Stimulated Emission Depletion Imaging Research Articles

Mechanical Tension of Biomembranes Can Be Measured by Super Resolution (STED) Microscopy of Force-Induced Nanotubes.

Membrane tension modulates the morphology of plasma-membrane tubular protrusions in cells but is difficult to measure. Here, we propose to use microscopy imaging to assess the membrane tension. We report direct measurement of membrane nanotube diameters with unprecedented resolution using stimulated emission depletion (STED) microscopy. For this purpose, we integrated an optical tweezers setup in a commercial microscope equipped for STED imaging and established micropipette aspiration of giant vesicles. Membrane nanotubes were pulled from the vesicles at specific membrane tension imposed by the aspiration pipet. Tube diameters calculated from the applied tension using the membrane curvature elasticity model are in excellent agreement with data measured directly with STED. Our approach can be extended to cellular membranes and will then allow us to estimate the mechanical membrane tension within the force-induced nanotubes.

Super-resolution microscopy unveils transmembrane\xa0domain-mediated internalization of cross-reacting material 197 into diphtheria toxin-resistant mouse J774A.1 cells and primary rat fibroblasts in vitro

Diphtheria toxin (DT) efficiently inhibits protein synthesis in human cells, resulting in severe disease diphtheria. The sensitivity towards DT varies between mammalian species. Mice and rats are resistant to DT. However, the reason underlying this insensitivity is controversially discussed and not well understood. Therefore, we investigated the steps of DT uptake, i.e. receptor binding and internalization into mouse J774A.1 macrophages and primary rat fibroblasts. We exploited the non-toxic DT-mutant cross-reacting material 197 (CRM197) and three additional receptor binding-deficient mutants (250 nM each) to investigate binding to cell surface and internalization into murine cells via flow cytometry and stimulated emission depletion (STED) super-resolution optical microscopy. Dual-color STED imaging unveiled CRM197 interacting with the murine precursor of the heparin-binding epidermal growth factor-like growth factor (HB-EGF). Moreover, we identified CRM197’s transmembrane domain as an additional HB-EGF binding site, which is also involved in the receptor-mediated internalization into murine cells. However, we do not find evidence for translocation of the catalytically active subunit (DTA) into the cytosol when 250 nM DT were applied. In conclusion, we provide evidence that the resistance of murine cells to DT is caused by an insufficiency of DTA to escape from endosomes and reach the cytosol. Possibly, a higher affinity interaction of DT and the HB-EGF is required for translocation, which highlights the role of the receptor in the endosomes during the translocation step. We extend the current knowledge about cellular uptake of the medically relevant DT and CRM197.

Deep adversarial network for super stimulated emission depletion imaging

Stimulated emission depletion (STED), as one of the emerging super-resolution techniques, defines a state-of-the-art image resolution method. It has been developed into a universal fluorescent imaging tool over the past several years. The currently best available lateral resolution offered by STED is around 20 nm, but in real live cell imaging applications, the regular resolution offered through this mechanism is around 100 nm, limited by phototoxicity. Many critical biological structures are below this resolution level. Hence, it will be invaluable to improve the STED resolution through postprocessing techniques. We propose a deep adversarial network for improving the STED resolution significantly, which takes an STED image as an input, relies on physical modeling to obtain training data, and outputs a “self-refined” counterpart image at a higher resolution level. In other words, we use the prior knowledge on the STED point spread function and the structural information about the cells to generate simulated labeled data pairs for network training. Our results suggest that 30-nm resolution can be achieved from a 60-nm resolution STED image, and in our simulation and experiments, the structural similarity index values between the label and output result reached around 0.98, significantly higher than those obtained using the Lucy–Richardson deconvolution method and a state-of-the-art UNet-based super-resolution network.

Spaser Nanoparticles for Ultranarrow Bandwidth STED Super-Resolution Imaging.

Super-resolution microscopy, as a powerful tool of seeing abundant spatial details, typically can only distinguish a few distinct targets at a time due to the spectral crosstalk between fluorophores. Spaser (i.e., surface plasmon laser) nanoprobes, which confine lasing emission into nanoscale, offer an opportunity to eliminate such obstacle. Here, realized is narrow band stimulated emission depletion (STED) nanoscopy on spaser nanoparticles by collecting the coherent spasing signals. Demonstrated are the physics concept and feasibility of erasing spaser emission by using a depletion beam to suppress the population inversion, which lays the foundation of spaser-based STED super-resolution. Thanks to the small size (47 nm) and narrow spectral linewidth (3.8 nm) of the spaser nanoparticles, a 74 nm spatial resolution in STED imaging within an acquisition bandwidth of 10 nm is finally obtained. These spaser nanoparticles, if multiplexing with different wavelengths, in principle, allow for spectral-multiplexed imaging, sensing, cytometry, and light operation of a large number of targets all at once.

