Stimulated Emission Depletion Research Articles

Ultrastructure Expansion Microscopy (U-ExM) of the Fission Yeast Schizosaccharomyces pombe.

Among widely used super-resolution microscopy techniques, including stimulated emission depletion (STED), photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM), expansion microscopy (ExM) is unique in achieving increased resolution through a physical manipulation of the actual sample rather than optics or postprocessing. Originally developed for applications in neuroscience, ExM now has a solid foothold across many fields and model systems, and has been adapted to work for organisms with cell walls, including budding and fission yeasts, through the inclusion of a pre-expansion enzymatic digestion step. A variant of the ExM technique optimized for preserving the architecture of protein complexes, ultrastructure expansion microscopy (U-ExM), enables super-resolution imaging of full 3D volumes at increased throughput using conventional microscopes and can be readily combined with commonly used antibodies, dyes, and stains. Here, we present its application to the fission yeast Schizosaccharomyces pombe.

Locally increased dysferlin expression improves left-ventricular function by stabilizing membrane nanodomains of cardiomyocytes in the myocardial infarction border zone

Abstract Introduction Dysferlin, a transmembrane protein with multiple Ca2+-binding C2-domains, is expressed in the sarcolemma and vesicle membranes of striated muscle cells. Upon Ca2+ entry, Dysferlin is thought to mediate vesicle fusion and membrane repair events in order to rapidly seal sarcolemmal lesions. Dysferlin mutations are associated with progressive muscular dystrophies and dilated cardiomyopathy. However, the precise role of Dysferlin in protection of membrane nanodomains in ventricular myocytes (VMs) after myocardial infarction (MI) remains elusive. Purpose We hypothesized that Dysferlin is crucial to prevent VMs from loss-of-function and cell death by stabilizing membrane nanodomains like the transverse-axial tubule (TAT) system and the intercalated disc (ICD) cell-cell contact sites post-MI. Methods and Results 1 week post-MI induced by LAD artery ligation, investigator-blinded echocardiography revealed increased infarct sizes and decreased left-ventricular (LV) ejection fraction of Dysferlin-knockout (KO) vs. WT mice (mean±SEM: 21±2 vs. 28±2%, n=20/30 mice, P<0.05). Immunoblotting of WT LV tissue lysates showed an upregulated Dysferlin expression to 148% compared to sham-operated controls. Importantly, immunohistology identified a maximal Dysferlin expression of 230% in the MI border zone that faces a high ischemic and mechanical stress. Hence, we used data-independent mass spectrometry (DIA-MS) to analyse the proteomic profile of the infarction zone, border zone and remote zone from WT vs. KO hearts 1 week post-MI (each n = 5 mice). Overall, we reproducibly quantified 4360 proteins across all LV samples, identifying local genotype-specific proteotypes post-MI. In WT vs. KO samples, DIA-MS revealed 725 differentially abundant proteins in the zone pairwise comparison, highlighting a vital role of Dysferlin for LV remodelling post-MI. In addition, superresolution stimulated emission depletion (STED) microscopy was applied to study the subcellular localization and function of Dysferlin in the MI border zone. Here, we observed VMs with disrupted TAT endomembrane networks and significantly extended ICD regions. Interestingly, STED imaging revealed Dysferlin clusters that highly decorated residual TAT structures and interdigitated ICD membrane folds in the MI border zone. DIA-MS analysis of anti-Dysferlin co-imunoprecipitation experiments from WT vs. KO LV tissues characterized the cardiac interactome of Dysferlin, confirming interactions with proteins of the TAT system and the gap junction hemichannel Connexin-43. Conclusions Our data demonstrate Dysferlin as an endogenous membrane repair protein necessary to stabilize sarcolemmal nanodomains in the MI border zone. Dysferlin not only prevents from loss of TAT structures, but also compensates for increased mechanical stress at the ICDs. Hence, Dysferlin emerges as a novel therapeutic target to control membrane integrity in VMs to prevent from loss-of-function and cell death post-MI.

