Stimulated Emission Depletion Research Articles

Human CASPR2 Antibodies Reversibly Alter Memory and the CASPR2 Protein Complex.

The encephalitis associated with antibodies against contactin-associated proteinlike 2 (CASPR2) is presumably antibody-mediated, but the antibody effects and whether they cause behavioral alterations are not well known. Here, we used a mouse model of patients' immunoglobulin G (IgG) transfer and super-resolution microscopy to demonstrate the antibody pathogenicity. IgG from patients with anti-CASPR2 encephalitis or healthy controls was infused into the cerebroventricular system of mice. The levels and colocalization of CASPR2 with transient axonal glycoprotein 1 (TAG1) were determined with stimulated emission depletion microscopy (40-70μm lateral resolution). Hippocampal clusters of Kv1.1 voltage-gated potassium channels (VGKCs) and GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were quantified with confocal microscopy. Behavioral alterations were assessed with standard behavioral paradigms. Cultured neurons were used to determine the levels of intracellular CASPR2 and TAG1 after exposure to patients' IgG. Infusion of patients' IgG, but not controls' IgG, caused memory impairment along with hippocampal reduction of surface CASPR2 clusters and decreased CASPR2/TAG1 colocalization. In cultured neurons, patients' IgG led to an increase of intracellular CASPR2 without affecting TAG1, suggesting selective CASPR2 internalization. Additionally, mice infused with patients' IgG showed decreased levels of Kv1.1 and GluA1 (two CASPR2-regulated proteins). All these alterations and the memory deficit reverted to normal after removing patients' IgG. IgG from patients with anti-CASPR2 encephalitis causes reversible memory impairment, inhibits the interaction of CASPR2/TAG1, and decreases the levels of CASPR2 and related proteins (VGKC, AMPAR). These findings fulfill the postulates of antibody-mediated disease and provide a biological basis for antibody-removing treatment approaches. ANN NEUROL 2022;91:801-813.

Rhodamine Fluorophores for STED Super‐Resolution Biological Imaging

AbstractAmong all fluorescent scaffolds, rhodamine and its derivatives have gained the greatest interest for super‐resolution imaging based on stimulated emission depletion (STED) microscopy, because of their typically high brightness, excellent photo‐stability and easy access to chemical derivatization. During the last few years, many successful examples have shown the imaging improvement by fine‐tuning the chemical structure of rhodamine scaffolds. However, there is still a lack of overall summarization, particularly during the most recent five years. Herein, the recent achievements in the design of rhodamine dyes toward STED super‐resolution bio‐imaging are summarized, by classifying them into four parts according to different biological targets including the cytoskeletons, membranous organelles, DNA/RNAs and other functional substances. The basic principle for structure design as well as the relationships between chemical modification and the function for STED imaging will be mainly focused.

Versatile Near‐Infrared Super‐Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD‐2

AbstractCRANAD‐2 is a fluorogenic curcumin derivative used for near‐infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD‐2 in two super‐resolution imaging techniques, namely stimulated emission depletion (STED) and single‐molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD‐2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45–55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super‐resolved image. The good performance and versatility of CRANAD‐2 provides a powerful tool for near‐infrared nanoscopic imaging of amyloids in vitro and in vivo.

High-resolution vibronic spectroscopy of a single molecule embedded in a crystal.

Vibrational levels of the electronic ground states in dye molecules have not been previously explored at a high resolution in solid matrices. We present new spectroscopic measurements on single polycyclic aromatic molecules of dibenzoterrylene embedded in an organic crystal made of para-dichlorobenzene. To do this, we use narrow-band continuous-wave lasers and combine spectroscopy methods based on fluorescence excitation and stimulated emission depletion to assess individual vibrational linewidths in the electronic ground state at a resolution of ∼30MHz dictated by the linewidth of the electronic excited state. In this fashion, we identify several exceptionally narrow vibronic levels with linewidths down to values around 2GHz. Additionally, we sample the distribution of vibronic wavenumbers, relaxation rates, and Franck-Condon factors, in both the electronic ground and excited states for a handful of individual molecules. We discuss various noteworthy experimental findings and compare them with the outcome of density functional theory calculations. The highly detailed vibronic spectra obtained in our work pave the way for studying the nanoscopic local environment of single molecules. The approach also provides an improved understanding of the vibrational relaxation mechanisms in the electronic ground state, which may help create long-lived vibrational states for applications in quantum technology.

