photosensitizers for Super-Resolution Imaging and Photodynamic Therapy.

Most aggregation-induced emission (AIE) luminogens exhibit high brightness, excellent photostability, and good biocompatibility, but these AIE-active agents, which kill two birds with one stone to result in applications in both stimulated emission depletion (STED) super-resolution imaging and photodynamic therapy (PDT), have not been reported yet but are urgently needed. To meet the requirements of STED nanoscopy and PDT, D-A-π-A-D type DTPABT-HP is designed by tuning conjugated π spacers. It exhibits red-shifted emission, high PLQY of 32.04%, and impressive 1O2 generation (9.24 fold compared to RB) in nanoparticles (NPs). Then, DTPABT-HP NPs are applied in cell imaging via STED nanoscopy, especially visualizing the dynamic changes of lysosomes in the PDT process at ultrahigh resolution. After that, in vivo PDT was also conducted by DTPABT-HP NPs, resulting in significantly inhibited tumor growth, with an inhibition rate of 86%. The work here is beneficial to the design of multifunctional agents and the deep understanding of their phototheranostic mechanism in biological research.

Pushing the Resolution Limit of Stimulated Emission Depletion Optical Nanoscopy.

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.

Photon-Weight-Reallocated Stimulated Emission Depletion Nanoscopy

Photon-Weight-Reallocated Stimulated Emission Depletion Nanoscopy

Quantitative super-resolution microscopy reveals the differences in the nanoscale distribution of nuclear phosphatidylinositol 4,5-bisphosphate in human healthy skin and skin warts.

Introduction: Imaging of human clinical formalin-fixed paraffin-embedded (FFPE) tissue sections provides insights into healthy and diseased states and therefore represents a valuable resource for basic research, as well as for diagnostic and clinical purposes. However, conventional light microscopy does not allow to observe the molecular details of tissue and cell architecture due to the diffraction limit of light. Super-resolution microscopy overcomes this limitation and provides access to the nanoscale details of tissue and cell organization. Methods: Here, we used quantitative multicolor stimulated emission depletion (STED) nanoscopy to study the nanoscale distribution of the nuclear phosphatidylinositol 4,5-bisphosphate (nPI(4,5)P2) with respect to the nuclear speckles (NS) marker SON. Results: Increased nPI(4,5)P2 signals were previously linked to human papillomavirus (HPV)-mediated carcinogenesis, while NS-associated PI(4,5)P2 represents the largest pool of nPI(4,5)P2 visualized by staining and microscopy. The implementation of multicolor STED nanoscopy in human clinical FFPE skin and wart sections allowed us to provide here the quantitative evidence for higher levels of NS-associated PI(4,5)P2 in HPV-induced warts compared to control skin. Discussion: These data expand the previous reports of HPV-induced increase of nPI(4,5)P2 levels and reveal for the first time the functional, tissue-specific localization of nPI(4,5)P2 within NS in clinically relevant samples. Moreover, our approach is widely applicable to other human clinical FFPE tissues as an informative addition to the classical histochemistry.

Solvatochromic Near-Infrared Aggregation-Induced Emission-Active Acrylonitriles by Acceptor Modulation for Low-Power Stimulated Emission Depletion Nanoscopy

Stimulated emission depletion (STED) nanoscopy has broadened our horizons to unravel mysterious functions of cellular structures on an unparalleled nanometer scale. Nevertheless, an intense depletion laser power is a general prerequisite for STED super-resolution imaging with a satisfactory resolution, inevitably leading to severe photobleaching of fluorophores, irreparable photodamage to biosamples, and impaired imaging quality. Herein, by modulating distinct acceptor units, a series of donor–acceptor–acceptor structured fluorescent acrylonitriles featured with aggregation-induced emission (AIE) for STED super-resolution imaging at a low depletion power were systematically developed. These AIE luminogens (AIEgens) exhibited tunable near-infrared emissions (650–733 nm) and high fluorescence quantum yields (QYs of up to 26.8%) in the solid state, significant Stokes shifts, and large two-photon absorption cross-sections. They also exhibited a typical solvatochromic effect and ultrahigh QYs of up to 98.4% in low-polarity solvents. Furthermore, these lipophilic solvatochromic acrylonitriles specifically lit up lipid droplets (LDs) with exceptionally high photostability in a wash-free manner. By taking TPA-BT-ANBI as an example, the STED super-resolution imaging of LDs with excellent resolution of 62 and 80 nm for cytosolic and nuclear LDs, respectively, at a low saturation depletion power of 0.83 MW/cm2 and extended time-lapse imaging of LD dynamics was achieved. Subsequent use of TPA-BT-ANBI for the optical discrimination of fatty liver tissues and two-photon deep-tissue imaging was also demonstrated. This study opens new avenues for versatile photostable materials at low depletion powers for target-specific STED super-resolution imaging.