Modern Methods of Fluorescence Nanoscopy in Biology

Optical microscopy has undergone significant changes in recent decades due to the breaking of the diffraction limit of optical resolution and the development of high-resolution imaging techniques, which are collectively known as fluorescence nanoscopy. These techniques allow researchers to observe biological structures and processes at a nanoscale level of detail, revealing previously hidden features and aiding in answering fundamental biological questions. Among the advanced methods of fluorescent nanoscopy are: STED (Stimulated Emission Depletion Microscopy), STORM (STochastic Optical Reconstruction Microscopy), PALM (Photo-activated Localization Microscopy), TIRF (Total Internal Reflection Fluorescence), SIM (Structured Illumination Microscopy), MINFLUX (Minimal Photon Fluxes), PAINT (Points Accumulation for Imaging in Nanoscale Topography) и RESOLFT (REversible Saturable Optical Fluorescence Transitions) and others. In addition, most of these methods make it possible to obtain volumetric (3D) images of the objects under study. In this review, we will look at the principles of these methods, their advantages and disadvantages, and their application in biological researches.

Bridging the resolution-sectioning gap in STED nanoscopy with coherent-hybrid depletion

Microscopes generally achieve better 2D imaging compared to 3D, and super-resolution microscopes often aggravate such axial-lateral resolution mismatch. A prime example is vortex phase-based stimulated emission depletion (STED) fluorescence microscopy, which only narrows the point-spread function laterally, thereby increasing the point-spread function (PSF) anisotropy. In this study, we developed a semi-analytical theory based on the Nijboer-Zernike expansion, developed a set of metrics and performed experiments to establish the merits of the alternative, bivortex phase-based, coherent-hybrid STED. We find that this mode reduces the axial-lateral resolution mismatch by providing access to PSF geometries that are strictly forbidden to the two conventional single-beam modes, emulating noisier multi-beam approaches. Both theory and experiment indicate that bivortex STED not only addresses the axial-lateral resolution gap but also delivers a higher signal-to-background ratio than the two more common STED modes that it bridges.

CGAS deficient mice display premature aging associated with de-repression of LINE1 elements and inflammation.

Aging-associated inflammation, or 'inflammaging" is a driver of multiple age-associated diseases. Cyclic GMP-AMP Synthase (cGAS) is a cytosolic DNA sensor that functions to activate interferon response upon detecting viral DNA in the cytoplasm. cGAS contributes to inflammaging by responding to endogenous signals such as damaged DNA or LINE1 (L1) cDNA which forms in aged cells. While cGAS knockout mice are viable their aging has not been examined. Unexpectedly, we found that cGAS knockout mice exhibit accelerated aging phenotype associated with induction of inflammation. Transcription of L1 elements was increased in both cGAS knockout mice and in cGAS siRNA knockdown cells associated with high levels of cytoplasmic L1 DNA and expression of ORF1 protein. Cells from cGAS knockout mice showed increased chromatin accessibility and decreased DNA methylation on L1 transposons. Stimulated emission depletion microscopy (STED) showed that cGAS forms nuclear condensates that co-localize with H3K9me3 heterochromatin marks, and H3K9me3 pattern is disrupted in cGAS knockout cells. Taken together these results suggest a previously undescribed role for cGAS in maintaining heterochromatin on transposable elements. We propose that loss of cGAS leads to loss of chromatin organization, de-repression of transposable elements and induction of inflammation resulting in accelerated aging.

ARF1 compartments direct cargo flow via maturation into recycling endosomes.

