Super-resolution Stimulated Emission Depletion Microscopy Research Articles

Polarity-Sensitive Probes for Superresolution Stimulated Emission Depletion Microscopy

The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed. Polarity-sensitive dyes are powerful tools to characterize such lipid membrane order, employing, for example, confocal and two-photon microscopy. The investigation of potential nanodomains, however, requires the use of superresolution microscopy. Here, we test the performance of the polarity-sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA, and NR12S in superresolution stimulated emission depletion microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles, show the high potential of these dyes for probing nanoscale membrane heterogeneity.

Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy

Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/−125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50 ± 15 nm and 120 ± 40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

Nanoscale clustering of the neurotrophin receptor TrkB revealed by super-resolution STED microscopy.

The nanoscale organization of the tropomyosin-related kinase receptor type B (TrkB), a promising therapeutic target for severe neurodegenerative and psychiatric disorders, is examined by stimulated emission depletion (STED) microscopy using the deconvoluted gated STED option. The performed immunofluorescence nanoscopic subdiffraction imaging of the membrane receptor localization reveals that clusters of oligomeric TrkB states and randomly organized nanodomains are formed in the membranes of differentiated human neuroblastoma SH-SY5Y cells, which are studied as an in vitro model of neurodegeneration. Despite that the monomeric (isolated) states of the receptor cannot be distinguished from its dimeric forms in such images, TrkB receptor dimers (or couple of individual monomers) are visualized at super-resolution as single pixels in the magnified Huygens-deconvoluted gated STED images. The clusters of higher-order TrkB oligomers are of dynamic nature rather than of a fixed stoichiometry. The propensity for membrane protein clustering as well as the dissociation of the TrkB receptors nanodomains can be modulated by neurotherapeutic formulations containing ω-3 polyunsaturated docosahexaenoic acid (DHA). Nanomolar concentrations of DHA change the receptor topology and lead to disruption of the cluster phases. This result is of therapeutic importance for TrkB receptor availability upon ligand binding as DHA favours the mobility and the dynamic distribution of the protein populations in the cell membranes.

In Vitro Modulation of TrkB Receptor Signaling upon Sequential Delivery of Curcumin-DHA Loaded Carriers Towards Promoting Neuronal Survival.

To in vitro investigate the capacity of carrier-free and lipid-nanoparticle (NP)-encapsulated phytochemical compounds to prevent neuronal damage through neurotrophin potentiating activities. Delivery of molecules promoting the neurotrophin receptor signaling in the central nervous system (CNS) present ongoing interest for combination therapy development. Super-resolution Stimulated Emission Depletion (STED) microscopy imaging and flow cytometry analysis were employed to study the expression of the neurotrophin TrkB receptor in a neuronal cell model, which is highly responsive to binding of brain-derived neurotrophic factor (BDNF). Dual drug-loaded nanoparticle formulations, prepared by self-assembly of lyotropic lipids and PEGylated amphiphile derivatives, were delivered to differentiated human neuroblastoma SH-SY5Y cells subjected to degenerative conditions. The expression of BDNF in the intra and extracellular domains was quantified by ELISA and flow cytometry after sequential treatment of the degenerating SH-SY5Y cells by neurotherapeutic formulations. Flow cytometry was also used to assess the phosphorylation of the transcription factor cAMP response element-binding protein (CREB) in the intracellular domain as a result of the treatment by nanoformulations. Over time, dual drug formulations (curcumin and docosahexaenoic acid (DHA)) promoted the neuronal survival and repair processes through enhanced BDNF secretion and increased phosphorylation of CREB as compared to untreated degenerating cells.

MHC I Expression Regulates Co-clustering and Mobility of Interleukin-2 and -15 Receptors in T Cells

MHC glycoproteins form supramolecular clusters with interleukin-2 and -15 receptors in lipid rafts of T cells. The role of highly expressed MHC I in maintaining these clusters is unknown. We knocked down MHC I in FT7.10 human T cells, and studied protein clustering at two hierarchic levels: molecular aggregations and mobility by Förster resonance energy transfer and fluorescence correlation spectroscopy; and segregation into larger domains or superclusters by superresolution stimulated emission depletion microscopy. Fluorescence correlation spectroscopy-based molecular brightness analysis revealed that the studied molecules diffused as tight aggregates of several proteins of a kind. Knockdown reduced the number of MHC I containing molecular aggregates and their average MHC I content, and decreased the heteroassociation of MHC I with IL-2Rα/IL-15Rα. The mobility of not only MHC I but also that of IL-2Rα/IL-15Rα increased, corroborating the general size decrease of tight aggregates. A multifaceted analysis of stimulated emission depletion images revealed that the diameter of MHC I superclusters diminished from 400–600 to 200–300 nm, whereas those of IL-2Rα/IL-15Rα hardly changed. MHC I and IL-2Rα/IL-15Rα colocalized with GM1 ganglioside-rich lipid rafts, but MHC I clusters retracted to smaller subsets of GM1- and IL-2Rα/IL-15Rα-rich areas upon knockdown. Our results prove that changes in expression level may significantly alter the organization and mobility of interacting membrane proteins.

