Stimulated Emission Depletion Research Articles

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.

Getting sharper: the brain under the spotlight of super-resolution microscopy.

Brain cells such as neurons and astrocytes exhibit an extremely elaborate morphology, and their functional specializations like synapses and glial processes often fall below the resolution limit of conventional light microscopy. This is a huge obstacle for neurobiologists because the nanoarchitecture critically shapes fundamental functions like synaptic transmission and Ca2+ signaling. Super-resolution microscopy can overcome this problem, offering the chance to visualize the structural and molecular organization of brain cells in a living and dynamic tissue context, unlike traditional methods like electron microscopy or atomic force microscopy. This review covers the basic principles of the main super-resolution microscopy techniques in use today and explains how their specific strengths can illuminate the nanoscale mechanisms that govern brain physiology.

A systematic review and comparison of automated tools for quantification of fibrous networks.

Fibrous networks are essential structural components of biological and engineered materials. Accordingly, many approaches have been developed to quantify their structural properties, which define their material properties. However, a comprehensive overview and comparison of methods is lacking. Therefore, we systematically searched for automated tools quantifying network characteristics in confocal, stimulated emission depletion (STED) or scanning electron microscopy (SEM) images and compared these tools by applying them to fibrin, a prototypical fibrous network in thrombi. Structural properties of fibrin such as fiber diameter and alignment are clinically relevant, since they influence the risk of thrombosis. Based on a systematic comparison of the automated tools with each other, manual measurements, and simulated networks, we provide guidance to choose appropriate tools for fibrous network quantification depending on imaging modality and structural parameter. These tools are often able to reliably measure relative changes in network characteristics, but absolute numbers should be interpreted with care. STATEMENT OF SIGNIFICANCE: Structural properties of fibrous networks define material properties of many biological and engineered materials. Many methods exist to automatically quantify structural properties, but an overview and comparison is lacking. In this work, we systematically searched for all publicly available automated analysis tools that can quantify structural properties of fibrous networks. Next, we compared them by applying them to microscopy images of fibrin networks. We also benchmarked the automated tools against manual measurements or synthetic images. As a result, we give advice on which automated analysis tools to use for specific structural properties. We anticipate that researchers from a large variety of fields, ranging from thrombosis and hemostasis to cancer research, and materials science, can benefit from our work.

Optical nanoscopy approach to unveil chromatin alterations implicated in neuroblastoma transformation.

Chromatin organization is a well-orchestrated mechanism regulating cellular physiology and its changes are associated with the onset of many diseases, including cancer. Neuroblastoma (NB) is the most common extracranial tumor in childhood, and it shows remarkable heterogeneity in the phenotypes and evolutionary patterns. Although the molecular mechanisms of this heterogeneity are still unclear, the impairment of the “epigenetic machinery” and the consequent chromatin reorganization might play an important role. We aim to assess the possible alterations in chromatin nanoscale architecture correlated with NB transformation using NB cells genetically engineered to overexpress the non-coding RNA NDM29, which promotes differentiation of tumor cells into a neuronal lineage. In both malignant NB cells (Mock) and neuron-like cells (S1.1), we explored chromatin organization and nuclear architecture by optical nanoscale microscopy using both confocal and stimulation emission depletion (STED) imaging methods, in parallel with molecular biology analyses. We characterized the nuclear 3D morphology of Mock vs. S1.1 cells by measuring volume, shape, and circularity of the nuclei as well as chromatin compaction and distribution. We imaged the immunolabeled histone H3 modifications (H3K9 trimethylation and H3K9 acetylation) to characterize chromatin regions typically associated with highly and poorly condensed chromatin, respectively. In parallel, we quantified the global DNA methylation status by measuring the levels of 5-methylcytosine (5-mC) using the enzyme-linked immunosorbent assay (ELISA) MethylFlash Global DNA. Comparing Mock vs. S1.1 cells, we observed (i) significant remodeling of nuclear morphometry; (ii) marked rearrangement of chromatin domains rich in highly-acetylated and -methylated H3K9; (iii) changes in average chromatin compaction and DNA methylation. Altogether, the results point to an involvement of epigenetic mechanisms which may sustain the chromatin remodeling during NB malignant transformation, paving the way towards new prognostic and therapeutic approaches and understanding chromatin remodeling in physiological/pathological processes.