Cellular membrane homoeostasis is maintained via a tightly regulated membrane and cargo flow between organelles of the endocytic and secretory pathways. Adaptor protein complexes (APs), which are recruited to membranes by the small GTPase ARF1, facilitate cargo selection and incorporation into trafficking intermediates. According to the classical model, small vesicles would facilitate bi-directional long-range transport between the Golgi, endosomes and plasma membrane. Here we revisit the intracellular organization of the vesicular transport machinery using a combination of CRISPR-Cas9 gene editing, live-cell high temporal (fast confocal) or spatial (stimulated emission depletion) microscopy as well as correlative light and electron microscopy. We characterize tubulo-vesicular ARF1 compartments that harbour clathrin and different APs. Our findings reveal two functionally different classes of ARF1 compartments, each decorated by a different combination of APs. Perinuclear ARF1 compartments facilitate Golgi export of secretory cargo, while peripheral ARF1 compartments are involved in endocytic recycling downstream of early endosomes. Contrary to the classical model of long-range vesicle shuttling, we observe that ARF1 compartments shed ARF1 and mature into recycling endosomes. This maturation process is impaired in the absence of AP-1 and results in trafficking defects. Collectively, these data highlight a crucial role for ARF1 compartments in post-Golgi sorting.

Anti-slit diaphragm antibodies on kidney biopsy identify pediatric patients with steroid-resistant nephrotic syndrome responsive to second-line immunosuppressants

Podocytopathies represent a group of glomerular disorders associated with minimal changes (MC) or focal segmental glomerulosclerosis (FSGS) lesion patterns at biopsy and heterogeneous responses to steroids. Anti-nephrin antibodies were previously found in such patients, suggesting an autoimmune form of podocytopathy. High resolution confocal microscopy on kidney biopsies of a cohort of 128 pediatric patients revealed localization of IgG along the slit diaphragm in 30% of patients with MC and 25% of those with FSGS, but not in other lesion patterns. Anti-nephrin IgG ELISA assay in the serum and stimulated emission depletion microscopy of kidney biopsies showed IgG-nephrin co-localization only in 77.8% of cases. Similar observations were obtained in a cohort of 48 adult patients with MC or FSGS at kidney biopsy, where IgG-nephrin colocalization was only 44.4%, suggesting the existence of autoantibodies binding to other slit proteins. Patients with anti-slit antibodies showed nephrotic syndrome at onset in 94.4% of cases. Patients with primary steroid-resistance had anti-slit antibodies in 27%, while those with secondary steroid-resistance in 87.5% of cases, irrespective of the histopathological lesion pattern. Steroid-resistant patients with anti-slit antibodies responded to second-line immunosuppressants in 92.3% vs. only 20% of patients that were anti-slit negative. No patient with anti-slit antibodies developed kidney failure vs. 51.7% of those negative for antibodies (66.7% with a genetic cause and 41.2% with a non-genetic cause). Thus, the detection of anti-slit antibodies can identify patients with an autoimmune podocytopathy responsive to treatment with second-line immunosuppressants, irrespective of the histopathological lesion pattern at biopsy.

Development of AI-assisted microscopy frameworks through realistic simulation with pySTED

The integration of artificial intelligence into microscopy systems significantly enhances performance, optimizing both image acquisition and analysis phases. Development of artificial intelligence-assisted super-resolution microscopy is often limited by access to large biological datasets, as well as by difficulties to benchmark and compare approaches on heterogeneous samples. We demonstrate the benefits of a realistic stimulated emission depletion microscopy simulation platform, pySTED, for the development and deployment of artificial intelligence strategies for super-resolution microscopy. pySTED integrates theoretically and empirically validated models for photobleaching and point spread function generation in stimulated emission depletion microscopy, as well as simulating realistic point-scanning dynamics and using a deep learning model to replicate the underlying structures of real images. This simulation environment can be used for data augmentation to train deep neural networks, for the development of online optimization strategies and to train reinforcement learning models. Using pySTED as a training environment allows the reinforcement learning models to bridge the gap between simulation and reality, as showcased by its successful deployment on a real microscope system without fine tuning.

