Biology subjects

Liu, X.

Publications and source records attributed to Liu, X..

At least 577 records · Page 32Linked to original sources

bioRxiv2025

Achievement-Based Differences in Cognitive-Emotional Interplay During Classroom Learning: A Multimodal Analysis with Experience Sampling Method

BackgroundThe relationship between cognition and emotion during learning is crucial for educational theories and has been under debate for a long time, from the ancient philosophy to current scientific analyses. However, the relationship may not be uniform and may vary across diverse individual groups. AimsTherefore, this study aims to explore whether the relationship between cognition and emotion varies across different student groups. Furthermore, it examines whether neurophysiological evidence can provide insights into such group differences. SampleThe study involved 22 high school students (11 high-performing and 11 low-performing) over 13 math lessons. Surveys were administered three times during each lesson, yielding a multilevel dataset with 777 samples. MethodsThe experience sampling method was employed to gather data on cognitive and emotional states periodically, as well as their regulations. Simultaneously, multimodal neurophysiological data, including electroencephalograph (EEG), electrodermal activity (EDA), and photoplethysmography (PPG), were collected during classroom learning to analyze the relationship further. ResultsCognitive state, cognitive regulation, emotional state, and emotional regulation were highly correlated overall. In high-performing students, the association was higher than that in low-performing students, especially at the level of within-subject repeated-measures. Multimodal data revealed the common neurophysiological basis of the correlated variation between cognition and emotion, and highlighted different neurophysiological patterns between high and low achievers. ConclusionsThe joint evidence from the experience sampling survey and neurophysiological measures reveals that the cognitive-emotional relationship differs by academic achievement levels, with high achievers revealing a more consistent variation between cognition and emotion. Such co-variation shares a common neurophysiological pattern with features associated with high student-teacher interaction and high concentration, further explaining the group differences.

neuroscience↗

bioRxiv2025

ST-FFPE-mIF: Integrating Spatial Transcriptomics and Multiplex Immunofluorescence in Formalin-Fixed Paraffin-Embedded Tissues Using Stereo-seq

Stereo-seq is the state-of-the-art technique, which allows capturing of spatial transcriptomic data at sub-cellular resolution and in large scale of view. Stereo-seq FFPE technique is recently launched with the capability to obtain spatially resolved gene expression in FFPE tissue sections. However, the omic information is limited to transcriptome. To delineate spatial molecular hierarchy from transcriptome to phenome, we present the protocol that implements nine-plex immunofluorescence staining with detection of spatial transcriptomics in one FFPE tissue section (short for ST-FFPE-mIF). This protocol details the modified biochemistry procedure and also provides analytical framework to achieve single-cell resolution through nuclear or membrane staining-based cell segmentation. We demonstrated the application of ST-FFPE-mIF in colorectal cancer sample to illustrate how ST-FFPE-mIF facilitates a robust analysis of spatially resolved multi-omic data at the single-cell level.

biochemistry↗

bioRxiv2025

Automatically Defining Protein Words for Diverse Functional Predictions Based on Attention Analysis of a Protein Language Model

Understanding the relationship between protein sequence and function remains a longstanding challenge in bioinformatics, and to date the lions share of related tools parse proteins at the domain or motif levels. Here, we define "protein words" as an alternative to "motif" for studying proteins and functional prediction applications. We first developed an unsupervised tool we term Protein Wordwise, which parses analyte protein sequences into protein words by analyzing attention matrices from a protein language model (PLM) through a community detection algorithm. We then developed a supervised sequence-function prediction model called Word2Function, for mapping protein words to GO terms through feature importance analysis. We compared the prediction performance of our protein word-based toolkit with a motif-based method (PROSITE) for multiple protein function datasets. We also assembled a functionally diverse data resource we term PWNet to support evaluation of protein words for predicting functional residues across 10 tasks (e.g., diverse biomolecular binding, catalysis, and ion-channel activity). Our toolkit outperforms PROSITE in all the examined datasets and tasks. By abandoning domains and instead using attention matrices from a PLM for automatic, systematic, and annotation-agnostic parsing of proteins, our toolkit both outperforms currently available tools for functional annotations at the residue and whole-protein levels and suggests innovative forms of protein analysis well-suited to the post-AlphaFold era of biochemistry.

