Stimulated Emission Depletion Microscopy Research Articles

DivIVA controls the dynamics of septum splitting and cell elongation in Streptococcus pneumoniae.

Bacterial shape and division rely on the dynamics of cell wall assembly, which involves regulated synthesis and cleavage of the peptidoglycan. In ovococci, these processes are coordinated within an annular mid-cell region with nanometric dimensions. More precisely, the cross-wall synthesized by the divisome is split to generate a lateral wall, whose expansion is insured by the insertion of the so-called peripheral peptidoglycan by the elongasome. Septum cleavage and peripheral peptidoglycan synthesis are, thus, crucial remodeling events for ovococcal cell division and elongation. The structural DivIVA protein has long been known as a major regulator of these processes, but its mode of action remains unknown. Here, we integrate click chemistry-based peptidoglycan labeling, direct stochastic optical reconstruction microscopy, and in silico modeling, as well as epifluorescence and stimulated emission depletion microscopy to investigate the role of DivIVA in Streptococcus pneumoniae cell morphogenesis. Our work reveals two distinct phases of peptidoglycan remodeling during the cell cycle that are differentially controlled by DivIVA. In particular, we show that DivIVA ensures homogeneous septum cleavage and peripheral peptidoglycan synthesis around the division site and their maintenance throughout the cell cycle. Our data additionally suggest that DivIVA impacts the contribution of the elongasome and class A penicillin-binding proteins to cell elongation. We also report the position of DivIVA on either side of the septum, consistent with its known affinity for negatively curved membranes. Finally, we take the opportunity provided by these new observations to propose hypotheses for the mechanism of action of this key morphogenetic protein.IMPORTANCEThis study sheds light on fundamental processes governing bacterial growth and division, using integrated click chemistry, advanced microscopy, and computational modeling approaches. It addresses cell wall synthesis mechanisms in the opportunistic human pathogen Streptococcus pneumoniae, responsible for a range of illnesses (otitis, pneumonia, meningitis, septicemia) and for one million deaths every year worldwide. This bacterium belongs to the morphological group of ovococci, which includes many streptococcal and enterococcal pathogens. In this study, we have dissected the function of DivIVA, which is a structural protein involved in cell division, morphogenesis, and chromosome partitioning in Gram-positive bacteria. This work unveils the role of DivIVA in the orchestration of cell division and elongation along the pneumococcal cell cycle. It not only enhances our understanding of how ovoid bacteria proliferate but also offers the opportunity to consider how DivIVA might serve as a scaffold and sensor for particular membrane regions, thereby participating in various cell cycle processes.

The ATTO 565 Dye and Its Applications in Microscopy.

ATTO 565, a Rhodamine-type dye, has garnered significant attention due to its remarkable optical properties, such as a high fluorescence quantum yield, and the fact that it is a relatively stable structure and has low biotoxicity. ATTO 565 has found extensive applications in combination with microscopy technology. In this review, the chemical and optical properties of ATTO 565 are introduced, along with the principles behind them. The functionality of ATTO 565 in confocal microscopy, stimulated emission depletion (STED) microscopy, single-molecule tracking (SMT) techniques, two-photon excitation-stimulated emission depletion microscopy (TPE-STED) and fluorescence correlation spectroscopy (FCS) is discussed. These studies demonstrate that ATTO 565 plays a crucial role in areas such as biological imaging and single-molecule localization, thus warranting further in-depth investigations. Finally, we present some prospects and concepts for the future applications of ATTO 565 in the fields of biocompatibility and metal ion detection. This review does not include theoretical calculations for the ATTO 565 molecule.

Advanced multiparametric image spectroscopy and super-resolution microscopy reveal a minimal model of CD95 signal initiation.

Unraveling the concentration-dependent spatiotemporal organization of receptors in the plasma membrane is crucial to understand cell signal initiation. A paradigm of this process is the oligomerization of CD95 during apoptosis signaling, with different oligomerization models being discussed. Here, we establish the molecular-sensitive approach cell lifetime Förster resonance energy transfer image spectroscopy to determine CD95 configurations in live cells. These data are corroborated by stimulated emission depletion microscopy, confocal photobleaching step analysis, and fluorescence correlation spectroscopy. We probed CD95 interactions for concentrations of ~10 to 1000 molecules per square micrometer, over nanoseconds to hours, and molecular to cellular scales. Quantitative benchmarking was achieved establishing high-fidelity monomer and dimer controls. While CD95 alone is primarily monomeric (~96%) and dimeric (4%), the addition of ligand induces oligomerization to dimers/trimers (~15%) leading to cell death. This study highlights molecular concentration effects and oligomerization dynamics. It reveals a minimal model, where small CD95 oligomers suffice to efficiently initiate signaling.

