Stimulated Emission Depletion Imaging Research Articles

Improving STED microscopy with SUPPOSe: enhancing resolution from a single-image

Here we apply the SUPPOSe algorithm on images acquired using Stimulated Emission Depletion (STED) microscopy with the aim of improving the resolution limit achieved. We processed images of the nuclear pore complex (NPC) from cell lines in which the Nup96 nucleoporin was endogenously labeled. This reference protein forms a ring whose diameter is ∼107 nm with 8 corners ∼42 nm apart from each other. The stereotypic arrangement of proteins in the NPC has been used as reference structures to characterize the performance of a variety of microscopy techniques. STED microscopy images resolve the ring arrangement but not the eightfold symmetry of the NPC. After applying the SUPPOSe algorithm to the STED images, we were able to solve the octagonal structure of the NPC. After processing 562 single NPC, the average radius of the NPC was found to be R = 54.2 ± 2.9 nm, being consistent with the theoretical distances of this structure. To verify that the solutions obtained are compatible with a NPC-type geometry, we rotate the solutions to optimally fit an eightfold-symmetric pattern and we count the number of corners that contain at least one localization. Fitting a probabilistic model to the histogram of the number of bright corners gives an effective labeling efficiency of 31%, which is in agreement with the values reported in for other cell lines and ligands used in Single Molecule Localization microscopy, showing that SUPPOSe can reliably retrieve sub-resolution, nanoscale objects from single acquisitions even in noisy conditions.

Auxochrome Dimethyl-Dihydroacridine Improves Fluorophores for Prolonged Live-Cell Super-Resolution Imaging.

Superior photostability, minimal phototoxicity, red-shifted absorption/emission wavelengths, high brightness, and an enlarged Stokes shift are essential characteristics of top-tier organic fluorophores, particularly for long-lasting super-resolution imaging in live cells (e.g., via stimulated emission depletion (STED) nanoscopy). However, few existing fluorophores possess all of these properties. In this study, we demonstrate a general approach for simultaneously enhancing these parameters through the introduction of 9,9-dimethyl-9,10-dihydroacridine (DMA) as an electron-donating auxochrome. DMA not only induces red shifts in emission wavelengths but also suppresses photooxidative reactions and prevents the formation of triplet states in DMA-based fluorophores, greatly improving photostability and remarkably minimizing phototoxicity. Moreover, the DMA group enhances the fluorophores' brightness and enlarges the Stokes shift. Importantly, the "universal" benefits of attaching the DMA auxochrome have been exemplified in various fluorophores including rhodamines, difluoride-boron complexes, and coumarin derivatives. The resulting fluorophores successfully enabled the STED imaging of organelles and HaloTag-labeled membrane proteins.

Monodispersed and Monofunctionalized DNA-Caged Au Nano-Clusters with Enhanced Optical Properties for STED Imaging.

The performance of Stimulated Emission Depletion (STED) microscopy depends critically on the fluorescent probe. Ultrasmall Au nanoclusters (Au NCs) exhibit large Stokes shift, and good stimulated emission response, which are potentially useful for STED imaging. However, Au NCs are polydispersed in size, sensitive to the surrounding environment, and difficult to control surface functional group stoichiometry, which results in reduced density and high heterogeneity in the labeling of biological structures. Here, this limitation is overcome by developing a method to encapsulate ultrasmall Au NCs with DNA cages, which yielded monodispersed, and monofunctionalized Au NCs that are long-term stable. Moreover, the DNA-caging also greatly improved the fluorescence quantum yield and photostability of Au NCs. In STED imaging, the DNA-caged Au NCs yielded ≈40nm spatial resolution and are able to resolve microtubule line shapes with good labeling density and homogeneity. In contrast, without caging, the Au NCs-DNA conjugatesonly achieved ≈55nm resolution and yielded spotted,poorly resolved microtubule structures, due to the presence of aggregates. Overall, a method is developed to achieve precise surface functionalization and greatly improve the monodispersity, stability, as well as optical properties of Au NCs, providing a promising class of fluorescent probes for STED imaging.

Rhodamine-based fluorescent probe for dynamic STED imaging of mitochondria.

