Stimulated Emission Depletion Microscopy Research Articles

Cell-Permeable Nanobodies Allow Dual-Color Super-Resolution Microscopy in Untransfected Living Cells.

Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi‐synthetic nanobodies are equipped with a cleavable cell‐penetrating peptide for efficient cellular entry, which enables super‐resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis.

Cortactin knockdown results in disruption of basal TBCs and alters turnover of Sertoli cell ESs in Rattus norvegicus

Here we explore the prediction that long-term knockdown of cortactin (CTTN), a component of tubulobulbar complexes (TBCs), disrupts TBCs in Sertoli cells and alters the turnover of basal ectoplasmic specializations (ESs). In rats, intratesticular injections of siRNA targeting CTTN (siCTTN) in one testis and nontargeting siRNA (siControl) in the contralateral testis were done on days 0, 2, 4, 6, and 8. The experiment was terminated on day 9 and testes were analyzed by either western blotting, or by stimulated emission depletion (STED), electron and/or conventional fluorescence microscopy. Levels of CTTN were successfully knocked down in experimental testes compared to controls. When cryo-sections were labeled for actin filaments, or CTTN, and oxysterol binding protein-related protein 9 (ORP9) and analyzed by STED microscopy, TBCs were "less distinct" than in tubules of the same stages from control testes. When analyzed by electron microscopy, redundant clumps of basal actin filament containing ESs were observed in experimental sections. Using labeling of actin filaments in ESs, thresholding techniques were used to calculate the number of pixels above threshold per unit length of tubule wall in seminiferous tubules at Stage VII. Median values were higher in experimental testes relative to controls in the four animals analyzed. Although we detected subtle differences in ES turnover, we were unable to demonstrate changes in spermatocyte translocation or in the levels of junction proteins at the sites. Our results are the first to demonstrate that perturbation of basal TBCs alters the turnover of actin-related junctions (ESs).

Coherent Interference Fringes of Two-Photon Photoluminescence in Individual Au Nanoparticles: The Critical Role of the Intermediate State.

The interaction between light and metal nanoparticles enables investigations of microscopic phenomena on nanometer length and ultrashort timescales, benefiting from strong confinement and enhancement of the optical field. However, the ultrafast dynamics of these nanoparticles are primarily investigated by multiphoton photoluminescence on picoseconds or photoemission on femtoseconds independently. Here, we presented two-photon photoluminescence (TPPL) measurements on individual Au nanobipyramids (AuNP) to reveal their ultrafast dynamics by double-pulse excitation on a global timescale ranging from subfemtosecond to tens of picoseconds. Two orders of magnitude photoluminescence enhancement, namely, coherent interference fringes, has been demonstrated. Power-dependent measurements uncovered the transform of the nonlinearity from 1 to 2 when the interpulse delay varied from tens of femtoseconds to tens of picoseconds. We proved that the real intermediate state plays a critical role in the observed phenomena, supported by numerical simulations with a three-state model. Our results provide insight into the role of intermediate states in the ultrafast dynamics of noble metal nanoparticles. The presence of the intermediate states in AuNP and the coherent control of state populations offer interesting perspectives for imaging, sensing, nanophotonics, and in particular, for preparing macroscopic superposition states at room temperature and low-power superresolution stimulated emission depletion microscopy.

Super-Resolution Fluorescence Microscopy Methods for Assessing Mouse Biology.

Super-resolution (diffraction unlimited) microscopy was developed 15 years ago; the developers were awarded the Nobel Prize in Chemistry in recognition of their work in 2014. Super-resolution microscopy is increasingly being applied to diverse scientific fields, from single molecules to cell organelles, viruses, bacteria, plants, and animals, especially the mammalian model organism Mus musculus. In this review, we explain how super-resolution microscopy, along with fluorescence microscopy from which it grew, has aided the renaissance of the light microscope. We cover experiment planning and specimen preparation and explain structured illumination microscopy, super-resolution radial fluctuations, stimulated emission depletion microscopy, single-molecule localization microscopy, and super-resolution imaging by pixel reassignment. The final section of this review discusses the strengths and weaknesses of each super-resolution technique and how to choose the best approach for your research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