bioinformatics↗

bioRxiv2025

Aging directs the differential evolution of KRAS-driven lung adenocarcinoma

Lung adenocarcinoma (LUAD), the most common histological subtype of lung cancer(1, 2), is a disease of the elderly, with an average age of diagnosis of about 70 years of age(3). Older age is associated with an increased incidence of KRAS-driven LUAD(4), a particularly deadly type of LUAD characterized by treatment resistance and relapse. Despite this, our understanding of how old age shapes KRAS-driven LUAD evolution remains incomplete. While the age-related increase in cancer risk was previously ascribed to the accumulation of mutations over time, we are now beginning to consider the role of host biology as an independent factor influencing cancer. Here, we use single-cell RNA-Sequencing of KP (KrasG12D/+; Trp53flox/flox) LUAD transplanted into young and old mice to define how old age affects LUAD evolution and map the changes that old age imposes onto LUADs microenvironment. Our data demonstrates that the aged lung environment steers LUAD evolution towards a primitive stem-like state that is associated with poor prognosis. We ascribe this differential evolution, at least in part, to a population of rare and highly secretory damage-associated alveolar differentiation intermediate (ADI) cells that accumulate in the aged tumor microenvironment (TME) and that dominate the niche signaling received by LUAD cells. Overall, our data puts aging center stage in coordinating LUAD evolution, highlighting the need to model LUAD in its most common context and creating a framework to tailor future cancer therapeutic strategies to the age of the patient to improve outcomes in the largest and most vulnerable LUAD patient population, the elderly.

cancer biology↗

bioRxiv2025

A pan-cancer atlas of T cell targets

T-cell-based immunotherapies have revolutionized cancer treatment, yet only a minority of patients are eligible for these approaches, significantly constrained by the limited knowledge of tumor-specific antigens. Here we present ImmunoVerse, a comprehensive map of T cell targets across 21 cancer types, revealing actionable tumor-specific targets in 89% of tumors analyzed. To define the repertoire of actionable T cell targets, we conducted an exhaustive pan-cancer analysis, integrating data from 7,188 RNA-Seq, 1,771 immunopeptidomes from 512 biological samples and 208 single-cell cancer datasets using novel AI methods, and compared these against 17,384 normal samples covering 51 tissues. Our analysis uncovered 62 viable surface protein targets and 28,446 tumor-specific HLA-presented antigens, deriving from 11 distinct molecular events, across 21 tumor types. Among these, we identified 5,928 previously uncharacterized neoantigens, new tumor self-antigens, peptides derived from tumor-specific cryptic ORFs, tumor-associated microbial targets and a novel splicing-derived PMEL peptide (sPMEL) with enhanced abundance and safety compared to the canonical clinical targets. We successfully expanded sPMEL-specific T cells, validating the therapeutic potential of these targets in functional assays. We highlight 153 promising new tumor targets and experimentally validate 19 targets representing six antigen classes. In addition to being the most comprehensive atlas of targets in scope, ImmunoVerse offers the most extensively annotated resource with key parameters for target selection, providing critical insights for therapeutic prioritization and clinical translation. To catalyze therapeutic development, we released our pan-cancer target atlas through an interactive web portal (https://www.immuno-verse.com) and made the accompanying toolkits available to the scientific community. This work redefines the landscape of therapeutic T cell targets and provides a foundational resource to unlock immunotherapy development across multiple cancers previously considered intractable.

cancer biology↗

bioRxiv2025

Tobacco smoke carcinogens exacerbate APOBEC mutagenesis and carcinogenesis

Mutational processes are thought to act independently and additively. We challenge this view by demonstrating that DNA-adducting agents sensitize the genome to mutagenesis by APOBEC enzymes. A model tobacco carcinogen (NQO) triggers a 100-fold increase in APOBEC3B- catalyzed signature mutations and elevates oral carcinoma levels in vivo. A hallmark of this unidirectional synergy is strand-coordinated pairs of APOBEC signature mutations within 32nt of each other (didyma). Biochemical experiments show that APOBEC3B can catalyze didyma formation, and genetic studies demonstrate requirements for APOBEC3B and functional nucleotide excision repair (XPA in human cells and Rad14 in yeast). Analyses of lung and head & neck cancers show that APOBEC mutagenesis and didyma are elevated in tumors from smokers compared to non-smokers. Additional tumor types with links to DNA-adducting agents also exhibit didyma. These studies support a model in which DNA-adducting carcinogens activate nucleotide excision repair and amplify mutagenesis by APOBEC enzymes in cancer.

cancer biology↗

bioRxiv2025

Decoding cellular transcriptional regulatory networks governing wheat inflorescence development

