Stimulated Emission Depletion Microscopy Research Articles

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.

3D Computational Modeling of Defective Early Endosome Distribution in Human iPSC-Based Cardiomyopathy Models.

Intracellular cargo delivery via distinct transport routes relies on vesicle carriers. A key trafficking route distributes cargo taken up by clathrin-mediated endocytosis (CME) via early endosomes. The highly dynamic nature of the endosome network presents a challenge for its quantitative analysis, and theoretical modelling approaches can assist in elucidating the organization of the endosome trafficking system. Here, we introduce a new computational modelling approach for assessment of endosome distributions. We employed a model of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with inherited mutations causing dilated cardiomyopathy (DCM). In this model, vesicle distribution is defective due to impaired CME-dependent signaling, resulting in plasma membrane-localized early endosomes. We recapitulated this in iPSC-CMs carrying two different mutations, TPM1-L185F and TnT-R141W (MUT), using 3D confocal imaging as well as super-resolution STED microscopy. We computed scaled distance distributions of EEA1-positive vesicles based on a spherical approximation of the cell. Employing this approach, 3D spherical modelling identified a bi-modal segregation of early endosome populations in MUT iPSC-CMs, compared to WT controls. Moreover, spherical modelling confirmed reversion of the bi-modal vesicle localization in RhoA II-treated MUT iPSC-CMs. This reflects restored, homogeneous distribution of early endosomes within MUT iPSC-CMs following rescue of CME-dependent signaling via RhoA II-dependent RhoA activation. Overall, our approach enables assessment of early endosome distribution in cell-based disease models. This new method may provide further insight into the dynamics of endosome networks in different physiological scenarios.

Visualization of cristae and mtDNA interactions via STED nanoscopy using a low saturation power probe

Mitochondria are crucial organelles closely associated with cellular metabolism and function. Mitochondrial DNA (mtDNA) encodes a variety of transcripts and proteins essential for cellular function. However, the interaction between the inner membrane (IM) and mtDNA remains elusive due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vivo probes specifically targeting the IM. Here, we have developed a novel fluorescence probe called HBmito Crimson, characterized by exceptional photostability, fluorogenicity within lipid membranes, and low saturation power. We successfully achieved over 500 frames of low-power stimulated emission depletion microscopy (STED) imaging to visualize the IM dynamics, with a spatial resolution of 40 nm. By utilizing dual-color imaging of the IM and mtDNA, it has been uncovered that mtDNA tends to habitat at mitochondrial tips or branch points, exhibiting an overall spatially uniform distribution. Notably, the dynamics of mitochondria are intricately associated with the positioning of mtDNA, and fusion consistently occurs in close proximity to mtDNA to minimize pressure during cristae remodeling. In healthy cells, >66% of the mitochondria are Class III (i.e., mitochondria >5 μm or with >12 cristae), while it dropped to <18% in ferroptosis. Mitochondrial dynamics, orchestrated by cristae remodeling, foster the even distribution of mtDNA. Conversely, in conditions of apoptosis and ferroptosis where the cristae structure is compromised, mtDNA distribution becomes irregular. These findings, achieved with unprecedented spatiotemporal resolution, reveal the intricate interplay between cristae and mtDNA and provide insights into the driving forces behind mtDNA distribution.

#369 Anti-nephrin antibodies identify patients with steroid-resistant nephrotic syndrome responsive to immunosuppressants