3D test sample for the calibration and quality control of stimulated emission depletion (STED) and confocal microscopes

Multiple samples are required to monitor and optimize the quality and reliability of quantitative measurements of stimulated emission depletion (STED) and confocal microscopes. Here, we present a single sample to calibrate these microscopes, align their laser beams and measure their point spread function (PSF) in 3D. The sample is composed of a refractive index matched colloidal crystal of silica beads with fluorescent and gold cores. The microscopes can be calibrated in three dimensions using the periodicity of the crystal; the alignment of the laser beams can be checked using the reflection of the gold cores; and the PSF can be measured at multiple positions and depths using the fluorescent cores. It is demonstrated how this sample can be used to visualize and improve the quality of STED and confocal microscopy images. The sample is adjustable to meet the requirements of different NA objectives and microscopy techniques and additionally can be used to evaluate refractive index mismatches as a function of depth quantitatively.

Optical Microscopy and the Extracellular Matrix Structure: A Review.

Biological tissues are not uniquely composed of cells. A substantial part of their volume is extracellular space, which is primarily filled by an intricate network of macromolecules constituting the extracellular matrix (ECM). The ECM serves as the scaffolding for tissues and organs throughout the body, playing an essential role in their structural and functional integrity. Understanding the intimate interaction between the cells and their structural microenvironment is central to our understanding of the factors driving the formation of normal versus remodelled tissue, including the processes involved in chronic fibrotic diseases. The visualization of the ECM is a key factor to track such changes successfully. This review is focused on presenting several optical imaging microscopy modalities used to characterize different ECM components. In this review, we describe and provide examples of applications of a vast gamut of microscopy techniques, such as widefield fluorescence, total internal reflection fluorescence, laser scanning confocal microscopy, multipoint/slit confocal microscopy, two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG, THG), coherent anti-Stokes Raman scattering (CARS), fluorescence lifetime imaging microscopy (FLIM), structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), ground-state depletion microscopy (GSD), and photoactivated localization microscopy (PALM/fPALM), as well as their main advantages, limitations.

IsoSTED microscopy with water-immersion lenses and background reduction

Fluorescence microscopy is an excellent tool to gain knowledge on cellular structures and biochemical processes. Stimulated emission depletion (STED) microscopy provides a resolution in the range of a few 10 nm at relatively fast data acquisition. As cellular structures can be oriented in any direction, it is of great benefit if the microscope exhibits an isotropic resolution. Here, we present an isoSTED microscope that utilizes water-immersion objective lenses and enables imaging of cellular structures with an isotropic resolution of better than 60 nm in living samples at room temperature and without CO2 supply or another pH control. This corresponds to a reduction of the focal volume by far more than two orders of magnitude as compared to confocal microscopy. The imaging speed is in the range of 0.8 s/μm3. Because fluorescence signal can only be detected from a diffraction-limited volume, a background signal is inevitably observed at resolutions well beyond the diffraction limit. Therefore, we additionally present a method that allows us to identify this unspecific background signal and to remove it from the image.

Three-dimensional nanothermometry below the diffraction limit.

Lanthanide-doped nanothermometers are used to measure temperature through changes in their emission characteristic with sensitivities of up to a few %/K. In contrast to their sensitivity, their spatial resolution, which is of critical importance for various applications, has not been thoroughly studied and optimized. We numerically investigated the improvement in spatial resolution of nanothermometers with a stimulated emission depletion microscopy approach. Fundamental relationships between spatial and temperature resolution were identified by using different beam parameters for the excitation and depletion beams. Our simulations predict contactless temperature measurement below the diffraction limit with temperature resolution of ±1.25K. We further studied the influence of sample thickness and position on both temperature and spatial resolution and showed the potential of three-dimensional measurements.