Temperature-responsive and biocompatible nanocarriers based on clay nanotubes for controlled anti-cancer drug release.

Administration of temperature-responsive drug carriers that release anticancer drugs at high temperatures can benefit hyperthermia therapies because of the synergistic effect of anticancer drug molecules and high temperature on killing the cancer cells. In this study, we design and characterize a new temperature-responsive nanocarrier based on a naturally occurring and biocompatible clay mineral, halloysite nanotubes. Poly(N-isopropylacrylamide) brushes were grown on the surface of halloysite nanotubes using a combination of mussel-inspired dopamine polymerization and surface-initiated atom transfer radical polymerization. The chemical structure of the hybrid materials was investigated using X-ray photoelectron spectroscopy, thermogravimetric analysis and energy-dispersive X-ray spectroscopy. The hybrid material was shown to have a phase transition temperature of about 32 °C, corresponding to a 40 nm thick polymer layer surrounding the nanotubes. Cell studies suggested that grafting of poly(N-isopropylacrylamide) brushes on the polydopamine-modified halloysite nanotubes suppresses the cytotoxicity caused by the polydopamine interlayer and drug release studies on nanotubes loaded with doxorubicin showed that thanks to the poly(N-isopropylacrylamide) brushes a temperature-dependent drug release is observed. Finally, a fluorescent dye molecule was covalently attached to the polymer-grafted nanotubes and stimulated emission depletion nanoscopy was used to confirm the internalization of the nanotubes in HeLa cells.

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Capturing mitochondria's intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to two-dimensional (2D) single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) to a benzo-fused cyanine dye, we report a mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, and markedly reduced phototoxicity. PKMO enables super-resolution (SR) recordings of IM dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell three-dimensional (3D) STED nanoscopy of mitochondria for 3D analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers, allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.

Accurate Background Reduction in Adaptive Optical Three-Dimensional Stimulated Emission Depletion Nanoscopy by Dynamic Phase Switching

Stimulated emission depletion (STED) fluorescence nanoscopy allows the three-dimensional (3D) visualization of nanoscale subcellular structures, providing unique insights into their spatial organization. However, 3D-STED imaging and quantification of dense features are obstructed by the low signal-to-background ratio (SBR) resulting from optical aberrations and out-of-focus background. Here, combining adaptive optics elements, we present an easy-to-implement, flexible, and effective method to improve the SBR by dynamic phase switching. By switching to a counterclockwise vortex phase mask and a top-hat one with an incorrect inner radius, the depletion pattern features a nonzero-intensity center, enabling accurate background recordings. When the recorded background is subtracted from the aberration-corrected 3D-STED image, the SBR in dense sample areas can be improved by a factor of 3–7 times. We demonstrate our method on various dense subcellular structures, showing more advantages than the software-based background subtraction algorithms.

Far Red-Shifted CdTe Quantum Dots for Multicolour Stimulated Emission Depletion Nanoscopy.

Stimulated emission depletion (STED) nanoscopy is a widely used nanoscopy technique. Two-colour STED imaging in fixed and living cells is standardised today utilising both fluorescent dyes and fluorescent proteins. Solutions to image additional colours have been demonstrated using spectral unmixing, photobleaching steps, or long-Stokes-shift dyes. However, these approaches often compromise speed, spatial resolution, and image quality, and increase complexity. Here, we present multicolour STED nanoscopy with far red-shifted semiconductor CdTe quantum dots (QDs). STED imaging of the QDs is optimized to minimize blinking effects and maximize the number of detected photons. The far-red and compact emission spectra of the investigated QDs free spectral space for the simultaneous use of fluorescent dyes, enabling straightforward three-colour STED imaging with a single depletion beam. We use our method to study the internalization of QDs in cells, opening up the way for future super-resolution studies of particle uptake and internalization.