Super-resolution 3D maps to study the antisecretory factor's effects on different GABAa receptor subunits in rat cerebellar granule cells in vitro.

The Antisecretory Factor (AF) is an endogenous protein that inhibits intestinal hypersecretion and various inflammation disorders in vivo. AF has been detected in many mammalian tissues and plasma, but its mechanisms of action are essentially unknown. Previously, we studied the pharmacological action of the AF on GABAA receptors in cerebellar granule cells, comparing the electrophysiological response evoked by two-photon mediated release of caged GABA compounds before and after the administration of AF-16, a 16 amino acids long peptide obtained from the amino-terminal end of the AF protein. After the treatment with AF-16, we observed an increase in the GABAA receptor responses, particularly in those containing the α6 subunit. To figure out the interactions of AF with GABAA receptors in the same cellular model (cerebellar granule cells), we combined immunofluorescence subunits’ staining with confocal and super-resolution microscopy. In particular, we took advantage of an innovative imaging technique that combines stimulated emission depletion (STED) with fluorescence lifetime microscopy (FLIM) to collect super-resolution 3D maps of different subunits distributed on the neuron cell membrane. We explored different approaches to analyze super-resolution fluorescence images obtained by labeling α1 and α6 subunits before and after 1-hour incubation with AF-16. Comparing pre- and post-treatment maps, we found differences in how different subunit populations respond to AF treatment. We critically analyzed these new experimental findings with our previous electrophysiological data to widen the knowledge of the mechanisms of interaction between GABAA receptor subunits and AF protein.

Actin rings form plasma membrane diffusion barriers.

In a fluid system like the plasma membrane, molecules will not enrich spontaneously. Since a high local concentration of specific molecules is vital for many membrane protein functions, a domain enriched in these molecules must be formed. This can be achieved by the generation of a physical diffusion barrier to the lateral movement of membrane components. One such barrier exists in the axon initial segment (AIS) of neurons, but the mechanism of the barrier so far remained elusive. Two candidates have emerged to be the cause of the membrane compartmentalization in the AIS: periodic actin rings (fence model) or large immobile transmembrane voltage-gated sodium channels (picket model). Here, we show that the diffusion barrier in the AIS is caused by the actin cytoskeleton. To do so, we combined in silico experiments with high-speed, high-density single-particle tracking and superresolution microscopy. In silico single-particle tracking (SPT) experiments clearly showed that the compartmentalization is compatible with the fence model but incompatible with the picket model. Furthermore, we show via SPT of membrane proteins in progenitor-derived neuronal cells that the diffusion barrier is also present in cells that do not exhibit an ion-channel enriched AIS, but do show actin rings along their processes. Further experiments using dual-colour live-cell stimulated emission depletion microscopy (STED) showed that periodic actin rings in cells are near (20 nm) the plasma membrane. Lastly, we show via correlative SPT/STORM that membrane molecules accumulate between actin rings. Taken together, we present here evidence for the compartmentalization of the plasma membrane by the actin cytoskeleton. Membrane proximal actin structures are a common feature of many cell types. Therefore, we speculate that the observed membrane compartmentalization is not specific to the neuronal cell lineage but rather a general mechanism of large-scale membrane organization.

Deciphering the chromatin organization and epigenomics involved in adipocyte differentiation and hypertrophy by multimodal nanoscopy.