Abstract Background and Aims The presence of circulating anti-nephrin antibodies has been recently reported in some patients with nephrotic syndrome (NS) and minimal change lesions, implying an autoimmune podocytopathy subset. We hypothesized that the presence of anti-nephrin antibodies would identify a subset of patients with steroid resistant NS (SRNS) and explain their response to second-line immunosuppressant therapy before and after kidney transplantation. Method We evaluated 31 patients with available kidney biopsy samples of our previously described prospective cohort of SRNS patients, genetically tested with whole exome sequencing at Meyer University Hospital IRCCS. By using high-resolution confocal microscopy, we assessed the co-localization between IgG and nephrin staining on kidney biopsy. To unequivocally establish anti-nephrin specificity of the deposited IgG, we performed super-resolution STimulated Emission Depletion (STED) microscopy. Results We observed anti-nephrin antibodies in 35.5% of patients with SRNS irrespective of podocytopathy lesion pattern. Antibody-positive (Antibody+) patients had a more severe NS at onset and better response to immunosuppression therapy compared to antibody-negative (Antibody-) patients. Only 1/11 Antibody+ patient developed chronic kidney disease compared to 9/20 Antibody- patients over a 10-year period. The single multidrug-resistant Antibody+ patient reached kidney failure had a pathogenic variant in the autosomal dominant-transmitted LMX1B. The patient experienced recurrence of NS after kidney transplantation with a positive response to plasmapheresis and rituximab. The allograft biopsy confirmed the presence of anti-nephrin antibodies. Conclusion The observed variability in patients with SRNS, influenced by a combination of genetic factors and nephrin autoimmunity, likely contributes to the diverse long-term outcomes and variations in responses to second-line immunosuppressant therapy.

A modular framework for multi-scale tissue imaging and neuronal segmentation.

The development of robust tools for segmenting cellular and sub-cellular neuronal structures lags behind the massive production of high-resolution 3D images of neurons in brain tissue. The challenges are principally related to high neuronal density and low signal-to-noise characteristics in thick samples, as well as the heterogeneity of data acquired with different imaging methods. To address this issue, we design a framework which includes sample preparation for high resolution imaging and image analysis. Specifically, we set up a method for labeling thick samples and develop SENPAI, a scalable algorithm for segmenting neurons at cellular and sub-cellular scales in conventional and super-resolution STimulated Emission Depletion (STED) microscopy images of brain tissues. Further, we propose a validation paradigm for testing segmentation performance when a manual ground-truth may not exhaustively describe neuronal arborization. We show that SENPAI provides accurate multi-scale segmentation, from entire neurons down to spines, outperforming state-of-the-art tools. The framework will empower image processing of complex neuronal circuitries.

Ultrastructural 3D Microscopy for Biomedicine: Principles, Applications, and Perspectives.

Modern biomedical research often requires a three-dimensional microscopic analysis of the ultrastructure of biological objects and materials. Conceptual technical and methodological solutions for three-dimensional structure reconstruction are needed to improve the conventional optical, electron, and probe microscopy methods, which to begin with allow one to obtain two-dimensional images and data. This review discusses the principles and potential applications of such techniques as serial section transmission electron microscopy; techniques based on scanning electron microscopy (SEM) (array tomography, focused ion beam SEM, and serial block-face SEM). 3D analysis techniques based on modern super-resolution optical microscopy methods are described (stochastic optical reconstruction microscopy and stimulated emission depletion microscopy), as well as ultrastructural 3D microscopy methods based on scanning probe microscopy and the feasibility of combining them with optical techniques. A comparative analysis of the advantages and shortcomings of the discussed approaches is performed.

Combined expansion and STED microscopy reveals altered fingerprints of postsynaptic nanostructure across brain regions in ASD-related SHANK3-deficiency.

Synaptic dysfunction is a key feature of SHANK-associated disorders such as autism spectrum disorder, schizophrenia, and Phelan-McDermid syndrome. Since detailed knowledge of their effect on synaptic nanostructure remains limited, we aimed to investigate such alterations in ex11|SH3 SHANK3-KO mice combining expansion and STED microscopy. This enabled high-resolution imaging of mosaic-like arrangements formed by synaptic proteins in both human and murine brain tissue. We found distinct shape-profiles as fingerprints of the murine postsynaptic scaffold across brain regions and genotypes, as well as alterations in the spatial and molecular organization of subsynaptic domains under SHANK3-deficient conditions. These results provide insights into synaptic nanostructure in situ and advance our understanding of molecular mechanisms underlying synaptic dysfunction in neuropsychiatric disorders.