Stimulated emission depletion (STED) microscopy holds tremendous potential and practical implications in the field of biomedicine. However, the weak anti-bleaching performance remains a major challenge limiting the application of STED fluorescent probes. Meanwhile, the main excitation wavelengths of most reported STED fluorescent probes were below 500 nm or above 600 nm, and few of them were between 500-600 nm. Herein, we developed a new tetraphenyl ethylene-functionalized rhodamine dye (TPERh) for mitochondrial dynamic cristae imaging that was rhodamine-based with an excitation wavelength of 560 nm. The TPERh probe exhibits excellent anti-bleaching properties and low saturating stimulated radiation power in mitochondrial STED super-resolution imaging. Given these outstanding properties, the TPERh probe was used to measure mitochondrial deformation, which has positive implications for the study of mitochondria-related diseases.

Fluorogenic Rhodamine Probes with Pyrrole Substitution Enables STED and Lifetime Imaging of Lysosomes in Live Cells.

Fluorogenic dyes with high brightness, large turn-on ratios, excellent photostability, favorable specificity, low cytotoxicity, and high membrane permeability are essential for high-resolution fluorescence imaging in live cells. In this study, we endowed these desirable properties to a rhodamine derivative by simply replacing the N, N-diethyl group with a pyrrole substituent. The resulting dye, Rh-NH, exhibited doubled Stokes shifts (54 nm) and a red-shift of more than 50 nm in fluorescence spectra compared to Rhodamine B. Rh-NH preferentially exists in a non-emissive but highly permeable spirolactone form. Upon binding to lysosomes, the collective effects of low pH, low polarity, and high viscosity endow Rh-NH with significant fluorescence turn-on, making it a suitable candidate for wash-free, high-contrast lysosome tracking. Consequently, Rh-NH enabled us to successfully explore stimulated emission depletion (STED) super-resolution imaging of lysosome dynamics, as well as fluorescence lifetime imaging of lysosomes in live cells.

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscopefor reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

Imaging dendritic spines in the hippocampus of a living mouse by 3D-stimulated emission depletion microscopy.

Stimulated emission depletion (STED) microscopy has been used to address a wide range of neurobiological questions in optically well-accessible samples, such as cell culture or brain slices. However, the application of STED to deeply embedded structures in the brain of living animals remains technically challenging. In previous work, we established chronic STED imaging in the hippocampus in vivo but the gain in spatial resolution was restricted to the lateral plane. In our study, we report on extending the gain in STED resolution into the optical axis to visualize dendritic spines in the hippocampus in vivo. Our approach is based on a spatial light modulator to shape the focal STED light intensity in all three dimensions and a conically shaped window that is compatible with an objective that has a long working distance and a high numerical aperture. We corrected distortions of the laser wavefront to optimize the shape of the bottle beam of the STED laser. We show how the new window design improves the STED point spread function and the spatial resolution using nanobeads. We then demonstrate the beneficial effects for 3D-STED microscopy of dendritic spines, visualized with an unprecedented level of detail in the hippocampus of a living mouse. We present a methodology to improve the axial resolution for STED microscopy in the deeply embedded hippocampus in vivo, facilitating longitudinal studies of neuroanatomical plasticity at the nanoscale in a wide range of (patho-)physiological contexts.

Long-wavelength excitation of carbon dots with dual-organelle targeting capability for live-cell imaging via STED nanoscopy

Mitochondria and lysosomes play important roles in maintaining normal cellular metabolism; thus, visualizing their morphology, structure, and interactions in living cells at the nanometer scale is essential for further understanding their functions. However, this remains challenging because of the current limitations of fluorescent probes, including poor photobleaching resistance, low biocompatibility, and lack of multi-organelle targeting. Herein, we report the development of biocompatible nitrogen-doped carbon dots (CNDs) with long excitation wavelengths, stable fluorescence, and dual-organelle targeting capabilities. The CNDs enter cells predominantly via energy-dependent transport (including clathrin- and caveolae-mediated endocytosis) and free diffusion and subsequently target the interior mitochondria and lysosomes; furthermore, they enable the stimulated emission depletion imaging of these organelles in living cells with resolutions of up to 134 and 55 nm, respectively. This study provides practical guidance for designing dual-organelle targeting fluorescent probes for the dynamic super-resolution imaging of cellular systems.