The adipose tissue is the physiological energy reservoir for the whole body, but it also acts as a central metabolic organ in the regulation of body energy homeostasis. In particular, fat is stored in adipocytes inside cytosolic lipid droplets (LDs). Adipocyte hypertrophy occurring in obesity refers to an increase in adipocyte size due to LD enlargement. We aim to investigate the nuclear architecture and epigenetics during adipocyte differentiation and hypertrophy using an in vitro model of 3T3-L1 pre-adipocytes firstly differentiated into mature adipocytes, then cultured with fatty acids to induce hypertrophy. On pre-adipocytes, mature and hypertrophic adipocytes, we assessed lipid accumulation as a hypertrophy marker. Chromatin plays a central role in controlling gene expression under different physiological and pathological mechanisms. We employed optical nanoscale microscopy, such as confocal, super-resolution stimulation emission depletion (STED), and molecular biology analyses (ELISA), to assess the remodeling of nuclear architecture, chromatin organization, and epigenetic modifications associated with adipocyte differentiation and hypertrophy. Confocal and STED analyses showed appreciable differences in nuclear architecture, chromatin condensation, distribution, and chromatin methylation/acetylation among pre-adipocytes, mature and hypertrophic adipocytes. Moreover, we quantified the average changes in the DNA methylation (5-methylcytosine) by ELISA immunoassay. These preliminary results indicate epigenome and nuclear organization remodeling during adipocyte differentiation and hypertrophy. We can conclude that nuclear architecture remodeling is a significant and central factor in the genome function regulation.

Novel insights about ribosomal DNA double strand break repair revealed by super-resolution imaging techniques and very fast CRISPR.

Ribosomal DNA (rDNA) is repetitive gene sequence that produces the RNA components for the translation machinery in eukaryotic cells - ribosomes. The maintenance of rDNA genome stability against double-strand breaks (DSBs) is critical regarding cellular aging related diseases and tumor development due to its repetitive nature and distribution over multiple chromosomes. Previous studies have shown that DSB within rDNA region will induce rDNA transcriptional repression and nucleolar segregation. However, it remains poorly understood regarding the kinetics of associated transcriptional regulation, the architecture of the nucleolar cap, and the link between them, likely due to the resolution limit of conventional imaging tools and the absence of proper system that can rapidly induce DSBs specific to rDNA region with accurately controlled timing. To resolve these limitations and provide deeper insights on how rDNA DSB repair response is regulated, we use self-built super-resolution fluorescence imaging systems, e.g., stimulated emission depletion microscopy, which allow to visualize nucleolar structures beyond the diffraction limit, and lattice light sheet microscopy, which enables to monitor fast kinetics of the repair process with minimal photo-induced disturbance, combined with a recently developed rapidly-inducible photo-caged CRISPR-Cas9 system (very fast CRISPR, vfCRISPR) in our lab, to study the spatiotemporal coordination of the DSB repair factor assembly upon rDNA DSB damage, and dissect its important role in regulating pathway choice and outcome fidelity.

Decrypting the spatial relationship between peripheral chromatin and nuclear lamina in Hutchinson-Gilford Progeria Syndrome using super-resolution microscopy techniques.

The genetic condition known as Hutchinson-Gilford Progeria Syndrome (HGPS) causes the early onset of aging symptoms and premature death. HGPS is caused by a single nucleotide mutation in the LMNA gene, resulting in a truncated form of the protein Lamin A known as ΔLA50 or Progerin. This protein modification causes aberrant lamina network organization inducing nuclear and DNA structure abnormalities, leading to the loss of peripheral heterochromatin. In this study, we examine variations in chromatin compaction in a cellular model of HPGS using Expansion Microscopy (ExM). This super-resolution microscopy technique is based on embedding biological samples into a water-absorbant polymer network that, following mechanical homogenization, enables a four-fold linear expansion in water, allowing nanoscale biophysical studies using conventional microscopes. The image analysis of the HGPS cell model shows a significant variation in chromatin compaction that seems to disrupt its physical association with the nuclear lamina. Furthermore, this loss of compaction leads to a detectable spatial separation between the lamina network, mainly composed of the mutated form Progerin, and the peripheral heterochromatin. Therefore, to quantify this distance variation caused by Progerin-induced chromatin disorganization, we analyzed a batch of control cells expressing wild-typer lamin A and HGPS cells, finding a one-order of magnitude difference between the two sets. In order to validate these results, we perform super-resolution Stimulated Emission Depletion (STED) imaging. The next step will be performing Correlative Light Electron Microscopy (CLEM) on non-expanded cells to push the resolution of our analysis even forward and to investigate chromatin compaction on a nanoscale level.

Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy.

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.

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.