Depletion of carbon dots in stimulated emission depletion microscopy developed with 405/532 nm continuous-wave lasers

Carbon dots (CDs) are emerging as new promising fluorescence probes for stimulated emission depletion microscopy, according to the excellent biocompatibility, low saturation intensity and broad excitation spectra to be compatible for different wavelength combinations. In this study, three kinds of CDs synthesized with phenylenediamines using a facile solvothermal approach have been characterized in detail. The influence of various organic solvents on suppression factors () is investigated. The results indicate that the curves of suppression factors in alcohol solvent are generally in linear relation with depletion power, rather than the conventional reciprocal relation. Moreover, CDs of 15 nm diameter display an ultralow saturation intensity ∼0.4 MW/cm2 in alcohol solvent at 492/10 nm fluorescence band, which is one-order improvement compared with common ATTO fluorescence dyes. The results indicate CDs have shown great promise as potential nanoprobes for stimulated emission depletion imaging.

Improved Ultrasound Localization Microscopy Based on Microbubble Uncoupling via Transmit Excitation.

Ultrasound localization microscopy (ULM) demonstrates great potential for visualization of tissue microvasculature at depth with high spatial resolution. The success of ULM heavily depends on robust localization of isolated microbubbles (MBs), which can be challenging in vivo especially within larger vessels where MBs can overlap and cluster close together. While MB dilution alleviates the issue of MB overlap to a certain extent, it drastically increases the data acquisition time needed for MBs to populate the microvasculature, which is already on the order of several minutes using recommended MB concentrations. Inspired by optical super-resolution imaging based on stimulated emission depletion (STED), here we propose a novel ULM imaging sequence based on MB uncoupling via transmit excitation (MUTE). MUTE “silences” MB signals by creating acoustic nulls to facilitate MB separation, which leads to robust localization of MBs especially under high concentrations. The efficiency of localization accomplished via the proposed technique was first evaluated in simulation studies with conventional ULM as a benchmark. Then, an in-vivo study based on the chorioallantoic membrane (CAM) of chicken embryos showed that MUTE could reduce the data acquisition time by half, thanks to the enhanced MB separation and localization. Finally, the performance of MUTE was validated in an in vivo mouse brain study. These results demonstrate the high MB localization efficacy of MUTE-ULM, which contributes to a reduced data acquisition time and improved temporal resolution for ULM.

Two-photon, fiber-coupled, super-resolution microscope for biological imaging

Imaging sub-diffraction dynamics of neural nanostructures involved in behaviors such as learning and memory in a freely moving animal is not possible with existing techniques. Here, we present a solution in the form of a two-photon (2P), fiber-coupled, stimulated emission depletion microscope and demonstrate its capabilities by acquiring super-resolution imaging of mammalian cells. A polarization-maintaining fiber is used to transport both the 2P excitation light (915 nm) and the donut-shaped depletion beam (592 nm), which is constructed by adding two temporally incoherent and orthogonally polarized Hermite–Gaussian fiber modes. The fiber output is insensitive to bending or temperature changes and is the first demonstration toward deep tissue super-resolution imaging in awake behaving animals.

Fluorescence Microscopy Methods for the Analysis and Characterization of Lignin.

Lignin is one of the most studied and analyzed materials due to its importance in cell structure and in lignocellulosic biomass. Because lignin exhibits autofluorescence, methods have been developed that allow it to be analyzed and characterized directly in plant tissue and in samples of lignocellulose fibers. Compared to destructive and costly analytical techniques, fluorescence microscopy presents suitable alternatives for the analysis of lignin autofluorescence. Therefore, this review article analyzes the different methods that exist and that have focused specifically on the study of lignin because with the revised methods, lignin is characterized efficiently and in a short time. The existing qualitative methods are Epifluorescence and Confocal Laser Scanning Microscopy; however, other semi-qualitative methods have been developed that allow fluorescence measurements and to quantify the differences in the structural composition of lignin. The methods are fluorescence lifetime spectroscopy, two-photon microscopy, Föster resonance energy transfer, fluorescence recovery after photobleaching, total internal reflection fluorescence, and stimulated emission depletion. With these methods, it is possible to analyze the transport and polymerization of lignin monomers, distribution of lignin of the syringyl or guaiacyl type in the tissues of various plant species, and changes in the degradation of wood by pulping and biopulping treatments as well as identify the purity of cellulose nanofibers though lignocellulosic biomass.

An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy.

In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.

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.