Dysferlin facilitates the de novo biogenesis of tubular membranes in hypertrophic remodelling

Abstract Introduction Ferlins are transmembrane proteins with multiple C2 domains that may mediate Ca2+ dependent membrane fusion and repair events. In ventricles, Dysferlin expression is dominant, and patient mutations have been associated with cardiomyopathies. Hence, we hypothesized that Dysferlin is essential for the functional integrity of the tubular endomembrane system in ventricular myocytes (VM), and may facilitate the cellular hypertrophic remodelling in pressure-overload. Results In 30 weeks old Dysferlin knockout (KO) mice, echocardiography revealed dilative cardiomyopathy with reduced left-ventricular ejection fractions (difference between means KO vs. wildtype (WT) −13.45±2.17%, p<0.001, n=16/20 mice). Asking for the subcellular relevance of Dysferlin, we investigated VM nanodomains by immunolabelling in STimulated Emission Depletion (STED) nanoscopy and visualized punctual Dysferlin clusters decorating the T-tubule system of mouse and human VM. Precisely, Dysferlin clusters localized to junctions of the tubular system and the sarcoplasmic reticulum (SR) in nanometric proximity to RyR2 Ca2+ release units. Complexome profiling (LC-MS/MS) also suggested interactions of Dysferlin with RyR2 and the SR membrane-tethering protein Junctophilin-2 (JP2) in high molecular weight complexes. In JP2-overexpressing mice, live-cell STED membrane imaging captured polyadic junctional membrane structures in VM, specifically surrounded by dense Dysferlin clusters, which proposes Dysferlin as stabilizing protein of tubule-SR junctions. Further, we studied the ultrastructural remodelling of the tubular system in a mouse model of cardiac hypertrophy and heart failure induced by transaortic constriction (TAC). Interestingly, mortality four weeks post TAC was not increased in KO mice. However, echocardiography uncovered that Dysf-KO prevents left-ventricular hypertrophy in response to pressure-overload, which was confirmed by single cell measurements (VM area: WT 3559±120 μm2 vs. KO 2671±87 μm2, n=163/158 VM, p<0.001). Thus, we assumed Dysferlin to mediate tubular network proliferation during hypertrophic remodelling. Indeed, WT VM showed an augmented Dysferlin expression post TAC (158±0,125%, p=0,005, n=7/7 mice), and immunofluorescence STED nanoscopy detected an increased Dysferlin network complexity. In detail, we visualized highly abundant Dysferlin clusters decorating newly shaped axial tubules, which were quantified by component-specific tubule network analysis. Conclusion Nanoscale imaging proposes Dysferlin as stabilizer of tubule-SR junctions, thereby promoting functional excitation-contraction coupling, and demonstrates the crucial importance of Dysferlin in pressure-overload induced hypertrophic remodelling, facilitating the de novo biogenesis of axial tubule membranes and tubule-SR junctions necessary for VM growth. Thus, Dysferlin may emerge as potential novel therapeutic rationale to control subcellular membrane remodelling in cardiac diseases. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Multiscale Bioimaging Cluster of Excellence: From Molecular Machines to Networks of Excitable Cells;German Research Foundation (DFG) - Collaborative Research Centre 1002 (SFB 1002): Modulatory Units in Heart Failure

Event-triggered STED imaging

Monitoring the proteins and lipids that mediate all cellular processes requires imaging methods with increased spatial and temporal resolution. STED (stimulated emission depletion) nanoscopy enables fast imaging of nanoscale structures in living cells but is limited by photobleaching. Here, we present event-triggered STED, an automated multiscale method capable of rapidly initiating two-dimensional (2D) and 3D STED imaging after detecting cellular events such as protein recruitment, vesicle trafficking and second messengers activity using biosensors. STED is applied in the vicinity of detected events to maximize the temporal resolution. We imaged synaptic vesicle dynamics at up to 24 Hz, 40 ms after local calcium activity; endocytosis and exocytosis events at up to 11 Hz, 40 ms after local protein recruitment or pH changes; and the interaction between endosomal vesicles at up to 3 Hz, 70 ms after approaching one another. Event-triggered STED extends the capabilities of live nanoscale imaging, enabling novel biological observations in real time.