Use of Super-Resolution Optical Microscopy To Reveal Direct Virus Binding at Hybrid Core-Shell Matrixes.

Polymer particles with antibody-like affinity, i.e., molecularly imprinted polymers, offer an ideal platform for biopharmaceutical virus purification. In recent years, attempts combining molecular imprinting technology with a variety of visualization and detection techniques have been reported for directly confirming the localized presence of the template. Direct target visualization is crucial for the characterization of molecularly imprinted polymers, especially if biological templates such as viruses are used. In the present study, for the first time the viral binding behavior at virus-imprinted polymers (VIPs) via stimulated emission depletion (STED) microscopy is shown by imaging individual, fluorescently labeled virus particles. STED microscopy achieves among various other super-resolution techniques the best temporal resolution at high spatial resolution. An innovative virus purification material selective for human adenovirus type 5 (AdV5) offered highly purified virus for the subsequent fluorescent labeling procedure, thus enabling STED imaging. Excellent binding affinities (150-fold higher versus control particles) and high selectivity toward the target virus (AdV5) were observed at those VIPs, even in competitive binding experiments with minute virus of mice using dual-label STED microscopy.

Long-term STED imaging of amyloid fibers with exchangeable Thioflavin T.

We report the use of the amyloid probe Thioflavin T (ThT) as a specific and exchangeable fluorophore for stimulated emission depletion (STED) super-resolution imaging of amyloid fibers. This method achieves a spatial resolution in the range of 60-70 nm, low image background and increased photostability that enables long-term STED imaging. These results expand the widespread uses of ThT and can be potentially extended to other common amyloid fluorescent probes, providing new tools for the study of amyloid diseases.

Numerically Enhanced Stimulated Emission Depletion Microscopy with Adaptive Optics for Deep-Tissue Super-Resolved Imaging.

In stimulated emission depletion (STED) nanoscopy, the major origin of decreased signal-to-noise ratio within images can be attributed to sample photobleaching and strong optical aberrations. This is due to STED utilizing a high-power depletion laser (increasing the risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here, we demonstrate a custom-built STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating tissue. We introduce and investigate a state of the art image denoising method by block-matching and collaborative 3D filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise and further enhance the image quality. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well established Fourier ring correlation metric. Results achieved through the combination of aberration correction and tailored image processing are experimentally validated through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under an 80 μm thick layer of tissue with lateral and axial resolution of 204 and 310 nm, respectively.

Recent advances in STED and RESOLFT super-resolution imaging techniques.

Stimulated emission depletion (STED) and reversible saturable optical fluorescence transition (RESOLFT) microscopy are the super-resolution imaging techniques that can acquire nanoscale spatial resolution. The spatial resolution of the other far-field optical microscopic techniques is bound by diffraction limit, however, STED/RESOLFT techniques eliminate the diffraction barrier. These microscopic techniques have taken the limits of optical image resolution down to the nanometer scale and opened new paths for biomedical and nanophosphor research. In this paper, we review the recent advancements of these techniques in the field of nanoscopy using continuous wave (CW) laser sources. Further, we discuss the main limitation of the STED microscopy in terms of essential requirements of higher depletion beam power and photobleaching issues. The RESOLFT microscopic technique can be considered as an alternate technique to overcome limitations of existing STED microscopy. Moreover, the Bessel and Gaussian-Bessel beam STED microscopic techniques are also reviewed to produce deep images with faster scanning of the samples. The organic molecules as well as the fluorescent doped nanoparticles like ZnSe:Mn having characteristics of excited state absorption can be investigated using RESOLFT microscopy.

Stable stimulated emission depletion imaging of extended sample regions

Stimulated emission depletion (STED) nanoscopy has become one of the most used nanoscopy techniques over the last decade. However, most recordings are done in specimen regions no larger than 10–30 × 10–30 μm2 due to aberrations, instability and manual mechanical stages. Here, we demonstrate automated 2D and 3D STED nanoscopy of extended sample regions up to 0.5 × 0.5 mm2 by using a scanning system that maintains stationary beams in the back focal plane. The setup allows up to 80–100 × 80–100 μm2 field of view (FOV) with uniform spatial resolution, a mechanical stage allowing sequential tiling to record larger sample areas, and a feedback system keeping the sample in focus at all times. Taken together, this allows automated recording of theoretically unlimited-sized sample areas and volumes, without compromising the achievable spatial resolution and image quality.