Neutrophil extracellular traps drive epithelial–mesenchymal transition of human colon cancer

Neutrophil extracellular traps (NETs) are extracellular structures, composed of nuclear DNA and various proteins released from neutrophils. Evidence is growing that NETs exert manifold functions in infection, immunity and cancer. Recently, NETs have been detected in colorectal cancer (CRC) tissues, but their association with disease progression and putative functional impact on tumourigenesis remained elusive. Using high-resolution stimulated emission depletion (STED) microscopy, we showed that citrullinated histone H3 (H3cit) is sufficient to specifically detect citrullinated NETs in colon cancer tissues. Among other evidence, this was supported by the close association of H3cit with de-condensed extracellular DNA, the hallmark of NETs. Extracellular DNA was reliably differentiated from nuclear condensed DNA by staining with an anti-DNA antibody, providing a novel and valuable tool to detect NETs in formalin-fixed paraffin-embedded tissues. Using these markers, the clinical association of NETs was investigated in a cohort of 85 patients with colon cancer. NETs were frequently detected (37/85, 44%) in colon cancer tissue sections and preferentially localised either only in the tumour centre or both in the tumour centre and the invasive front. Of note, citrullinated NETs were significantly associated with high histopathological tumour grades and lymph node metastasis. In vitro, purified NETs induced filopodia formation and cell motility in CRC cell lines. This was associated with increased expression of mesenchymal marker mRNAs (vimentin [VIM], fibronectin [FN1]) and epithelial-mesenchymal transition promoting transcription factors (ZEB1, Slug [SNAI2]), as well as decreased expression of the epithelial markers E-cadherin (CDH1) and epithelial cell adhesion molecule (EPCAM). These findings indicated that NETs activate an epithelial-mesenchymal transition-like process in CRC cells and may contribute to the metastatic progression of CRC. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

Human Aquaporin-4 orthogonal array of particles in biomimetic membranes: aggregation, protein tracking, and STED

Aquaporin-4 (AQP4) is an integral water channel protein located in the plasma membrane of astrocyte cells in the central nervous system (CNS) and helps to regulate water-ion homeostasis. Some isoforms of AQP4 further order themselves into aggregates known as orthogonal arrays of particles (OAPs). AQP4 exists in two main isoforms, M1 and M23, with M23 being the isoform that favors stabilization by aggregation into OAPs. What role these OAPs play in the membrane of cells is still largely unknown. This study takes these AQP4 aggregates and incorporates them in a supported CNS biomimetic and images them using stimulated emission depletion (STED) superresolution microscopy.

ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the lamprey central canal.

Cerebrospinal fluid-contacting (CSF-c) neurons line the central canal of the spinal cord and a subtype of CSF-c neurons expressing somatostatin, forms a homeostatic pH regulating system. Despite their importance, their intricate spatial organization is poorly understood. The function of another subtype of CSF-c neurons expressing dopamine is also investigated. Imaging methods with a high spatial resolution (5-10 nm) are used to resolve the synaptic and ciliary compartments of each individual cell in the spinal cord of the lamprey to elucidate their signalling pathways and to dissect the cellular organization. Here, light-sheet and expansion microscopy resolved the persistent ventral and lateral organization of dopamine- and somatostatin-expressing CSF-c neuronal subtypes. The density of somatostatin-containing dense-core vesicles, resolved by stimulated emission depletion microscopy, was shown to be markedly reduced upon each exposure to either alkaline or acidic pH and being part of a homeostatic response inhibiting movements. Their cilia symmetry was unravelled by stimulated emission depletion microscopy in expanded tissues as sensory with 9 + 0 microtubule duplets. The dopaminergic CSF-c neurons on the other hand have a motile cilium with the characteristic 9 + 2 duplets and are insensitive to pH changes. This novel experimental workflow elucidates the functional role of CSF-c neuron subtypes in situ paving the way for further spatial and functional cell-type classification.

ISM-assisted tomographic STED microscopy.

Stimulated emission depletion (STED) microscopy theoretically provides unlimited resolution. However, in practice the achievable resolution in biological samples is essentially limited by photobleaching. One method which overcomes this problem is tomographic STED (tomoSTED) microscopy. In tomoSTED microscopy, one-dimensional depletion patterns facing in different directions are successively applied in order to acquire a highly-resolved image in two dimensions. In this context, the number of addressed directions depends on the desired angular homogeneity of the point spread function or the optical transfer function and thus on the resolution increase as compared to diffraction-limited imaging. At a reasonable angular homogeneity the light dose and thus bleaching can be reduced, as compared to conventional STED microscopy. Here, we propose and demonstrate for the first time, to our knowledge, that the number of required depletion pattern orientations can be reduced by combining tomoSTED microscopy with the concept of image scanning microscopy (ISM). With our realization of an ISM-tomoSTED microscope, we show that approximately a factor of 2 lower number of orientations are required to achieve the same resolution and image quality as in tomoSTED microscopy.