Modulated illumination microscopy: Application perspectives in nuclear nanostructure analysis.

The structure of the cell nucleus of higher organisms has become a major topic of advanced light microscopy. So far, a variety of methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well as different types of patterned illumination microscopy, modulated either laterally (in the object plane) or axially (along the optical axis). Based on our experience, we discuss here some application perspectives of Modulated Illumination Microscopy (MIM) and its combination with single-molecule localisation microscopy (SMLM). For example, spatially modulated illumination microscopy/SMI (illumination modulation along the optical axis) has been used to determine the axial extension (size) of small, optically isolated fluorescent objects between ≤ 200nm and ≥ 40nm diameter with a precision down to the few nm range; it also allows the axial positioning of such structures down to the 1nm scale; combined with laterally structured illumination/SIM, a 3D localisation precision of ≤1nm is expected using fluorescence yields typical for SMLM applications. Together with the nanosizing capability of SMI, this can be used to analyse macromolecular nuclear complexes with a resolution approaching that of cryoelectron microscopy.

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides.

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.

Association of poly(rC)-binding protein-2 with sideroflexin-3 through TOM20 as an iron entry pathway to mitochondria

Iron is essential for all the lives and mitochondria integrate iron into heme and Fe-S clusters for diverse use as cofactors. Here, we screened mitochondrial proteins in KU812 human chronic myelogenous leukemia cells by glutathione S-transferase pulldown assay with PCBP2 to identify mitochondrial receptors for PCBP2, a major cytosolic Fe(II) chaperone. LC–MS analyses identified TOM20, sideroflexin-3 (SFXN3), SFXN1 and TOM70 in the affinity-score sequence. Stimulated emission depletion microscopy and proteinase-K digestion of mitochondria in HeLa cells revealed that TOM20 is located in the outer membrane of mitochondria whereas SFXN3 is located in the inner membrane. Although direct association was not observed between PCBP2 and SFXN3 with co-immunoprecipitation, proximity ligation assay demonstrated proximal localization of PCBP2 with TOM20 and there was a direct binding between TOM20 and SFXN3. Single knockdown either of PCBP2 and SFXN3 in K562 leukemia cells significantly decreased mitochondrial catalytic Fe(II) and mitochondrial maximal respiration. SFXN3 but not MFRN1 knockout (KO) in mouse embryonic fibroblasts decreased FBXL5 and heme oxygenase-1 (HO-1) but increased transferrin uptake and induced ferritin, indicating that mitochondrial iron entry through SFXN3 is distinct. MFRN1 KO revealed more intense mitochondrial Fe(II) deficiency than SFXN3 KO. Insufficient mitochondrial heme synthesis was evident under iron overload both with SFXN3 and MFRN KO, which was partially reversed by HO-1 inhibitor. Conversely, SFXN3 overexpression caused cytosolic iron deficiency with mitochondrial excess Fe(II), which further sensitized HeLa cells to RSL3-induced ferroptosis. In conclusion, we discovered a novel pathway of iron entry into mitochondria from cytosol through PCBP2-TOM20-SFXN3 axis.

Dynamic Mode Decomposition of Multiphoton and Stimulated Emission Depletion Microscopy Data for Analysis of Fluorescent Probes in Cellular Membranes.