Theoretical insight on the saturated stimulated emission intensity of a squaraine dye for STED nanoscopy

Stimulated emission depletion nanoscopy (STED) is increasingly applied for the insights into the ultra-structures of organelles in live cells because of the bypassing of the Abbe’s optical diffraction limit. Theoretically, with the increase of excitation and depletion laser power, the imaging resolution can be accordingly enhanced and even close to the infinity. Unfortunately, powerful laser illuminations usually produce severe phototoxicity and photobleaching, which will lead to more extra-interference with biological events in live cells and accelerate the decomposition of the fluorescent probes. In view of the trade-off of cell viability and imaging resolution, excellent probes with superior photophysical properties are great in demand. For a qualified STED probes, the saturated stimulated emission intensity (Isat) is considered as a key evaluating factor. According to the formula, Isat is inversely proportional to the stimulated emission cross section (σsti) of the fluorescent probe. However, the relationship between the σsti and chemical structure of the STED probe remain to be unclear. In this work, we explore the influence factors by theoretical calculations on a squaraine dye (MitoEsq-635) and a commercial dye (Atto647N). The results indicate that the increase of transition dipole moment (μ) are beneficial for the increase of σsti, thereafter reducing Isat. Furthermore, we firstly proposed that stimulated emission depletion was qualitatively interpreted by the investigation on the potential energy surfaces of ground states (S0) and the first excited states (S1) of the dyes.

Dynamic Heterochromatin States in Anisotropic Nuclei of Cells on Aligned Nanofibers.

The cancer cell nucleus deforms as it invades the interstitial spaces in tissues and the tumor microenvironment. While alteration of the chromatin structure in a deformed nucleus is expected and documented, the chromatin structure in the nuclei of cells on aligned matrices has not been elucidated. In this work we elucidate the spatiotemporal organization of heterochromatin in the elongated nuclei of cells on aligned nanofibers with stimulated emission depletion nanoscopy and fluorescence correlation spectroscopy. We show that the anisotropy of nuclei is sufficient to drive H3K9me3-heterochromatin alterations, with enhanced H3K9me3 nanocluster compaction and aggregation states that otherwise are indistinguishable from diffraction-limited microscopy. We interrogated the higher-order heterochromatin structures within major chromatin compartments in anisotropic nuclei and discovered a wider spatial dispersion of nanodomain clusters in the nucleoplasm and condensed larger nanoclusters near the periphery and pericentromeric heterochromatin. Upon examining the spatiotemporal dynamics of heterochromatin in anisotropic nuclei, we observed reduced mobility of the constitutive heterochromatin mark H3K9me3 and the associated heterochromatin protein 1 (HP1α) at the nucleoplasm and periphery regions, correlating with increased viscosity and changes in gene expression. Since heterochromatin remodeling is crucial to genome integrity, our results reveal an unconventional H3K9me3 heterochromatin distribution, providing cues to an altered chromatin state due to perturbations of the nuclei in aligned fiber configurations.

Dual-modulation difference stimulated emission depletion microscopy to suppress the background signal

Stimulated emission depletion (STED) nanoscopy is one of the most well-developed nanoscopy techniques that can provide subdiffraction spatial resolution imaging. Here, we introduce dual-modulation difference STED microscopy (dmdSTED) to suppress the background noise in traditional STED imaging. By applying respective time-domain modulations to the two continuous-wave lasers, signals are distributed discretely in the frequency spectrum and thus are obtained through lock-in demodulation of the corresponding frequencies. The background signals can be selectively eliminated from the effective signal without compromise of temporal resolution. We used nanoparticle, fixed cell, and perovskite coating experiments, as well as theoretical demonstration, to confirm the effectiveness of this method. We highlight dmdSTED as an idea and approach with simple implementation for improving the imaging quality, which substantially enlarges the versatility of STED nanoscopy.