Green emitted CdSe@ZnS quantum dots for FLIM and STED imaging applications

Inorganic quantum dots (QDs) have excellent optical properties, such as high fluorescence intensity, excellent photostability and tunable emission wavelength, etc., facilitating them to be used as labels and probes for bioimaging. In this study, CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope (FLIM) and stimulated emission depletion (STED) nanoscopy imaging. The emission peak of CdSe@ZnS QDs centered at 526[Formula: see text]nm with a narrow width of 19[Formula: see text]nm and the photoluminescence quantum yield (PLQY) was 64%. The QDs presented excellent anti-photobleaching property which can be irradiated for 400[Formula: see text]min by STED laser with 39.8[Formula: see text]mW. The lateral resolution of 42.0[Formula: see text]nm is demonstrated for single QDs under STED laser (27.5[Formula: see text]mW) irradiation. Furthermore, the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5[Formula: see text]nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes. Meanwhile, Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM, and their fluorescence lifetime was around 3.1[Formula: see text]ns, consistent with the in vitro value, suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.

Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon (RTN) and cytoskeleton-linking membrane protein 63 (CLIMP-63), respectively. Here, stimulated emission depletion (STED) super-resolution microscopy shows that reticulon4a (RTN4a) and CLIMP-63 also regulate the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ER monomeric oxidizing environment-optimized green fluorescent protein (ERmoxGFP), membrane Sec61βGFP, knock-in calreticulin-GFP, and antibody-labeled ER-resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. RTN4a segregates away from and restricts lumenal blob length, while CLIMP-63 associates with and increases lumenal blob length. RTN4a and CLIMP-63 also regulate the nanodomain distribution of ER-resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that RTN4a and CLIMP-63 regulate dynamic nanoscale lumenal compartmentalization along peripheral ER tubules. RTN4a enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER-shaping proteins RTN and CLIMP-63 therefore regulate lumenal ER nanodomain heterogeneity, interaction with ER-resident proteins, and dynamics in peripheral ER tubules.

Multiplexed Superresolution CRISPR Imaging of Chromatin in Living Cells

Multiplexed Superresolution CRISPR Imaging of Chromatin in Living Cells

A Fiber-Coupled Stimulated Emission Depletion Microscope for Bend-Insensitive Through-Fiber Imaging

We present results for a new type of fiber-coupled stimulated emission depletion (STED) microscope which uses a single fiber to transport STED and excitation light, as well as collect the fluorescence signal. Our method utilizes two higher-order eigenmodes of polarization maintaining (PM) fiber to generate the doughnut-shaped STED beam. The modes are excited with separate beams that share no temporal coherence, yielding output that is independent of fiber bending. We measured the resolution using 45 nm fluorescent beads and found a median bead image size of 116 nm. This resolution does not change as function of fiber bending radius, demonstrating robust operation. We report, for the first time, STED images of fixed biological samples collected in the epi-direction through fiber. Our microscope design shows promise for future use in super-resolution micro-endoscopes and in vivo neural imaging in awake and freely-behaving animals.

Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens.

Super-resolution fluorescence microscopy has become an important catalyst for discovery in the life sciences. In STimulated Emission Depletion (STED) microscopy, a pattern of light drives fluorophores from a signal-emitting on-state to a non-signalling off-state. Only emitters residing in a sub-diffraction volume around an intensity minimum are allowed to fluoresce, rendering them distinguishable from the nearby, but dark fluorophores. STED routinely achieves resolution in the few tens of nanometers range in biological samples and is suitable for live imaging. Here, we review the working principle of STED and provide general guidelines for successful STED imaging. The strive for ever higher resolution comes at the cost of increased light burden. We discuss techniques to reduce light exposure and mitigate its detrimental effects on the specimen. These include specialized illumination strategies as well as protecting fluorophores from photobleaching mediated by high-intensity STED light. This opens up the prospect of volumetric imaging in living cells and tissues with diffraction-unlimited resolution in all three spatial dimensions.

Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy.

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems.

Loss of Protein Phosphatase 1 Regulatory Subunit PPP1R3A Promotes Atrial Fibrillation.

Abnormal calcium (Ca2+) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN. Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln-knockout mice. Ppp1r3a-knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a-knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca2+ imaging. Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF. PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.

AFM-STED correlative nanoscopy reveals a dark side in fluorescence microscopy imaging.

It is known that the presence of fluorophores can influence the dynamics of molecular processes. Despite this, an affordable technique to control the fluorophore distribution within the sample, as well as the rise of unpredictable anomalous processes induced by the fluorophore itself, is missing. We coupled a stimulated emission depletion (STED) microscope with an atomic force microscope to investigate the formation of amyloid aggregates. In particular, we studied the in vitro aggregation of insulin and two alloforms of β amyloid peptides. We followed standard methods to induce the aggregation and to label the molecules at different dye-to-protein ratios. Only a fraction of the fibrillar aggregates was displayed in STED images, indicating that the labeled molecules did not participate indistinctly to the aggregation process. This finding demonstrates that labeled molecules follow only selected pathways of aggregation, among the multiple that are present in the aggregation reaction.