A combination of surface-initiated controlled radical polymerization (SET-LRP) and click-chemistry for the chemical modification and fluorescent labeling of cellulose nanofibrils: STED super-resolution imaging of a single fibril and a single fibril embedded in a composite

A strategy is developed to modify cellulose nanofibril (CNF) surfaces with a combination of Cu0-mediated radical polymerization (SET-LRP) and CuI-catalyzed azide-alkyne click-chemistry (CuAAC). CNFs were grafted with statistical copolymers of di(ethylene glycol) ethyl ether acrylate (DEGEEA) and acrylic acid 3-trimethylsilyl-prop-2-ynyl ester (TMSPgA) that allows labeling of multiple fluorescent dyes, e.g. AF488 and ATTO633, special dyes for confocal laser scanning microscopy and stimulated emission depletion (STED) microscopy. Through our strategy and these microscopic techniques, we visualized isolated fibrils and fibrils embedded in a PVA composite in a high resolution. This work also provides new insight into the effect of the clickable entity/precursor on the compatibility of modified fibrils with the composite matrix.Graphical abstract

Sub-100 nm pixel pitch via STED photolithography with a nanoprinting-at-expansion/employments-at-recovery strategy.

Featured with its extraordinary super-resolution capability, the advent of stimulated emission depletion (STED) lithography has allowed for vastly reduced minimum feature size of a single pixel down to the deep sub-diffraction scale so as to produce unprecedented nanofeatures. However, the anticipated sub-diffraction pixel pitch down below 100 nm remains out of reach due to redundant polymerization of adjacent exposures at a short distance, so called memory effect. In this work, a nanoprinting-at-expansion/employments-at-recovery strategy is applied in the dual-beam STED lithography technique to surmount the memory effect and break adjacent-exposure limit imposed on minimizing the pixel pitch. The implementation of a femtosecond laser at a wavelength of 532 nm, the same as the inhibition laser beam, working as the initiation laser beam, can drastically reduce the saturated inhibition laser intensity by 74% for abating redundant polymerization subjected to multiple exposures in realizing nanoscale pixel pitch. The adjacent-exposure zone can be separated by isotropically expanding an elastic PDMS substrate for further diminishing redundant polymerization. Applying stretching ratio of 30%, a minimum super-resolved nanodots pixel pitch of 96 nm was achieved with single-dot size of 34 nm on both planar and hierarchical substrate, which offers a record-close distance for printing adjacent pixels. With its nanometer discernibility, this method holds great promise for future versatile utilization in advanced nanoimprinting, high density data storage, etc.

Photoactivatable Large Stokes Shift Fluorophores for Multicolor Nanoscopy.

We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and ∼100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells, and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted tetrazine ligation and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of an imaging channel with another large Stokes shift dye have been demonstrated.

Discriminating between different proteins in the microstructure of acidified milk gels by super-resolution microscopy

Super-resolution microscopy (Stimulated Emission Depletion, STED) combined with quantitative image analysis and rheology was used to study acidified milk gels with the addition of both endogenous milk proteins (i.e., liquid casein and whey protein concentrate, LCC and LWPC) and different types of whey protein ingredients. Casein and whey protein were stained separately using two different dyes prior to mixing. STED micrographs showed that added whey protein concentrate (WPC) and nano-particulated whey protein (NWP) were able to self-aggregate and attach to casein assemblies by inter-chain crosslinking. The behavior of NWP was shown to be similar to the behavior of LWPC, except that NWP formed larger aggregates with increased connectivity to the gel network. Micro-particulated whey protein (MWP) did not appear to interact with any other proteins and scattered MWP particles were visible in the mixed gels. The spatial colocalization of the fluorescence emission stemming from casein and whey protein, respectively, was highest for the system that contained only endogenous proteins. The lowest colocalization level was found for the systems with added MWP which also showed the highest image coarseness in accordance with the lowest observed G’. The systems with added LWPC or NWP exhibited thicker aggregate strands, which correlated to higher G’ compared to the other systems with WPC.