Reorganization of Lipid Diffusion by Myelin Basic Protein as Revealed by STED Nanoscopy

Myelin is a multilayered membrane that ensheathes axonal fibers in the vertebrate nervous system, allowing fast propagation of nerve action potentials. It contains densely packed lipids, lacks an actin-based cytocortex, and requires myelin basic protein (MBP) as its major structural component. This protein is the basic constituent of the proteinaceous meshwork that is localized between adjacent cytoplasmic membranes of the myelin sheath. Yet, it is not clear how MBP influences the organization and dynamics of the lipid constituents of myelin. Here, we used optical stimulated emission depletion super-resolution microscopy in combination with fluorescence correlation spectroscopy to assess the characteristics of diffusion of different fluorescent lipid analogs in myelin membrane sheets of cultured oligodendrocytes and in micrometer-sized domains that were induced by MBP in live epithelial PtK2 cells. Lipid diffusion was significantly faster and less anomalous both in oligodendrocytes and inside the MBP-rich domains of PtK2 cells compared with undisturbed live PtK2 cells. Our data show that MBP reorganizes lipid diffusion, possibly by preventing the buildup of an actin-based cytocortex and by preventing most membrane proteins from entering the myelin sheath region. Yet, in contrast to myelin sheets in oligodendrocytes, the MBP-induced domains in epithelial PtK2 cells demonstrate no change in lipid order, indicating that segregation of long-chain lipids into myelin sheets is a process specific to oligodendrocytes.

392. Super-Resolution (STED) Imaging Reveals Simultaneous Co-Docking of Tandem Chimeric Antigen Receptors to Two Target Antigens Enhancing T Cell Functionality and Mitigating Antigen Escape

Background: Tandem chimeric antigen receptors (CARs) are bispecific CAR molecules with two different target recognition exodomains joined in tandem. Tandem CARs mediate distinct T cell activation to either target antigen and super-additive functionality upon encounter of both targets simultaneously. Tandem CAR T cells successfully mitigate antigen escape and exhibit enhanced antitumor activity compared to their unispecific counterparts. Stimulated emission depletion (STED) microscopy can achieve a sub-diffractive lateral resolution of ~ 50 nm enabling imaging of the immunological synapse at near single molecule levels.Hypothesis: Super resolution STED Imaging can differentiate heterodimers created by Tandem CAR molecules simultaneously co-docking to both target antigens at the immunological synapse (IS).Methods: Human T cells were retrovirally transduced to express a Tandem CAR molecule specific for HER2(ErbB2) and IL13Rα2. Conjugates of Tandem CAR T cells and the human glioblastoma cell line U373 were fixed after 30 minutes of incubation then labelled with primary unconjugated monoclonal antibodies against HER2 and IL13Rα2 tumor ligands. The IS was examined at the T cell/ U373 interface using confocal and STED microscopy then confirmed with in situ proximity ligation assay (PLA). Unispecific CAR T cells, T cells co-expressing HER2 and IL13Rα2 CARs and non-transduced T cells were used as experimental controls.Results: Confocal microscopy showed co-clustering of HER2 and IL13Rα2 at the IS in contrast to only HER2 or IL13Rα2 localized to the IS for the IL13Rα2 CAR and HER2 CAR T cell/GBM conjugates, respectively. Using a fixed intensity threshold for all conjugates analyzed, quantification of receptor accumulation at the IS revealed that there was increased collective clustering of target molecules at the IS (p= 0.0002). Super-resolution STED microscopy was used to interrogate the quality and quantity of co-localized Tan CAR target ligands at the immune synapse. Measurement of co-localized HER2 and IL13Rα2 aggregate diameters showed a distribution of 80-200 nm aggregate size. The aggregate size has high correlation to the predicted size of CAR-tumor ligand-antibody-antigen complex, indicating increased probability of co-docking of the target ligands at the Tan CAR synapse. Co-localization of the two tumor antigens was present with a very low frequency in the bi-specific T cell - tumor synapse with their average aggregate size ranging from 300-500 nm, suggesting that these ligands formed independent and not “co-docked” conjugates. Co-localization was absent in the single CAR -tumor synapse. In situ PLA with a maximum distance of 30-40 nm showed accumulation of HER2/IL13Rα2 heterodimer signals at the immunological synapse of Tandem CAR Tcell/ GBM IS.Conclusion: Tandem CAR molecules simultaneously engage two target antigens mediating significantly enhances T cell activation through a bifunctional immunological synapse.

Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state.

Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.

The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter.

The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P2 and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca2+ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P2 complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca2+ concentrations is likely to be important for Ca2+-regulated secretion.

Superresolution Imaging of Optical Vortices in a Speckle Pattern.

We characterize, experimentally, the intensity minima of a polarized high numerical aperture optical speckle pattern and the topological charges of the associated optical vortices. The negative of a speckle pattern is imprinted in a uniform fluorescent sample by photobleaching. The remaining fluorescence is imaged with superresolution stimulated emission depletion microscopy, which reveals subdiffraction fluorescence confinement at the center of optical vortices. The intensity statistics of saturated negative speckle patterns are predicted and measured. The charge of optical vortices is determined by controlling the handedness of circular polarization, and the creation or annihilation of a vortex pair along propagation is shown.

Focus on microscopy and photonics

Focus on microscopy and photonics

A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics

Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software.

Super-resolution imaging of the natural killer cell immunological synapse on a glass-supported planar lipid bilayer.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

Super-resolution optical microscopy of lipid plasma membrane dynamics.

Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid-protein interactions and the traditional lipid 'raft' theory.

Quantification of Internalized Silica Nanoparticles via STED Microscopy.

The development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2 × 1010 particles mL−1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to the in vitro sedimentation, diffusion, and dosimetry (ISDD) model 20–27% of the particles sedimented. In comparison, 102-103 NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 1012 particles mL−1) of the smaller particles induced cytotoxicity.

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca2+) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca2+ from the sarcoplasmic reticulum (SR). The Ca2+ leak out of the SR is an important process for cellular Ca2+ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca2+ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca2+ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca2+ sparks and robust Ca2+ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca2+ spark and nonspark-based SR Ca2+ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca2+-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca2+ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca2+ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca2+ release properties due to variations in inter-RyR coupling via local subspace Ca2+ concentration ([Ca2+]ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.

Ultrafast photon counting applied to resonant scanning STED microscopy.

To take full advantage of fast resonant scanning in super-resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue-to-digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (∼50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time-gated continuous wave STED technology to the usage of resonant scanning with hardware-based time-gating. The assembled system provides superb signal-to-noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant-scanning continuous wave STED microscopy with online time-gated detection.

Spatial Distribution of DARPP-32 in Dendritic Spines

The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3´, 5´-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons. Primary culture of striatal neurons were immunofluorescently labeled for DARPP-32 with Alexa-594 and for the dopamine D1 receptor (D1R) with atto-647N. Dual-color STED microscopy revealed discrete localizations of DARPP-32 and D1R in the spine structure, with clustered distributions in both head and neck. Dissected spine structures reveal that the DARPP-32 signal rarely overlapped with the D1R signal. The D1R receptor is positioned in an “aggregated” manner primarily in the spine head and to some extent in the neck, while DARPP-32 forms several neighboring small nanoclusters spanning the whole spine structure. The DARPP-32 clusters have a mean size of 52 +/- 6 nm, which is close to the resolution limit of the microscope and corresponds to the physical size of a few individual phosphoprotein immunocomplexes. Dissection of synaptic proteins using superresolution microscopy gives possibilities to reveal in better detail biologically relevant information, as compared to diffraction-limited microscopy. In this work, the dissected postsynaptic topology of the DARPP-32 phosphoprotein provides strong evidence for a compartmentalized and confined distribution in dendritic spines. The protein topology and the relatively low copy number of phosphoprotein provides a conception of DARPP-32’s possibilities to fine-tune the regulation of synaptic signaling, which should have an impact on the performance of the neuronal circuits in which it is expressed.

New Insight into Lipid-Protein Membrane Organization and its Functionality with Super-Resolution STED Microscopy

Stimulated Emission Depletion (STED) far-field microscopy allows the study of living cells with nanoscale resolution, otherwise impeded by the limited spatial resolution of conventional microscopes. Besides the recording of images, the combination of STED with single-molecule sensitive spectroscopic tools such as Fluorescence Correlation Spectroscopy (FCS) discloses complex dynamical processes hidden to the conventional observations. For example, STED-FCS offers novel insights into important cellular processes, such as lipid-lipid, lipid-protein interactions or the formation of so-called “lipid-rafts” in the cellular plasma membrane, and their role in cellular functionality. Improved insights are realized by the implementation of gated detection or by recording STED-FCS data during scanning.