Bleaching protection and axial sectioning in fluorescence nanoscopy through two-photon activation at 515 nm

Activation of caged fluorophores in microscopy has mostly relied on the absorption of a single ultraviolet (UV) photon of ≲400 nm wavelength or on the simultaneous absorption of two near-infrared (NIR) photons >700 nm. Here, we show that two green photons (515 nm) can substitute for a single photon (~260 nm) to activate popular silicon-rhodamine (Si-R) dyes. Activation in the green range eliminates the chromatic aberrations that plague activation by UV or NIR light. Thus, in confocal fluorescence microscopy, the activation focal volume can be matched with that of confocal detection. Besides, detrimental losses of UV and NIR light in the optical system are avoided. We apply two-photon activation (2PA) of three Si-R dyes in different superresolution approaches. STED microscopy of thick samples is improved through optical sectioning and photobleaching reduced by confining active fluorophores to a thin layer. 2PA of individualized fluorophores enables MINSTED nanoscopy with nanometer-resolution.

Fundamental precision limits of fluorescence microscopy: a perspective on MINFLUX.

Over the past years, fluorescence microscopy (FM) has steadily progressed in increasing the localization precision of fluorescent emitters in biological samples and led to new claims, whose rigorous validation remains an outstanding problem. We present a novel, to the best of our knowledge, multi-parameter estimation framework that captures the full complexity of a single-emitter FM localization experiment. We showcase our method with Minimum Flux (MINFLUX) microscopy, among the highest-resolution approaches, demonstrating that (i) the localization precision can be increased only by turning the illumination intensity up, thus increasing the risk of photo-bleaching, and it is independent from the beams' separation, and (ii) in presence of background noise, the localization precision decreases with the beams' separation. Finally, we apply our method to Minimum Flux Stimulated Emission Depletion (MINSTED) microscopy, showing that a reduction of the beam width can provide similar performance to MINFLUX.

A comparison of super-resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium.

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyze their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

Fast finite difference solver for optical microscopy in deep biological tissue

Optical scattering poses a significant challenge to high-resolution microscopy within deep tissue. To accurately predict the performance of various microscopy techniques in thick samples, we present a computational model that efficiently solves Maxwell’s equation in highly scattering media. This toolkit simulates the deterioration of the laser beam point spread function (PSF) without making a paraxial approximation, enabling accurate modeling of high-numerical-aperture (NA) objective lenses commonly employed in experiments. Moreover, this framework is applicable to a broad range of scanning microscopy techniques including confocal microscopy, stimulated emission depletion (STED) microscopy, and ground-state depletion microscopy. Notably, the proposed method requires only readily obtainable macroscopic tissue parameters. As a practical demonstration, we investigate the performance of Laguerre–Gaussian (LG) versus Hermite–Gaussian (HG) depletion beams in STED microscopy.

β-Galactosidase- and Photo-Activatable Fluorescent Probes for Protein Labeling and Super-Resolution STED Microscopy in Living Cells.

We report on the synthesis of two fluorescent probes which can be activated by β-Galactosidase (β-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with β-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin-Halo fusion protein in live cells with overexpressed β-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science.

The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part I: Super-Resolution’s STED, SIM, SMI, and SMLM

Abstract As technology advances, the field of microscopy offers unique opportunities for scientific exploration. However, the complex terminology used to describe these cutting-edge techniques often appears as a jumble of letters, akin to alphabet soup. Among the plethora of acronyms, some of the most prevalent are associated with super-resolution microscopy, including Stimulated Emission Depletion Microscopy (STED), Structured Illumination Microscopy (SIM), Spatially Modulated Illumination (SMI), and Single-Molecule Localization Microscopy (SMLM). These techniques push past the limitations of conventional light microscopy, achieving resolutions down to the tens of nanometers. This article aims to decode these common super-resolution techniques, fostering a deeper understanding of microscopy and igniting interest in their application.

Investigation of the effects of polarisation and alignment method of retarders in isoSTED microscopy

Stimulated emission depletion (STED) microscopy is a powerful tool for observing subcellular structures beyond the diffraction-limited resolution. To achieve improved isotropic three-dimensional (3D) resolution in STED microscopy, 4Pi-geometry, which is equipped with two opposing objectives, is adopted; this approach is known as isoSTED microscopy. In isoSTED microscopy, constructive and destructive interference occur at the shared focal plane for lateral and axial depletion, respectively. The performance of isoSTED microscopy depends critically on the polarisation status and optical power differences of the two beams of the 4Pi cavity, which are adjusted using retarders. Misalignment of the retarders creates a nonzero core of the depletion focus owing to partial destructive interference. We analytically investigated the effects of retarder misalignments and proposed a simple and accurate alignment method for retarders. We also demonstrate that the proposed method can generate a completely destructive interference pattern and consequently achieve a sub-40-nm 3D resolution.