Wheat inflorescence architecture, particularly spikelet and floret development, is critical for grain yield. To decode the cellular transcriptional regulatory network (cTRN) underlying wheat inflorescence development, we integrated multiple single-cell omics technologies to construct a spatiotemporal atlas of transcriptional and chromatin accessibility dynamics. This comprehensive analysis identified 20 cell types, 7,211 cell type-specific genes and 152,333 cell type-specific accessible chromatin regions (csACRs) in the wheat inflorescence. Trajectory analysis identified two sub-clusters of proliferating cells as the origins of spikelet and floret formation, deviating from the traditional developmental model. Key transcription factors and hormone-related genes in the cTRN, along with the csACRs, providing new targets for modulating wheat inflorescence architecture. Our findings provide a high-resolution resource for crop inflorescence research and establish a paradigm for applying spatiotemporal single-cell omics analysis to plant biology.

plant biology↗

bioRxiv2025

The Power of Three: Dynactin associates with three dyneins under load for greater force production

Cytoplasmic dynein is an essential microtubule motor protein that powers organelle transport and mitotic spindle assembly. Its activity depends on dynein-dynactin-cargo adaptor complexes, such as dynein-dynactin-BicD2 (DDB), which typically function with two dynein motors. We show that mechanical tension recruits a third dynein motor via an auxiliary BicD adaptor binding the light intermediate chain of the third dynein, stabilizing multi-dynein assemblies and enhancing force generation. Lis1 prevents dynein from transitioning into a force-limiting phi-like conformation, allowing single-dynein DDB to sustain forces up to [~]4.5 pN, whereas force generation often ends at [~]2.5 pN without Lis1. Complexes with two or three dyneins generate [~]7 pN and [~]9 pN, respectively, consistent with a staggered motor arrangement that enhances collective output. Under load, DDB primarily takes [~]8 nm steps, challenging existing dynein coordination models. These findings reveal adaptive mechanisms that enable robust intracellular transport under varying mechanical demands.

biophysics↗

bioRxiv2025

Sequencing-based Spatial Transcriptomics with scRNA-seq Sensitivity

The advent of spatial transcriptomics has dramatically expanded our ability to study the vast network of cell-cell interactions at the molecular level in tissue. Among current methods, sequencing-based approaches have great potential in discovering because of its unbiased capture. In the last couple of years, the spatial resolution for the capture addresses has been significantly improved from 100 m to <1 m, well below the size of a mammalian cell. However, the capture efficiency has always been a pain point, [~]one order of magnitude lower than that of single cell RNA sequencing (scRNA-seq). The low capture efficiency limits the depth and breadth of its applications in the study of complex biological systems and diseases. Here, we introduce Salus Spatial transcriptomic system (Salus-STS), which provides [~]1 m capture resolution and a capture efficiency [~]1 order of magnitude higher than other current methods. Analysis with sub-cellular resolution becomes practical for sequencing-based spatial transcriptomics.

molecular biology↗

bioRxiv2025

A multilamellar organelle for chemosymbiosis in an aplacophoran mollusc adapted to anoxic cold seep sediment

Symbiosis with chemoautotrophic bacteria has evolved in many animal lineages inhabiting reducing habitats such as hydrothermal vents, allowing these holobionts to thrive in dark biospheres1. In certain instances, the symbionts have become intracellular, residing within specialised bacteriocytes2. The integration of microbial symbionts with eukaryotic cells vary across known animals; however, no specialised organelle dedicated to chemosynthesis has been identified yet2. Here, we report a mode of symbiosis where sulphur-oxidising bacteria cultured within spherical multilamellar compartments (~12 m) in the cold-seep aplacophoran mollusc Chaetoderma shenloong. This organelle, which we name dracosphera, is ubiquitous within the hypertrophied and intricately reticulate digestive gland of C. shenloong, which has otherwise lost most of its gut. Given that the symbionts are strictly anaerobic and the host resides in anoxic sediments tens of centimetres below the surface, the dracosphera may serve to minimise oxygen diffusion to the bacteria, akin to mechanisms observed in microbial diazotrophs3 or termite hindguts4, as supported by our genomic and spatial transcriptomic analyses. Hypoxic conditions have been known to induce radical adaptations in meiofauna, exemplified by the acquisition of hydrogenosomes5. Our discovery of similarly exceptional adaptations in C. shenloong provides new insights into the evolution of such organelles also in larger animals.