68 Characterization of Bovine Extracellular Vesicles Isolated from Colostrum

Abstract Bovine colostrum is composed of a variety of biologically active compounds including microRNAs (miRNA), which are found within extracellular vesicles (EVs). Newborn calves are agammaglobulinemic. Hence, the ability of the bovine neonate to fight off pathogens is therefore almost entirely dependent upon colostrum ingestion. Extracellular vesicle-derived miRNAs may have a crucial role in the regulation of the immune response. Thus, we aimed to characterize the EVs morphology from bovine colostrum using stimulated emission depletion (STED) super resolution microscopy. Bovine colostrum samples (approx. 3-4 h after parturition) were collected from primiparous Holstein dairy cows (n = 10) at a commercial dairy farm. Colostrum samples (15 mL) were subjected to a series of centrifugation steps to achieve defatted supernatant and to label EV pellets with EV specific markers. The EV pellets were resuspended in 100µL of 1 x PBS for EV staining methods or resuspended in 1 mL of trizol reagent for further miRNA isolation. The EVs suspensions were incubated with a primary polyclonal antibody anti-CD63 and with a secondary antibody (Alexa Fluor 594) for approximately 90 minutes each. Labeled EV-antibody complex were analyzed using confocal and STED microscopy. Based on the confocal and super resolution STED imaging capability obtained from the EVs-derived from bovine colostrum (Figure 1), labeled EVs with anti-CD63 and the enhanced resolution of STED over conventional confocal imaging, allowed to image the EVs at their actual size (30-200 nm). Further in-depth view on EV morphology, marker proteins expressed on the membrane, and size will contribute to our understanding of EVs-derived from colostrum and their functions. This will be crucial in studies to evaluate EVs uptake by intestinal cells for the correct identification of compartments involved in subcellular trafficking.

STED imaging of endogenously tagged ARF GTPases reveals their distinct nanoscale localizations.

ADP-ribosylation factor (ARF) GTPases are major regulators of cellular membrane homeostasis. High sequence similarity and multiple, possibly redundant functions of the five human ARFs make investigating their function a challenging task. To shed light on the roles of the different Golgi-localized ARF members in membrane trafficking, we generated CRISPR-Cas9 knockins (KIs) of type I (ARF1 and ARF3) and type II ARFs (ARF4 and ARF5) and mapped their nanoscale localization with stimulated emission depletion (STED) super-resolution microscopy. We find ARF1, ARF4, and ARF5 on segregated nanodomains on the cis-Golgi and ER-Golgi intermediate compartments (ERGIC), revealing distinct roles in COPI recruitment on early secretory membranes. Interestingly, ARF4 and ARF5 define Golgi-tethered ERGIC elements decorated by COPI and devoid of ARF1. Differential localization of ARF1 and ARF4 on peripheral ERGICs suggests the presence of functionally different classes of intermediate compartments that could regulate bi-directional transport between the ER and the Golgi. Furthermore, ARF1 and ARF3 localize to segregated nanodomains on the trans-Golgi network (TGN) and are found on TGN-derived post-Golgi tubules, strengthening the idea of distinct roles in post-Golgi sorting. This work provides the first map of the nanoscale organization of human ARF GTPases on cellular membranes and sets the stage to dissect their numerous cellular roles.

Morphological feature recognition of different differentiation stages of induced ADSCs based on deep learning

In order to accurately identify the morphological features of different differentiation stages of induced Adipose Derived Stem Cells (ADSCs) and judge the differentiation types of induced ADSCs, a morphological feature recognition method of different differentiation stages of induced ADSCs based on deep learning is proposed. Using the super-resolution image acquisition method of ADSCs differentiation based on stimulated emission depletion imaging, after obtaining the super-resolution images at different stages of inducing ADSCs differentiation, the noise of the obtained image is removed and the image quality is optimized through the ADSCs differentiation image denoising model based on low rank nonlocal sparse representation; The denoised image is taken as the recognition target of the morphological feature recognition method for ADSCs differentiation image based on the improved Visual Geometry Group (VGG-19) convolutional neural network. Through the improved VGG-19 convolutional neural network and class activation mapping method, the morphological feature recognition and visual display of the recognition results at different stages of inducing ADSCs differentiation are realized. After testing, this method can accurately identify the morphological features of different differentiation stages of induced ADSCs, and is available.