Abstract 2886: The mechanistic effect of DHA on FABP7 associated membrane lipid order & nanodomain distribution in glioblastoma migration

Abstract Glioblastoma (GBM) is the most common primary brain malignancy. Brain fatty acid binding protein (B-FABP, FABP7) promotes glioblastoma (GBM) migration and correlates to dismal prognosis in GBM patients. Long chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and arachidonic acid (AA) are abundant in brain cell membranes. DHA and AA are preferred ligands of FABP7, with DHA having the stronger affinity for FABP7. Previous studies have shown that DHA inhibits GBM migration in an FABP7-dependent manner. In the current study, we have demonstrated that DHA dramatically altered membrane FABP7 nanoscale distribution pattern, which resulting from plasma membrane lipid ordered domain disruption in GBM cells and neurosphere cultures. We used quantitative membrane order assay to show that FABP7-expressing cells has increased membrane lipid order which is tightly correlated with higher GBM migration property. Using stimulated emission depletion (STED) microscopy, we observed lipid ordered domain was associated with FABP7 nanoscale domain formation. Interestingly, super-resolution microscopy quantitative image analysis revealed that FABP7 membrane nanodomains distribution was dramatically altered upon DHA treatment, which is associated with DHA inhibitory effect on membrane lipid order. Furthermore, STED microscopy has also revealed that reduced FABP7 nanodomain formation in slow migrating GBM cells are similar as FABP7-expressing GBM cells upon DHA treatment. Therefore, we hypothesize that these alterations are likely to be the mechanism of FABP7-mediated DHA inhibitory role in GBM migration. Citation Format: Xia Xu, Yixiong Wang, Wonshik Choi, Roseline Godbout. The mechanistic effect of DHA on FABP7 associated membrane lipid order & nanodomain distribution in glioblastoma migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2886.

Ratiometric photon reassignment based on fluorescence lifetime to improve resolution in pulse STED microscopy.

Depletion beams with long pulse durations (∼600ps) have recently been applied in stimulated emission depletion (STED) microscopy to reduce photobleaching and phototoxicity. Therefore, improving the resolution of pulse STED at lower depletion power for the super-resolution imaging of live biological specimens has attracted increasing interest. Herein, we present a simple method termed ratiometric photon reassignment based on the fluorescence lifetime, in which highly spatially resolved long-lifetime fluorophore components in the center are extracted, and short-lifetime fluorophore components in the periphery are discarded to improve resolution without increasing the depletion power. The experimental results demonstrate improved resolution and signal-to-noise ratio compared with traditional time-gated STED. Our proposed method requires lower budget and data processing because, in contrast to the separation of photons by lifetime tuning), lifetime measurements are not required.

Stimulated Emission Depletion Microscopy with Color Centers in Hexagonal Boron Nitride

Stimulated emission depletion, or STED microscopy is a well-established super-resolution technique, but is ultimately limited by the chosen flourophore. Here we demonstrate STED microscopy with color centers in nanoscale flakes of hexagonal boron nitride using time-gated continuous wave STED. For color centers with zero phonon line emission around 580 nm we measure a STED cross-section of (5.5 $\pm$ 3.2) x $10^{-17} \mbox{cm}^{2}$, achieve a resolution of $\sim$ 50 nm and resolve two color centers separated by 250 nm, which is less than the diffraction limit. The achieved resolution is limited by the numerical aperture of the objective lens (0.8) and the available laser power, and we predict that a resolution of sub-10 nm can be achieved with an oil immersion objective lens, similar to state-of-the-art resolution obtained with nitrogen vacancy centers in diamond.

Fusion of clathrin and caveolae endocytic vesicles revealed by line-switching dual-color STED microscopy

Clathrin- and caveolae-mediated endocytosis are the most commonly used pathways for the internalization of cell membrane receptors. However, due to their dimensions are within the diffraction limit, traditional fluorescence microscopy cannot distinguish them and little is known about their interactions underneath cell membrane. In this study, we proposed the line-switching scanning imaging mode for dual-color triplet-state relaxation (T-Rex) stimulated emission depletion (STED) super-resolution microscopy. With this line-switching mode, the cross-talk between the two channels, the side effects from pulse picker and image drift in frame scanning mode can be effectively eliminated. The dual-color super-resolution imaging results in mixed fluorescent beads validated the excellent performance. With this super-resolution microscope, not only the ring-shaped structure of clathrin and caveolae endocytic vesicles, but also their semi-fused structures underneath the cell membrane were distinguished clearly. The resultant information will greatly facilitate the study of clathrin- and caveolae-mediated receptor endocytosis and signaling process and also our home-built dual-color T-Rex STED microscope with this line-switching imaging mode provides a precise and convenient way to study subcellular-scale protein interactions.

Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons.

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGluu that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.

Pupil function design for multifocal confocal, STED, and isoSTED microscopy.

Point scanning super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy are powerful tools to observe biological samples at sub-diffraction limited resolution in three dimensions. However, scanning the sample with only a single beam limits the imaging speed in these microscopes. Here, we propose a concept to increase this speed by introducing highly flexible multifocal illumination and detection. We introduce phase patterns in the objectives’ pupil planes to create arrays of foci in the sample plane with negligible loss of laser power. High uniformity of these foci’s intensities is achieved by iteratively applying a weighted Gerchberg–Saxton phase retrieval algorithm. We characterize the performance of this iterative approach numerically and present simulation results that demonstrate the high quality of the focus arrays for future implementations in laser-scanning STED and isoSTED microscopes. The same approach can also be applied in diffraction-limited confocal laser scanning microscopy.

Aberration correction in stimulated emission depletion microscopy to increase imaging depth in living brain tissue.

.Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue.Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth.Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices.Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of inside the biological tissue and obtain a 60% signal increase after correction.Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue.

Continuous-wave near-infrared stimulated-emission depletion microscopy using downshifting lanthanide nanoparticles.

Stimulated-emission depletion (STED) microscopy has profoundly extended our horizons to the subcellular level1-3. However, it remains challenging to perform hours-long, autofluorescence-free super-resolution imaging in near-infrared (NIR) optical windows under facile continuous-wave laser depletion at low power4,5. Here we report downshifting lanthanide nanoparticles that enable background-suppressed STED imaging in all-NIR spectral bands (λexcitation = 808 nm, λdepletion = 1,064 nm and λemission = 850-900 nm), with a lateral resolution of below 20 nm and zero photobleaching. With a quasi-four-level configuration and long-lived (τ > 100 μs) metastable states, these nanoparticles support near-unity (98.8%) luminescence suppression under 19 kW cm-2 saturation intensity. The all-NIR regime enables high-contrast deep-tissue (~50 μm) imaging with approximately 70 nm spatial resolution. These lanthanide nanoprobes promise to expand the application realm of STED microscopy and pave the way towards high-resolution time-lapse investigations of cellular processes at superior spatial and temporal dimensions.

The subcellular arrangement of alpha-synuclein proteoforms in the Parkinson\u2019s disease brain as revealed by multicolor STED microscopy

Various post-translationally modified (PTM) proteoforms of alpha-synuclein (aSyn)—including C-terminally truncated (CTT) and Serine 129 phosphorylated (Ser129-p) aSyn—accumulate in Lewy bodies (LBs) in different regions of the Parkinson’s disease (PD) brain. Insight into the distribution of these proteoforms within LBs and subcellular compartments may aid in understanding the orchestration of Lewy pathology in PD. We applied epitope-specific antibodies against CTT and Ser129-p aSyn proteoforms and different aSyn domains in immunohistochemical multiple labelings on post-mortem brain tissue from PD patients and non-neurological, aged controls, which were scanned using high-resolution 3D multicolor confocal and stimulated emission depletion (STED) microscopy. Our multiple labeling setup highlighted a consistent onion skin-type 3D architecture in mature nigral LBs in which an intricate and structured-appearing framework of Ser129-p aSyn and cytoskeletal elements encapsulates a core enriched in CTT aSyn species. By label-free CARS microscopy we found that enrichments of proteins and lipids were mainly localized to the central portion of nigral aSyn-immunopositive (aSyn+) inclusions. Outside LBs, we observed that 122CTT aSyn+ punctae localized at mitochondrial membranes in the cytoplasm of neurons in PD and control brains, suggesting a physiological role for 122CTT aSyn outside of LBs. In contrast, very limited to no Ser129-p aSyn immunoreactivity was observed in brains of non-neurological controls, while the alignment of Ser129-p aSyn in a neuronal cytoplasmic network was characteristic for brains with (incidental) LB disease. Interestingly, Ser129-p aSyn+ network profiles were not only observed in neurons containing LBs but also in neurons without LBs particularly in donors at early disease stage, pointing towards a possible subcellular pathological phenotype preceding LB formation. Together, our high-resolution and 3D multicolor microscopy observations in the post-mortem human brain provide insights into potential mechanisms underlying a regulated LB morphogenesis.