Water-soluble AIE photosensitizer in short-wave infrared region for albumin-enhanced and self-reporting phototheranostics

Organic photosensitizers (PSs) play important roles in phototheranostics, and contribute to the fast development of precision medicine. However, water-soluble and highly emissive organic PSs, especially those emitting in the short-wave infrared region (SWIR), are still challenging. Also, it's difficult to prepare self-reporting PSs for visualizing the treatment via stimulated emission depletion (STED) nanoscopy. Thus, in this work, a water-soluble molecule of DTPAP-TBZ-I with aggregation-induced emission features is designed for the self-reporting photodynamic therapy (PDT) in an ultra-high resolution. In contrast to single molecule, its complex (DTPAP-TBZ-I@BSA) shows much enhanced fluorescence properties and reactive oxygen species (ROS) generation in SWIR window. Their photoluminescence quantum yield is determined to be ∼20.6 % and the enhancement of ROS generation is ∼18-fold. During the PDT, immigration of the complex from cytoplasm to nucleus is also observed via STED nanoscopy with a resolution of 66.11 nm, which allows self-report in the PDT treatment. DTPAP-TBZ-I@BSA is finally utilized for the imaging-guided PDT in vivo with a tumor inhibition rate of 84 %. This is the first work in albumin-enhanced water-soluble organic PSs in SWIR window for self-reporting phototheranostics at ultra-high resolutions, providing an ideal solution for the next generation of photosensitizers for precise medicine.

Bacterial Cellulose In Vitro Uptake by Macrophages, Epithelial Cells, and a Triculture Model of the Gastrointestinal Tract.

Bacterial cellulose (BC) has a long-standing human consumption history in different geographies without any report of adverse effects. Despite its unique textural and functional properties, the use of BC in food products in Europe is still restricted due to concerns over its nanosize. Here, we evaluated the potential uptake of celluloses (from plant and microbial sources, processed using different blenders) by macrophages (differentiated THP-1 cells) and human intestinal epithelial cells (Caco-2 and HT29-MTX cells) without (coculture) or with (triculture) Raji-B cells. A carbohydrate-binding module coupled to a green fluorescent protein was employed to observe cellulose in the cell cultures by confocal laser scanning microscopy and stimulated emission depletion microscopy. The methodology demonstrated excellent sensitivity, allowing detection of single nanocrystals within cells. All celluloses were taken up by the macrophages, without significantly compromising the cell's metabolic viability. The viability of the cocultures was also not affected. Furthermore, no internalization was observed in the triculture cell model that was exposed 24 h to BC and Avicel LM310. When (rarely) detected, cellulose particles were found on the apical side of the membrane. Overall, the obtained results suggest that BC should not be absorbed into the human gut.

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.

Mitochondrial STED Imaging and Membrane Potential Monitoring with a Cationic Molecular Probe.

Mitochondria are essential organelles that not only undergo dynamic morphological changes but also exhibit functional activities such as mitochondrial membrane potential (MMP). While super-resolution techniques such as stimulated emission depletion (STED) nanoscopy can visualize the ultrastructure of mitochondria and the MMP probe can monitor mitochondria function, few dyes meet both demands. Here, a small molecule (MitoPDI-90) based on perylene diimide with cationic groups is reported and used for mitochondrial STED imaging and MMP indication. Characterized by excellent photostability, biocompatibility, and high quantum yield, MitoPDI-90 exhibits STED imaging compatibility, facilitating visualization of mitochondrial cristae and time-lapse imaging of highly dynamic mitochondria in living cells. Besides, MitoPDI-90 targets the mitochondria through electrical potential, also enabling live-cell MMP monitoring. MitoPDI-90 allows for super-resolution visualization and time-lapse imaging of mitochondria, and more importantly, indication of changes in MMP, providing insight into the functional activity of live-cell mitochondria.

Influence of Host Lattice Ions on the Dynamics of Transient Multiband Upconversion in Yb-Er Codoped NaLnF4 and LiLnF4 Microcrystals (Ln: Y, Lu, Gd).