microbiology↗

bioRxiv2025

Fast and accurate modeling of TCR-peptide-MHC complexes using tFold-TCR

Alpha-beta T cell receptor ({beta}TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is a corner- stone of the adaptive immune system. Fast and accurate modeling of TCR-pMHC structures is crucial for understanding TCR recognition of pMHCs at the molecular level, which is essential for the development of TCR-based therapeutics and vaccines. Despite significant interest, this challenge remains unresolved due to the diversity of TCR-pMHC interactions and limited structural data. Here, we present tFold-TCR, a high-throughput, end-to-end universal model for predicting three-dimensional (3D) atomic-level structures of TCR-pMHC complexes, capable of predicting TCRs of different classes and MHC structures from diverse systems. tFold-TCR leverages a specially trained, protein-protein interaction-sensitive large protein language model to extract intra- and inter-chain residue contact information and evolutionary relationships, bypassing the need for multiple sequence alignment (MSA) searches. It also features innovative structure prediction and flexible docking modules to enhance accuracy, particularly for interacting contacts. Compared to existing methods, including AlphaFold-3, tFold-TCR demonstrates a 30.7% increase in prediction success rate evaluated by DockQ and is over 25 times faster. These advancements enable large-scale structural characterization of TCRs and their interactions with pMHCs. Utilizing this capability, we constructed TCRStructDB, the largest database of TCR-pMHC structures to date, encompassing 2.2 million TCRs, 0.8 million pMHCs, and 45,000 TCR-pMHC complexes. TCRStructDB provides unprecedented insights into one of the most diverse receptor-ligand interactions in biology.

bioinformatics↗

bioRxiv2025

Lower slow wave sleep and rapid eye-movement sleep are associated with brain atrophy of AD-vulnerable regions

Study objectivesSleep deficiency is associated with Alzheimers disease (AD) pathogenesis. We examined the association of sleep architecture with anatomical features observed in AD: (1) atrophy of hippocampus, entorhinal, inferior parietal, parahippocampal, precuneus, and cuneus regions ("AD-vulnerable regions") and (2) cerebral microbleeds. MethodsIn 271 participants of the Atherosclerosis Risk in the Communities Study, we examined the association of baseline sleep architecture with anatomical features identified on brain MRI 13[~]17 years later. Sleep architecture was quantified as the proportion of slow wave sleep (SWS), proportion of rapid eye-movement sleep (REM), and arousals index using polysomnography. Outcomes included (1) volumetric measurements of each AD-vulnerable region and (2) the presence of any cerebral microbleeds (CMBs) and that of lobar CMBs, which are more specifically associated with AD. We analyzed the association of each sleep predictor with each MRI outcome, adjusting for covariates. ResultsHaving less SWS was associated with smaller inferior parietal region ({beta}=-44.19 mm3 [95%CI=-76.63,-11.76]) and cuneus ({beta}=-11.99 mm3 [-20.93,-3.04]) after covariate adjustment. Having less REM was associated with smaller inferior parietal region ({beta}=-75.52 mm3 [-129.34, -21.70]) and precuneus ({beta}=-31.93 mm3 [-63.79,-0.07]). After FDR adjustments, lower SWS and REM, respectively, were associated with smaller inferior parietal region. Arousal index was not associated with the volumes of AD-vulnerable regions. None of the sleep architecture variables were associated with CMBs or lobar CMBs. ConclusionsSleep deficiency is associated with the atrophy of the inferior parietal region, which is observed in early AD. Sleep architecture may be a modifiable risk factor for AD. Brief summarya. Current Knowledge/Study Rationale: two sentences summarizing why the study was doneWhile impaired sleep architecture has been associated with Alzheimers disease [AD] diagnosis and cognitive decline. To better understand the impact of sleep on AD pathogenesis, this study examined the association of sleep architecture with anatomical features observed in AD, including the atrophy of AD-vulnerable regions and CMBs. b. Study Impact: two sentences summarizing how the study impacts the fieldOur study shows that lower slow wave sleep and rapid eye movement sleep may be precipitating factors of inferior parietal region atrophy, which is associated with AD risk. Importantly, the current studys findings can help characterize underlying mechanisms of how sleep deficiency, a prevalent disturbance among middle-aged and older adults, may facilitate AD pathogenesis and cognitive impairment.

neuroscience↗

bioRxiv2025

VZV gE Promotes STING Degradation via Midnolin-Proteasome Pathway to Inhibit cGAS-STING Signaling