An analysis of the membrane organization and intracellular trafficking of lipids often relies on multiphoton (MP) and super-resolution microscopy of fluorescent lipid probes. A disadvantage of particularly intrinsically fluorescent lipid probes, such as the cholesterol and ergosterol analogue, dehydroergosterol (DHE), is their low MP absorption cross-section, resulting in a low signal-to-noise ratio (SNR) in live-cell imaging. Stimulated emission depletion (STED) microscopy of membrane probes like Nile Red enables one to resolve membrane features beyond the diffraction limit but exposes the sample to a lot of excitation light and suffers from a low SNR and photobleaching. Here, dynamic mode decomposition (DMD) and its variant, higher-order DMD (HoDMD), are applied to efficiently reconstruct and denoise the MP and STED microscopy data of lipid probes, allowing for an improved visualization of the membranes in cells. HoDMD also allows us to decompose and reconstruct two-photon polarimetry images of TopFluor-cholesterol in model and cellular membranes. Finally, DMD is shown to not only reconstruct and denoise 3D-STED image stacks of Nile Red-labeled cells but also to predict unseen image frames, thereby allowing for interpolation images along the optical axis. This important feature of DMD can be used to reduce the number of image acquisitions, thereby minimizing the light exposure of biological samples without compromising image quality. Thus, DMD as a computational tool enables gentler live-cell imaging of fluorescent probes in cellular membranes by MP and STED microscopy.

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.

Structural basis for the regulatory mechanism of mammalian mitochondrial respiratory chain megacomplex-I2III2IV2

Mammalian mitochondrial electron transport chain complexes are the most important and complicated protein machinery in mitochondria. Although this system has been studied for more than a century, its composition and molecular mechanism are still largely unknown. Here we report the high-resolution cryo-electron microscopy (Cryo-EM) structures of porcine respiratory chain megacomplex-I2III2IV2 (MCI2III2IV2) in five different conformations, including State 1, State 2, Mid 1, Mid 2, and Mid 3. High-resolution Cryo-EM imaging, combined with super-resolution gated stimulated emission depletion microscopy (gSTED), strongly supports the formation of MCI2III2IV2 in live cells. Each MCI2III2IV2 structure contains 141 subunits (70 different kinds of peptides, 2.9 MDa) in total with 240 transmembrane helices. The mutual influence among CI, CIII, and CIV shown in the MCI2III2IV2 structure suggests this megacomplex could act as an integral unit in electron transfer and proton pumping. The conformational changes from different states suggest a plausible regulatory mechanism for the MCI2III2IV2 activation/deactivation process.

MINSTED tracking of single biomolecules

Here we show that MINSTED localization, a method whereby the position of a fluorophore is identified with precisely controlled beams of a STED microscope, tracks fluorophores and hence labeled biomolecules with nanometer/millisecond spatiotemporal precision. By updating the position for each detected photon, MINSTED recognizes fluorophore steps of 16 nm within <250 μs using about 13 photons. The power of MINSTED tracking is demonstrated by resolving the stepping of the motor protein kinesin-1 walking on microtubules and switching protofilaments.

Differential Mitochondrial, Oxidative Stress and Inflammatory Responses to SARS-CoV-2 Spike Protein Receptor Binding Domain in Human Lung Microvascular, Coronary Artery Endothelial and Bronchial Epithelial Cells.

Recent evidence indicates that the SARS-CoV-2 spike protein affects mitochondria with a cell type-dependent outcome. We elucidate the effect of the SARS-CoV-2 receptor binding domain (RBD) on the mitochondrial network and cristae morphology, oxygen consumption, mitoROS production, and inflammatory cytokine expression in cultured human lung microvascular (HLMVECs), coronary artery endothelial (HCAECs), and bronchial epithelial cells (HBECs). Live Mito Orange staining, STED microscopy, and Fiji MiNa analysis were used for mitochondrial cristae and network morphometry; an Agilent XFp analyser for mitochondrial/glycolytic activity; MitoSOX fluorescence for mitochondrial ROS; and qRT-PCR plus Luminex for cytokines. HLMVEC exposure to SARS-CoV-2 RBD resulted in the fragmentation of the mitochondrial network, mitochondrial swelling, increased cristae area, reduced cristae density, and suppressed mitochondrial oxygen consumption and glycolysis. No significant mitochondrial morphology or oxygen consumption changes were observed in HCAECs and HBECs. SARS-CoV-2 RBD induced mitoROS-mediated expression of cytokines GM-CSF and IL-1β in all three investigated cell types, along with IL-8 expression in both endothelial cell types. The findings suggest mitochondrial ROS control SARS-CoV-2 RBD-induced inflammation in HLMVECs, HCAECs, and HBECs, with the mitochondria of HLMVECs being more sensitive to SARS-CoV-2 RBD.