Super-resolution STED imaging in the inner and outer whole-mount mouse retina

Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.

An advanced organic molecular probe for multimodal fluorescence imaging of cellular lipid droplets

Lipid droplets (LDs) are critical cellular organelles associated with various physiological processes in eukaryotic cells. To visualize LDs and research their complex physiological functions, various fluorescence imaging models including the confocal imaging, the lambda scan imaging and the stimulated emission depletion (STED) super-resolution imaging have been successfully employed in recent years. However, since different fluorescence imaging models have different requirements for fluorescent probes, it is very challenging to employ single fluorescent probe for multimodal fluorescence imaging. Herein, an advanced organic fluorescent probe Lipi-DSBOMe has been sophisticatedly developed for purpose. Featuring with the advantages of high staining selectivity towards LDs, polarity sensitive luminescence, high photostability and low STED saturation intensity, this fluorescent probe is well suited for multimodal fluorescence imaging. For instance, employed with this probe for three-dimensional (3D) confocal imaging to quantitatively determine the change of number of LDs under the oleic acid stimulation, lambda scan imaging to precisely measure the polarity of LDs in different types of cells, and STED imaging to measure the size of LDs with the state-of-the-art resolution (42 nm). In overall, employing this single fluorescent probe for multimodal fluorescence imaging has allowed to provide abundant information about LDs, thus effectively promoting the LDs biology studies.

Increased Mitochondrial Calcium Fluxes in Hypertrophic Right Ventricular Cardiomyocytes from a Rat Model of Pulmonary Artery Hypertension

Pulmonary artery hypertension causes right ventricular hypertrophy which rapidly progresses to heart failure with underlying cardiac mitochondrial dysfunction. Prior to failure, there are alterations in cytosolic Ca2+ handling that might impact mitochondrial function in the compensatory phase of RV hypertrophy. Our aims, therefore, were (i) to measure beat-to-beat mitochondrial Ca2+ fluxes, and (ii) to determine mitochondrial abundance and function in non-failing, hypertrophic cardiomyocytes. Male Wistar rats were injected with either saline (CON) or monocrotaline (MCT) to induce pulmonary artery hypertension and RV hypertrophy after four weeks. Cytosolic Ca2+ ([Ca2+]cyto) transients were obtained in isolated right ventricular (RV) cardiomyocytes, and mitochondrial Ca2+ ([Ca2+]mito) was recorded in separate RV cardiomyocytes. The distribution and abundance of key proteins was determined using confocal and stimulated emission depletion (STED) microscopy. The RV mitochondrial function was also assessed in RV homogenates using oxygraphy. The MCT cardiomyocytes had increased area, larger [Ca2+]cyto transients, increased Ca2+ store content, and faster trans-sarcolemmal Ca2+ extrusion relative to CON. The MCT cardiomyocytes also had larger [Ca2+]mito transients. STED images detected increased mitochondrial protein abundance (TOM20 clusters per μm2) in MCT, yet no difference was found when comparing mitochondrial respiration and membrane potential between the groups. We suggest that the larger [Ca2+]mito transients compensate to match ATP supply to the increased energy demands of hypertrophic cardiomyocytes.

Identifying STEDable BF2-Azadipyrromethene Fluorophores.

BF2-azadipyrromethenes are highly versatile fluorophores used for cellular and in vivo imaging in the near-infrared and far-red regions of the spectrum. As of yet, their use in conjunction with super-resolution imaging methodologies has not been explored. In this report, a series of structurally related BF2-azadipyrromethenes has been examined for their suitability for use with stimulated emission depletion (STED) nanoscopy. The potential for STED imaging was initially evaluated using aqueous solutions of fluorophores as an effective predictor of fluorophore suitability. For live cell STED imaging in both 2D and 3D, several far-red emitting BF2-azadipyrromethenes were successfully employed. Image resolution below the diffraction limit of a confocal microscope was demonstrated through measurement of distinct intracellular features including the nuclear membrane, nuclear lamina invaginations, the endoplasmic reticulum, and vacuoles. As the STED ability of BF2-azadipyrromethene fluorophores has now been established, their use with this super-resolution method may be expected to increase in the future.