Self-Quenched Fluorophore-DNA Labels for Super-Resolution Fluorescence Microscopy.

Protein labeling through transient and repetitive hybridization of short, fluorophore-labeled DNA oligonucleotides has become widely applied in various optical super-resolution microscopy methods. The main advantages are multitarget imaging and molecular quantification. A challenge is the high background signal originating from the presence of unbound fluorophore-DNA labels in solution. Here, we report the self-quenching of fluorophore dimers conjugated to DNA oligonucleotides as a general concept to reduce the fluorescence background. Upon hybridization, the fluorescence signals of both fluorophores are restored. We expand the toolbox of fluorophores suitable for self-quenching and report their spectra and hybridization equilibria. We apply self-quenched fluorophore-DNA labels to stimulated emission depletion microscopy and single-molecule localization microscopy and report improved imaging performances.

Development of Two-Photon Super-Resolution Microscopy

Two-photon excitation microscopy enables in vivo deep-tissue imaging within organisms. This technique is based on two-photon excitation, a nonlinear optical process that uses near-infrared light for excitation, resulting in high tissue permeability. Notably, two-photon excitation occurs only near the focal plane; therefore, minimally invasive tomographic images can be obtained. Owing to these features, two-photon excitation microscopy is currently widely used in medical and life-science research, particularly in the domain of neuroscience for in vivo visualization of deep tissues. However, the use of long-wavelength excitation light in two-photon excitation microscopy has resulted in a larger focused spot size and relatively low spatial resolution, which is a limitation of this technique for further applications. Recent studies have described super-resolution microscopy techniques applied to two-photon excitation microscopy in an attempt to observe living organisms "as they are in their natural state" with high spatial resolution. We have also addressed this topic using an optical approach (two-photon stimulated emission depletion microscopy) and an image analysis approach (two-photon super-resolution radial fluctuation). Here, we describe these approaches together with a discussion of our recent accomplishments.

Recent Advances in Fluorescent Nanoparticles for Stimulated Emission Depletion Imaging.

Stimulated emission depletion (STED) microscopy, as a popular super-resolution imaging technique, has been widely used in bio-structure analysis and resolving the dynamics of biological processes beyond the diffraction limit. The performance of STED critically depends on the optical properties of the fluorescent probes. Ideally, the probe should process high brightness and good photostability, and exhibit a sensitive response to the depletion beam. Organic dyes and fluorescent proteins, as the most widely used STED probes, suffer from low brightness and exhibit rapid photobleaching under a high excitation power. Recently, luminescent nanoparticles (NPs) have emerged as promising fluorescent probes in biological imaging due to their high brightness and good photostability. STED imaging using various kinds of NPs, including quantum dots, polymer dots, carbon dots, aggregation-induced emission dots, etc., has been demonstrated. This review will comprehensively review recent advances in fluorescent NP-based STED probes, discuss their advantages and pitfalls, and outline the directions for future development.

Theoretical design of a single-mode fiber-based bi-order Bessel beam for a STED system

Stimulated emission depletion (STED) microscopy has attracted great research attention due to its applications to breaking diffraction limits for imaging and lithography. However, its implementation based on single-mode fibers often encounters challenges such as complex structural integration, costly fabrication processes, and the need for specific fiber designs. Herein, a low-cost bi-order Bessel beam based on one single-mode fiber integrated with a structurally simple wavelength-scale microstructure (WSM) on fiber end was proposed for STED system. Through simulation study for full-scale WSM optimization, we have successfully developed a bicolor laser beam (BLB) consisting of a zero-order Bessel beam at a wavelength of 405 nm and a donuts high-order Bessel beam at a wavelength of 532 nm. This fiber-based configuration allows us to achieve a diffraction-limited spot size with a working distance of 0.67λpump and a minimum FWHM of 0.395λpump. By combining wavelength division multiplexing technology with power modulation of the donuts beam, this work provides a promising way for achieving super-resolution imaging or lithography with only one single-mode fiber.

OpenSTED: open-source dynamic intensity minimum system for stimulated emission depletion microscopy.