Sample Preparation for Multicolor STED Microscopy.

Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.

Multi‐Level Approach for Comprehensive Enamel Phenotyping

Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 µm for in vivo imaging to 1.5 µm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium-carbon ratio. However, the methods mentioned above only show the result-already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels-starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high-resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: In vivo MicroCT scanning of mouse Basic Protocol 2: Ex vivo HR-MicroCT of the teeth Basic Protocol 3: SEM for teeth microstructure Basic Protocol 4: Stimulated emission depletion (STED) microscopy.

Facilely prepared aggregation-induced emission (AIE) nanocrystals with deep-red emission for super-resolution imaging.

Organic nanocrystals (NCs) with high brightness are highly desirable for biological imaging. However, the preparation of NCs by a facile and fast method is still challenging. Herein, an aggregation-induced emission (AIE) luminogen of 4,4′-(5,6-difluorobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(N,N-bis(4-methoxyphenyl)aniline) (DTPA-BT-F) in the deep-red region is designed with intensive crystalline features to obtain NCs by kinetically controlled nanoprecipitation. The prepared AIE NCs with high brightness and good photo-stability are then applied in super-resolution imaging via stimulated emission depletion (STED) nanoscopy. As observed, the nanostructures in lysosomes of both fixed and live cells are well visualized with superior lateral resolutions under STED nanoscopy (full width at half maximum values, 107 and 108 nm) in contrast to that in confocal imaging (548 and 740 nm). More importantly, dynamic monitoring and long-term tracking of lysosomal movements in live HeLa cells, such as lysosomal contact, can also be carried out by using DTPA-BT-F NCs at a superior resolution. To the best of our knowledge, this is the first case of AIE NCs prepared by nanoprecipitation for STED nanoscopy, thus providing a new strategy to develop high performance imaging agents for super-resolution imaging.

Halogen-modified carbazole derivatives for lipid droplet-specific bioimaging and two-photon photodynamic therapy

Lipid droplets (LDs) are dynamic multifunctional organelles that participate in the regulation of many metabolic processes, visualization of which is necessary for biological research. In this work, a series of two-photon responsive fluorescent probes (C-H, C-Br, and C-I) based on carbazole units were designed and synthesized. Thereinto, an iodine-modified carbazole derivative C-I exhibited an exciting lipid droplet targeting ability due to its excellent lipophilicity. Meanwhile, benefiting from its larger Stokes shift and two-photon absorption cross-section, C-I was employed for two-photon confocal laser scanning microscopy (CLSM) and stimulated emission depletion (STED) microscopy imaging to observe LDs more accurately. In addition, given the heavy atom effect, C-I can effectively generate reactive oxygen species (ROS) leading to cancer cell apoptosis under near-infrared light irradiation. Notably, we explained the process of cell apoptosis through in vitro simulation experiments. This study provides a promising platform for visualization of lipid droplets.

Fast dual-beam alignment method for stimulated emission depletion microscopy using aggregation-induced emission dye resin

A stimulated emission depletion is capable of breaking the diffraction limit by exciting fluorescent molecules with a solid Gaussian beam and quenching the excited molecules with another donut beam through stimulated emission. The coincidence degree of these two beams in three dimensions will significantly influence the spatial resolution of the microscope. However, the conventional alignment approach based on raster scanning of gold nanoparticles by the two laser beams separately suffers from a mismatch between fluorescence and scattering modes. To circumvent the above problems, we demonstrate a fast alignment design by scanning the second beam over the fabricated sample, which is made of aggregation-induced emission (AIE) dye resin. The relative positions of solid and donut laser beams can be represented by the fluorescent AIE from the labeled spots in the dye resin. This design achieves ultra-high resolutions of 22 nm in the x/y relative displacement and 27 nm in the z relative displacement for fast spatial matching of the two laser beams. This study has potential applications in scenarios that require the spatial matching of multiple laser beams, and the field of views of different objectives, for example, in a microscope with high precision.