A simple empirical algorithm for optimising depletion power and resolution for dye and system specific STED imaging.

Here we show an easy method for determining an effective dye saturation factor ('PSTED ') for STED (Stimulated Emission Depletion) microscopy. We define PSTED to be a combined microscope system plus dye factor (analogous to the traditional ground truth Is measurement, which is microscope independent) that is functionally defined as the power in the depletion beam that provides a resolution enhancement of 41% compared to confocal, according to the modified Abbe's formula for STED resolution enhancement. We show that the determination of PSTED provides insight not only into the suitability of a particular dye and the best imaging parameters to be used for an experiment, but also sets the critical value for correctly determining the point spread function (PSF) used in deconvolution of STED images. PSTED can be a function of many experimental variables, both microscope and sample related. Here we show the utility of doing PSTED determinations by (1) exploiting the simple relationship between width and a threshold-defined area provided by a Gaussian PSF, for either linear or spherical objects and (2) linearising the normally inverse hyperbolic function of resolution versus power that can determine PSTED . We show that this rearrangement allows us to determine PSTED using only a few measurements: either at a few relatively low depletion powers, on traditional bead size measurements or by finding the total area of a naturally occurring sub-limit sized biological feature (in this case, microtubules). We show the derivation of these equations and methods and the utility of its use by characterising several dyes and a local imaging parameter relevant to STED microscopy. This information is used to predict the enhancement of resolution of the point spread function necessary for post-processing deconvolution. LAY DESCRIPTION: Stimulated Emission Depletion (STED) microscopy is a fluorescence imaging superresolution technique that achieves tens of nanometres resolution. This is done by utilising a depletion laser to effectively quench (deplete) fluorescence in a donut shape overlapping the normally excited fluorescence spot. The size of the remaining (undepleted) central fluorescence spot is power dependent allowing 'tunable' resolution with the power of the STED depletion laser. This depletion power versus resolution relationship is dye and instrument dependent. We have developed a method for quickly measuring this relationship to optimise experiments based on individual dyes and microscope specific parameters. This allows for quickly optimising microscope settings and for correctly postprocessing images.

Titanium Dioxide Nanoparticles Induce Mitochondrial Dynamic Imbalance and Damage in HT22 Cells

Mitochondria, as dynamic organelles, are precisely regulated by fusion and fission. The dynamic balance of fusion and fission controls mitochondrial morphology and their subcellular location and function. Exposure to titanium dioxide nanoparticles (TiO2NPs) may cause serious health problems. However, how TiO2NPs affect the mitochondrial dynamics remains unclear. In the present study, we investigated the changes of mitochondrial dynamics in the TiO2NPs-treated HT22 cells by confocal and stimulated emission depletion (STED) microscopy. The confocal images demonstrated obvious changes in the average length and density of the mitochondria after TiO2NPs treatment, while STED images further obtained the nanoscale submitochondrial structures of the mitochondria under TiO2NPs insult. The fluorescence intensity distributions suggested that mitochondria fragmented in the TiO2NPs-treated cells. TiO2NPs treatment caused mitochondrial dynamic imbalance due to the imbalanced expression of dynamin-related protein 1 (Drp1) and optic atrophy 1 (Opa1). Furthermore, we examined the levels of oxidative stress and mitochondrial membrane potential (MMP) and the generation of adenosine triphosphate (ATP), which revealed the damage of mitochondria under TiO2NPs exposure. Meanwhile, the significant changes of expressions of B-cell lymphoma 2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cytochrome c (Cyt C), and caspase 9 demonstrated that TiO2NPs treatment activated the mitochondrial-related apoptosis pathway. These cellular events can be largely prevented via cell incubation with mitoTEMPO, a mitochondria-targeted superoxide scavenger. Our results confirm that TiO2NPs targeted the mitochondria, inducing mitochondrial dynamic imbalance and damage in HT22 cells. Our study provides an insightful understanding of the mechanisms underlying TiO2NPs cytotoxicity.

Spectrin in mammalian hearing mechanisms

Structural Biology Stereocilia are hair-like structures in the inner ear that transform sound vibration into a neural signal. These structures stop growing in humans early in fetal development, so their resilience is critical to lifelong hearing. Liu et al. used stimulated emission depletion imaging of the inner ear hair cells of mice to observe the arrangement of cytoskeletal proteins at the base of stereocilia rootlets. The elasticity of the protein spectrin may allow long-term survival of stereocilia under constant mechanical stress, analogous to the role of spectrin in red blood cell deformability. Damage to the observed ring-like structures of spectrin was associated with hearing loss in mice. Sci. Adv. 10.1126/sciadv.aav7803 (2019).