Tracking down fluorophores to the angstrom level by MINSTED.

The ability to localize a fluorophore down to its physical size is a unique feature of state of the art nanoscopy methods like MINFLUX and MINSTED. MINSTED achieves this localization precision with minimal number of detected photons by stimulated emission depletion (STED) and detection-driven repositioning of the central minimum of the STED donut towards the fluorophore position. STED, as one of the key principles of MINSTED, makes use of a dedicated light pattern to inhibit the fluorophore's ability to fluoresce. The intensity needed is proportional to the stimulated emission cross-section of the fluorophore at the laser wavelength. This wavelength is a compromise between cross-section and direct excitation of the fluorophore. Conventional STED imaging cannot benefit much from shifting the STED wavelength closer to the emission maximum, as peripheral fluorophores are excited by the STED laser. By contrast, MINSTED, operating on singularized emitters, can make use of STED light at shorter wavelength to localize emitters down to the angstrom level. Using switchable fluorophores, this allows imaging of both reference DNA origami samples and biological specimens, with localization precisions smaller than the physical size of the fluorophore. Such high localization precisions pave the way for new insights into biological systems, as the instrument is not the resolution-limiting factor anymore.

Spatial distribution of αs1-caseins and β-caseins in milk gels acidified with glucono-δ-lactone

Acid-induced destabilization of casein micelles leads to coagulation of milk which plays an important role in the formation of yogurt. The resulting protein network is formed by aggregation and reconfiguration of casein micelles, including the release of a portion of caseins from these micelles. It is so far unknown how individual αs1-and β-caseins are organized within this complex network, and how their distribution depends on yogurt composition and processing. Here, we imaged the spatial distribution of caseins using stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM). We used fluorescently tagged antibodies against αs1-and β-caseins to localize them inside glucono-δ-lactone (GDL)-acidified milk gels. We conducted quantitative skeleton analysis of STED images and showed that αs1-and β-caseins contribute with different levels of connectivity to the acid induced milk network.

Spatial resolution enhancement in photon-starved STED imaging using deep learning-based fluorescence lifetime analysis.

As a super-resolution imaging method, stimulated emission depletion (STED) microscopy has unraveled fine intracellular structures and provided insights into nanoscale organizations in cells. Although image resolution can be further enhanced by continuously increasing the STED-beam power, the resulting photodamage and phototoxicity are major issues for real-world applications of STED microscopy. Here we demonstrate that, with 50% less STED-beam power, the STED image resolution can be improved up to 1.45-fold using the separation of photons by a lifetime tuning (SPLIT) scheme combined with a deep learning-based phasor analysis algorithm termed flimGANE (fluorescence lifetime imaging based on a generative adversarial network). This work offers a new approach for STED imaging in situations where only a limited photon budget is available.

An approach for quantitative mapping of synaptic periactive zone architecture and organization.

Following exocytosis at active zones, synaptic vesicle membranes and membrane-bound proteins must be recycled. The endocytic machinery that drives this recycling accumulates in the periactive zone (PAZ), a region of the synapse adjacent to active zones, but the organization of this machinery within the PAZ, and how PAZ composition relates to active zone release properties, remains unknown. The PAZ is also enriched for cell adhesion proteins, but their function at these sites is poorly understood. Here, using Airyscan and stimulated emission depletion imaging of Drosophila synapses, we develop a quantitative framework describing the organization and ultrastructure of the PAZ. Different endocytic proteins localize to distinct regions of the PAZ, suggesting that subdomains are specialized for distinct biochemical activities, stages of membrane remodeling, or synaptic functions. We find that the accumulation and distribution of endocytic but not adhesion PAZ proteins correlate with the abundance of the scaffolding protein Bruchpilot at active zones-a structural correlate of release probability. These data suggest that endocytic and exocytic activities are spatially correlated. Taken together, our results identify novel relationships between the exocytic and endocytic apparatus at the synapse and provide a new conceptual framework to quantify synaptic architecture.

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

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