Stable but not rigid: Chronic in vivo STED nanoscopy reveals extensive remodeling of spines, indicating multiple drivers of plasticity.

Excitatory synapses on dendritic spines of pyramidal neurons are considered a central memory locus. To foster both continuous adaption and the storage of long-term information, spines need to be plastic and stable at the same time. Here, we advanced in vivo STED nanoscopy to superresolve distinct features of spines (head size and neck length/width) in mouse neocortex for up to 1 month. While LTP-dependent changes predict highly correlated modifications of spine geometry, we find both, uncorrelated and correlated dynamics, indicating multiple independent drivers of spine remodeling. The magnitude of this remodeling suggests substantial fluctuations in synaptic strength. Despite this high degree of volatility, all spine features exhibit persistent components that are maintained over long periods of time. Furthermore, chronic nanoscopy uncovers structural alterations in the cortex of a mouse model of neurodegeneration. Thus, at the nanoscale, stable dendritic spines exhibit a delicate balance of stability and volatility.

The plant nuclear lamina proteins NMCP1 and NMCP2 form a filamentous network with lateral filament associations.

Plant genomes lack genes encoding intermediate filament proteins, including lamins; however, functional lamin analogues are presumed to exist in plants. Plant-specific coiled-coil proteins, that is, nuclear matrix constituent proteins (NMCPs), are the most likely candidates as the structural elements of the nuclear lamina because they exhibit a lamin-like domain arrangement. They are exclusively localized at the nuclear periphery and have functions that are analogous to those of lamins. However, their assembly into filamentous polymers has not yet been confirmed. In this study, we examined the higher-order structure of NMCP1 and NMCP2 in Apium graveolens cells by using stimulated emission depletion microscopy combined with immunofluorescence cell labelling. Our analyses revealed that NMCP1 and NMCP2 form intricate filamentous networks, which include thick segments consisting of filament bundles, forming a dense filamentous layer extending across the nuclear periphery. Furthermore, the outermost chromatin distribution was found to be in the nucleoplasm-facing region of the nuclear lamina. Recombinant Daucus carota NMCP1 with a His-tag produced in Escherichia coli refolded into dimers and self-assembled into filaments and filament bundles. These results suggest that NMCP1 and NMCP2 organize into the nuclear lamina by forming a filamentous network with filament bundles that localize at the nuclear periphery.

Multi-label invivo STED microscopy by parallelized switching of reversibly switchable fluorescent proteins.

Despite the tremendous success of super-resolution microscopy, multi-color invivo applications are still rare. Here we present live-cell multi-label STED microscopy invivo and invitro by combining spectrally separated excitation and detection with temporal sequential imaging of reversibly switchable fluorescent proteins (RSFPs). Triple-label STED microscopy resolves pre- and postsynaptic nano-organizations invivo in mouse visual cortex employing EGFP, Citrine, and the RSFP rsEGP2. Combining the positive and negative switching RSFPs Padron and Dronpa-M159T enables dual-label STED microscopy. All labels are recorded quasi-simultaneously by parallelized on- and off-switching of the RSFPs within the fast-scanning axis. Depletion is performed by a single STED beam so that all channels automatically co-align. Such an addition of a second or third marker merely requires a switching laser, minimizing setup complexity. Our technique enhances invivo STED microscopy, making it a powerful tool for studying multiple synaptic nano-organizations or the tripartite synapse invivo.