Stimulated emission depletion (STED) is a powerful super-resolution microscopy technique that can be used for imaging live cells. However, the high STED laser powers can cause significant photobleaching and sample damage in sensitive biological samples. The dynamic intensity minimum (DyMIN) technique turns on the STED laser only in regions of the sample where there is fluorescence signal, thus saving significant sample photobleaching. The reduction in photobleaching allows higher resolution images to be obtained and longer time-lapse imaging of live samples. A stand-alone module to perform DyMIN is not available commercially. In this work, we developed an open-source design to implement three-step DyMIN on a STED microscope and demonstrated reduced photobleaching for timelapse imaging of beads, cells, and tissue. The DyMIN system uses a fast multiplexer circuit and inexpensive field-programmable gate array controlled by Labview software that operates as a stand-alone module for a STED microscope. All software and circuit diagrams are freely available. We compared time-lapse images of bead samples using our custom DyMIN system to conventional STED and recorded a higher signal when using DyMIN after a 50-image sequence. We further demonstrated the DyMIN system for time-lapse STED imaging of live cells and brain tissue slices. Our open-source DyMIN system is an inexpensive add-on to a conventional STED microscope that can reduce photobleaching. The system can significantly improve signal to noise for dynamic time-lapse STED imaging of live samples.

The Expression of Cell Cycle Cyclins in a Human Megakaryoblast Cell Line Exposed to Simulated Microgravity.

The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space.

Amphiphilic coumarin-based probes for live-cell STED nanoscopy of plasma membrane

Plasma membranes are vital biological structures, serving as protective barriers and participating in various cellular processes. In the field of super-resolution optical microscopy, stimulated emission depletion (STED) nanoscopy has emerged as a powerful method for investigating plasma membrane-related phenomena. However, many applications of STED microscopy are critically restricted by the limited availability of suitable fluorescent probes. This paper reports on the development of two amphiphilic membrane probes, SHE-2H and SHE-2N, specially designed for STED nanoscopy. SHE-2N, in particular, demonstrates quick and stable plasma membrane labelling with negligible intracellular redistribution. Both probes exhibit outstanding photostability and resolution improvement in STED nanoscopy, and are also suited for two-photon excitation microscopy. Furthermore, microscopy experiments and cytotoxicity tests revealed no noticeable cytotoxicity of probe SHE-2N at concentration used for fluorescence imaging. Spectral analysis and fluorescence lifetime measurements conducted on probe SHE-2N using giant unilamellar vesicles, revealed that emission spectra and fluorescence lifetimes exhibited minimal sensitivity to lipid composition variations. These novel probes significantly augment the arsenal of tools available for high-resolution plasma membrane research, enabling a more profound exploration of cellular processes and dynamics.

Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy

Fluorescence microscopy is limited by photoconversion due to continuous illumination, which results in not only photobleaching but also conversion of fluorescent molecules into species of different spectral properties through photoblueing. Here, we determined different fluorescence parameters of photoconverted products for various fluorophores under standard confocal and stimulated emission depletion (STED) microscopy conditions. We observed changes in both fluorescence spectra and lifetimes that can cause artifacts in quantitative measurements, which can be avoided by using exchangeable dyes.

In Situ Structural Characterization of Cardiomyocyte Microenvironment by Multimodal STED Microscopy

Within the myocardium, cardiomyocytes reside in a complex and dynamic extracellular matrix (ECM) consisting of a basement membrane (BM) and interstitial matrix. The interactions between cardiomyocytes and the myocardial ECM play a critical role in maintaining cardiac geometry and function throughout cardiac development and in adult hearts. Understanding how the structural changes of the myocardial ECM affect cardiomyocyte function requires knowledge of pericellular structures. These structures are of a size beyond the resolution of conventional optical microscopy. Here, we demonstrated multi-scale and multi-aspect characterization of the cardiomyocyte microenvironment in myocardial tissue sections using multimodal stimulated emission depletion (STED) microscopy. Second harmonic generation and autofluorescence facilitated multiplexed imaging, enabling the interpretation of protein distribution in 3D. STED imaging modality revealed BM structures of cardiomyocytes and myocardial capillaries at the subdiffractional level. Moreover, meaningful measurements retrieved from acquired images, such as sarcomere length and capillary density, enabled quantitative assessment of myocardial structures.

Single image Fourier ring correlation

We address resolution assessment for (light super-resolution) microscopy imaging. In modalities where imaging is not diffraction limited, correlation between two noise independent images is the standard way to infer the resolution. Here we take away the need for two noise independent images by computationally splitting one image acquisition into two noise independent realizations. This procedure generates two Poisson noise distributed images if the input is Poissonian distributed. As most modern cameras are shot-noise limited this procedure is directly applicable. However, also in the presence of readout noise we can compute the resolution faithfully via a correction factor. We evaluate our method on simulations and experimental data of widefield microscopy, STED microscopy, rescan confocal microscopy, image scanning microscopy, conventional confocal microscopy, and transmission electron microscopy. In all situations we find that using one image instead of two results in the same computed image resolution.