The murine ortholog of Kaufman oculocerebrofacial syndrome gene Ube3b is crucial for the maintenance of the excitatory synapses in the young adult stage

Kaufman oculocerebrofacial syndrome (KOS) is an autosomal recessive developmental disorder. Inactivating mutations in UBE3B, an E3 ubiquitin ligase gene are causative for KOS. We have reported that towards postnatal week three, its murine ortholog, Ube3b, acts as a negative regulator of the number of dendritic spines. In this study, we investigated the role of Ube3b at the synapse in the young adult mice. With an improved estimation method, images from the hippocampal CA1 and CA2 regions acquired with 3D Stimulated Emission Depletion (3D-STED) microscopy were used to quantify the excitatory synapse numbers. In the young adult mice, the excitatory synapse density was decreased in brain-specific Ube3b conditional knockout mice as compared to the control. Our results indicate the novel role of Ube3b in the maintenance of synapse numbers in the young adult period.

A new organic molecular probe as a powerful tool for fluorescence imaging and biological study of lipid droplets.

Background: Lipid droplets (LDs) are critical organelles associated with many physiological processes in eukaryotic cells. To visualize and study LDs, fluorescence imaging techniques including the confocal imaging as well as the emerging super-resolution imaging of stimulated emission depletion (STED), have been regarded as the most useful methods. However, directly limited by the availability of advanced LDs fluorescent probes, the performances of LDs fluorescence imaging are increasingly unsatisfied with respect to the fast research progress of LDs. Methods: We herein newly developed a superior LDs fluorescent probe named Lipi-QA as a powerful tool for LDs fluorescence imaging and biological study. Colocalization imaging of Lipi-QA and LDs fluorescent probe Ph-Red was conducted in four cell lines. The LDs staining selectivity and the photostability of Lipi-QA were also evaluated by comparing with the commercial LDs probe Nile Red. The in-situ fluorescence lifetime of Lipi-QA in LDs was determined by time-gated detection. The cytotoxicity of Lipi-QA was assessed by MTT assay. The STED saturation intensity as well as the power- and gate time-dependent resolution were tested by Leica SP8 STED super-resolution nanoscopy. The time-lapse 3D confocal imaging and time-lapse STED super-resolution imaging were then designed to study the complex physiological functions of LDs. Results: Featuring with the advantages of the super-photostability, high LDs selectivity, long fluorescence lifetime and low STED saturation intensity, the fluorescent probe Lipi-QA was capable of the long-term time-lapse three-dimensional (3D) confocal imaging to in-situ monitor LDs in 3D space and the time-lapse STED super-resolution imaging (up to 500 STED frames) to track the dynamics of LDs with nanoscale resolution (37 nm). Conclusions: Based on the state-of-the-art fluorescence imaging results, some new biological insights into LDs have been successfully provided. For instance, the long-term time-lapse 3D confocal imaging has surely answered an important and controversial question that the number of LDs would significantly decrease rather than increase upon starvation stimulation; the time-lapse STED super-resolution imaging with the highest resolution has impressively uncovered the fission process of nanoscale LDs for the first time; the starvation-induced change of LDs in size and in speed has been further revealed at nanoscale by the STED super-resolution imaging. All of these results not only highlight the utility of the newly developed fluorescent probe but also significantly promote the biological study of LDs.

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.

Parallel illumination for depletion microscopy through acousto-optic spatial light modulation

Several types of super-resolution microscopy, such as Stimulated Emission Depletion (STED), Reversible Saturable Optical Fluorescence Transitions (RESOLFT) or Switching Laser Mode (SLAM) microscopies, employ Laguerre-Gaussian beams (also called vortex or doughnut beams) to obtain fluorescence information within a sub-wavelength region of the specimen under observation, thus breaking the diffraction limit and producing images of greatly improved quality. However, in general, these techniques operate on a point-by-point basis, so we need to raster scan the sample in order to build a full, meaningful image, which takes time. Parallelization of the illumination is the only way to make these microscopies more suitable for live cell imaging applications. Here, we demonstrate the parallel production of arbitrary arrays of Gaussian and Laguerre-Gaussian lasers foci suitable for super-resolution microscopy, together with the possibility to fast scan through the sample, by means of acousto-optic spatial light modulation, a technique that we have pioneered in the past in several other fields.