Inorganic host matrices provide a tunable luminescence environment for lanthanide ions, allowing for the modulation of upconversion luminescence (UCL) properties. AREF4 (A = alkali metal, RE = rare earth) have a low phonon energy and a high optical damage threshold, making them widely used as the host matrix for UCL materials. However, the impact mechanism of alkali metal ions and lanthanide lattice ions on transient UCL dynamics in AREF4 remains unclear. This study utilized a high-power nanosecond-pulsed laser at 976 nm to excite Yb-Er codoped NaLnF4 and LiLnF4 (Ln: Y, Lu, and Gd) microcrystals (MCs). All samples exhibit multiband emission, and the transient UC dynamics are discussed in detail. Compared with LiLnF4, NaLnF4 has higher UC efficiency and red to green (R/G) ratio. Lanthanide ions (Y, Lu, and Gd) affect the energy transfer (ET) distance in Yb-Er codoped systems, thereby altering UC efficiency and the R/G ratio. The energy level coupling between Gd3+ and Er3+ prolongs the duration of the UC emission. Specifically, the red emission lifetime of NaGdF4 is five times longer than that of NaYF4. Our research contributes to exploring excellent alternative host matrices for NaYF4 in the fields of rapid-response optoelectronic devices, micro-nano lasers, and stimulated emission depletion (STED) microscopy.

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.

Supramolecular Complex of Cucurbit[7]uril with Diketopyrrolopyrole Dye: Fluorescence Boost, Biolabeling and Optical Microscopy.

New photostable and bright supramolecular complexes based on cucurbit[7]uril (CB7) host and diketopyrrolopyrole (DPP) guest dyes having two positively charged 4-(trimethylammonio)phenyl groups were prepared and characterized. The dye core displays large Stokes shift (in H2O, abs./emission max. 480/550 nm; ϵ~19 000, τfl>4 ns), strong binding with the host (~560 nM Kd) and a linker affording fluorescence detection of bioconjugates with antibody and nanobody. Combination of protein-functionalized DPP dye with CB7 improves photostability and affords up to 12-fold emission gain. Two-color confocal and stimulated emission depletion (STED) microscopy with 595 nm or 655 nm STED depletion lasers shows that the presence of CB7 not only leads to improved brightness and image quality, but also results in DPP becoming cell-permeable.

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.

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.

Polarization-modulated dual-wavelength metalens to overcome the diffraction limit

Two-photon stimulated emission depletion (STED) fluorescence microscopy has demonstrated advantages in ultrahigh resolution biological imaging. However, the bulky setup for generating excitation and depletion beams hinders the system development. Here, a miniaturized single-layer polarization-modulated dual-wavelength metalens is proposed for applications in STED. The metalens can achieve achromatic focusing at 532 nm and 632 nm, and can generate both a Gaussian-shaped spot and a donut-shaped depletion spot across different polarization states. Based on the theoretical analysis, we obtained an effective fluorescence spot that overcomes the diffraction limit. The multifunctional imaging capability of the metalens is also demonstrated, including bright-field and edge-detection imaging. The proposed metalens offers several advantages such as flexibility, single-layer, light-weight and multifunctionality. These features make it an ideal candidate for enabling super-resolution in minimally invasive imaging.

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.

Enhancement of Signal‐to‐Background Ratio in Molecular Vibrational Signal Extraction by Stimulated Emission Depletion Mechanism

Herein, a novel approach is presented to mitigate the fluorescence interference during the detection of vibrational signal via the stimulated emission depletion (STED). STED is the mechanism commonly employed in optical imaging; however, its application should not be confined solely to this field. To explore additional possibilities, a novel application of STED in vibrational spectroscopy detection is introduced. Vibrational spectroscopy is a widely used technique for the material detection and identification, but its sensitivity is influenced by impurity signals, especially the fluorescence. The proposed method is capable of suppressing fluorescence without influencing vibrational signal. At the low concentration of fluorescent impurities, the signal‐to‐background ratio of vibrational spectroscopy is 2.6 times as high as that without this method. The introduction of depletion light can enhance the detection of vibrational signals, resulting in more optimal signal detection. A promising new application of STED other than super‐resolution imaging is investigated.