Phosphodiesterases 4B and 4D Differentially Regulate cAMP Signaling in Calcium Handling Microdomains of Mouse Hearts.

The ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling (ECC) by signaling in discrete subcellular microdomains. Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations of PDE4 activity in human hearts has been shown to result in arrhythmias and heart failure. Here, we sought to systematically investigate specific roles of PDE4B and PDE4D in the regulation of cAMP dynamics in three distinct subcellular microdomains, one of them located at the caveolin-rich plasma membrane which harbors the L-type calcium channels (LTCCs), as well as at two sarco/endoplasmic reticulum (SR) microdomains centered around SR Ca2+-ATPase (SERCA2a) and cardiac ryanodine receptor type 2 (RyR2). Transgenic mice expressing Förster Resonance Energy Transfer (FRET)-based cAMP-specific biosensors targeted to caveolin-rich plasma membrane, SERCA2a and RyR2 microdomains were crossed to PDE4B-KO and PDE4D-KO mice. Direct analysis of the specific effects of both PDE4 subfamilies on local cAMP dynamics was performed using FRET imaging. Our data demonstrate that all three microdomains are differentially regulated by these PDE4 subfamilies. Whereas both are involved in cAMP regulation at the caveolin-rich plasma membrane, there are clearly two distinct cAMP microdomains at the SR formed around RyR2 and SERCA2a, which are preferentially controlled by PDE4B and PDE4D, respectively. This correlates with local cAMP-dependent protein kinase (PKA) substrate phosphorylation and arrhythmia susceptibility. Immunoprecipitation assays confirmed that PDE4B is associated with RyR2 along with PDE4D. Stimulated Emission Depletion (STED) microscopy of immunostained cardiomyocytes suggested possible co-localization of PDE4B with both sarcolemmal and RyR2 microdomains. In conclusion, our functional approach could show that both PDE4B and PDE4D can differentially regulate cardiac cAMP microdomains associated with calcium homeostasis. PDE4B controls cAMP dynamics in both caveolin-rich plasma membrane and RyR2 vicinity. Interestingly, PDE4B is the major regulator of the RyR2 microdomain, as opposed to SERCA2a vicinity, which is predominantly under PDE4D control, suggesting a more complex regulatory pattern than previously thought, with multiple PDEs acting at the same location.

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.

Fluorescence microscopy shadow imaging for neuroscience.

Fluorescence microscopy remains one of the single most widely applied experimental approaches in neuroscience and beyond and is continuously evolving to make it easier and more versatile. The success of the approach is based on synergistic developments in imaging technologies and fluorophore labeling strategies that have allowed it to greatly diversify and be used across preparations for addressing structure as well as function. Yet, while targeted labeling strategies are a key strength of fluorescence microscopy, they reciprocally impose general limitations on the possible types of experiments and analyses. One recent development that overcomes some of these limitations is fluorescence microscopy shadow imaging, where membrane-bound cellular structures remain unlabeled while the surrounding extracellular space is made to fluoresce to provide a negative contrast shadow image. When based on super-resolution STED microscopy, the technique in effect provides a positive image of the extracellular space geometry and entire neuropil in the field of view. Other noteworthy advantages include the near elimination of the adverse effects of photobleaching and toxicity in live imaging, exhaustive and homogeneous labeling across the preparation, and the ability to apply and adjust the label intensity on the fly. Shadow imaging is gaining popularity and has been applied on its own or combined with conventional positive labeling to visualize cells and synaptic proteins in their parenchymal context. Here, we highlight the inherent limitations of fluorescence microscopy and conventional labeling and contrast these against the pros and cons of recent shadow imaging approaches. Our aim is to describe the brief history and current trajectory of the shadow imaging technique in the neuroscience field, and to draw attention to its ease of application and versatility.

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.