Aggregation-Induced Emission (AIE) in Super-resolution Imaging: Cationic AIE Luminogens (AIEgens) for Tunable Organelle-Specific Imaging and Dynamic Tracking in Nanometer Scale.

Organelle-specific imaging and dynamic tracking in ultrahigh resolution is essential for understanding their functions in biological research, but this remains a challenge. Therefore, a facile strategy by utilizing anion-π+ interactions is proposed here to construct an aggregation-induced emission luminogen (AIEgen) of DTPAP-P, not only restricting the intramolecular motions but also blocking their strong π-π interactions. DTPAP-P exhibits a high photoluminescence quantum yield (PLQY) of 35.04% in solids, favorable photostability and biocompatibility, indicating its potential application in super-resolution imaging (SRI) via stimulated emission depletion (STED) nanoscopy. It is also observed that this cationic DTPAP-P can specifically target to mitochondria or nucleus dependent on the cell status, resulting in tunable organelle-specific imaging in nanometer scale. In live cells, mitochondria-specific imaging and their dynamic monitoring (fission and fusion) can be obtained in ultrahigh resolution with a full-width-at-half-maximum (fwhm) value of only 165 nm by STED nanoscopy. This is about one-sixth of the fwhm value in confocal microscopy (1028 nm). However, a migration process occurs for fixed cells from mitochondria to nucleus under light activation (405 nm), leading to nucleus-targeted super-resolution imaging (fwhm= 184 nm). These findings indicate that tunable organelle-specific imaging and dynamic tracking by a single AIEgen at a superior resolution can be achieved in our case here via STED nanoscopy, thus providing an efficient method to further understand organelle's functions and roles in biological research.

Environmental enrichment enhances patterning and remodeling of synaptic nanoarchitecture as revealed by STED nanoscopy.

Synaptic plasticity underlies long-lasting structural and functional changes to brain circuitry and its experience-dependent remodeling can be fundamentally enhanced by environmental enrichment. It is however unknown, whether and how the environmental enrichment alters the morphology and dynamics of individual synapses. Here, we present a virtually crosstalk-free two-color in vivo stimulated emission depletion (STED) microscope to simultaneously superresolve the dynamics of endogenous PSD95 of the post-synaptic density and spine geometry in the mouse cortex. In general, the spine head geometry and PSD95 assemblies were highly dynamic, their changes depended linearly on their original size but correlated only mildly. With environmental enrichment, the size distributions of PSD95 and spine head sizes were sharper than in controls, indicating that synaptic strength is set more uniformly. The topography of the PSD95 nanoorganization was more dynamic after environmental enrichment; changes in size were smaller but more correlated than in mice housed in standard cages. Thus, two-color in vivo time-lapse imaging of synaptic nanoorganization uncovers a unique synaptic nanoplasticity associated with the enhanced learning capabilities under environmental enrichment.

Surface-Engineered Gold Nanoclusters for Stimulated Emission Depletion and Correlated Light and Electron Microscopy Imaging.

Stimulated emission depletion (STED) nanoscopy is an emerging super-resolution imaging platform for the study of the cellular structure. Developing suitable fluorescent probes of small size, good photostability, and easy functionalization is still in demand. Herein, we introduce a new type of surface-engineered gold nanoclusters (Au NCs) that are ultrasmall (1.7 nm) and ultrabright (QY = 60%) for STED bioimaging. A rigid shell formed by l-arginine (l-Arg) and 6-aza-2-thiothymine (ATT) on the Au NC surface enables not only its strong fluorescence in aqueous solution but also its easy chemical modification for specific biomolecule labeling. Au NCs show remarkable performance as STED nanoprobes, including high depletion efficiency, good photobleaching resistance, and low saturation intensity. Super-resolution imaging has been achieved with these Au NCs, and targeted nanoscopic imaging of cellular tubulin has been demonstrated. Moreover, the circular structure of lysosomes in live cells has been revealed. As a Au NC is also an ideal probe for electron microscopy, dual imaging of Aβ42 aggregates with the single labeling probe of Au NCs has been realized in correlative light and electron microscopy (CLEM). This work reports, for the first time, the application of Au NCs as a novel probe in STED and CLEM imaging. With their excellent properties, Au NCs show promising potential for nanoscale bioimaging.