Varicella-zoster virus (VZV) infects 85 million individuals annually and it is known for its intricate interplay with host cellular mechanisms, significantly impacting antiviral immune responses. Previous studies have highlighted the critical role of VZV glycoprotein E (gE) in virus-host interactions, but the precise mechanisms remain to be elucidated. Our results demonstrate that VZV gE interacts with STING, specifically through the gE (350-480aa) and STING (151-160aa) regions. This interaction predominantly involves incompletely glycosylated gE. Functional assays revealed that gE inhibits the cGAS-STING signaling pathway. gE enhances VZV proliferation and promotes STING degradation via the proteasome pathway, without affecting STING ubiquitination. We identified Midnolin as a mediator in this degradation process, with gE facilitating the interaction between STING and Midnolin. In vivo, gE expression in mice led to diminished antiviral responses upon HSV-1 infection, highlighting gEs role in modulating immune signaling. Our findings provide significant insights into VZVs evasion of host immune responses. By promoting STING degradation through an unconventional Midnolin-proteasome pathway, VZV gE effectively suppresses antiviral signaling, facilitating enhanced viral proliferation. The findings contribute significantly to the understanding of VZV pathogenesis and provide new therapeutic strategies against VZV.

microbiology↗

bioRxiv2025

Viscoelastic HyA Hydrogel Promotes Recovery of Muscle Quality and Vascularization in a Murine Model of Delayed Rotator Cuff Repair

Rotator cuff tears are among the most common musculotendinous injuries with high risk of permanent functional disability. Following surgical repair, sub-optimal patient outcomes are directly correlated with poor muscle quality; namely, injury site fatty infiltration (FI), fibrosis, and muscle atrophy. Muscle resident fibro-adipogenic progenitor cells (FAPs) have been identified as key regulators of post-injury skeletal muscle regeneration and repair by maintaining a promyogenic environment. In this work, human-derived FAPs (hFAPs) were encapsulated into hyaluronic acid (HyA)-based hydrogels functionalized with bsp-RGD(15) cell adhesion peptide, heparin, and a matrix metalloproteinase (MMP)-cleavable crosslinker. Hydrogel-encapsulated hFAPs increased expression of the promyogenic marker UCP1 and production of the anti-inflammatory cytokine IL-10, while downregulating the expression of the fibrotic marker SMA over time. A murine model of unilateral rotator cuff transection, denervation, and delayed repair was treated with the HyA hydrogel or PBS and compared to a contralateral, non-injured control limb. Muscle histology 6 weeks post-repair revealed that the hydrogel reduced fibrosis, FI, and muscle atrophy while supporting vascularization of the injured tissue region. Collectively, these results suggest that the hydrogel alone can promote muscle regeneration in a clinically relevant delayed repair model of rotator cuff tear, which we hypothesize is due to controlled FAP differentiation into promyogenic lineages.

bioengineering↗

bioRxiv2025

The Ralstonia solanacearum effector RipAV targets Plant U-box proteins and induces proteasomal-dependent degradation of BIK1

Plants have developed a complex immune system to detect and respond to invading pathogens. A critical aspect of this defence relies on regulatory mechanisms that control the activation of immune responses, ensuring these are efficient yet do not compromise overall plant performance. Many pathogens secrete proteins called effectors to manipulate plant cellular functions, suppressing plant immunity and promoting infection. Here, we show that the bacterial effector RipAV, secreted by the soil-borne bacterial pathogen Ralstonia solanacearum, hijacks a major immune signalling hub to suppress immune responses. RipAV targets members of the plant-specific ubiquitin ligase (PUB) family and calcium-dependent protein kinase 28 (CPK28), which has been shown to phosphorylate a set of PUBs to enhance their activity and regulate the stability of the key immune regulator BIK1. RipAV association enhances the CPK28-mediated phosphorylation of PUBs, inducing the proteasome-mediated degradation of BIK1 and the suppression of immunity. Importantly, we found that RipAV is required for maintaining a low accumulation of BIK1 during a R. solanacearum infection. These findings provide new insights into the sophisticated strategies employed by pathogens to subvert plant immunity.

plant biology↗

bioRxiv2025

Three-dimensional quantitative characterization of Coxiella burnetii infection using focused ion beam-scanning electron microscopy

Coxiella burnetii is a highly virulent intracellular pathogen that causes acute and chronic Q fever in humans. The bacterium utilizes a Dot/Icm type IV secretion system (T4SS) to translocate over 100 effectors into host cells to facilitate biogenesis of Coxiella-containing vacuoles (CCVs), which are specialized lysosome-derived organelles that support bacterial replication. During replication in CCVs, C. burnetii undergoes a unique biphasic developmental cycle defined by two distinct cellular forms: the infectious and metabolically dormant small cell variant (SCV) that converts to a replicative large cell variant (LCV). This developmental cycle was believed to be intimately coupled with biogenesis of CCVs, but the underlying mechanisms remained unclear. Here, we combine advanced focused ion beam-scanning electron microscopy (FIB-SEM) with machine learning-based data analyses to visualize host cells infected by C. burnetii. We reveal significant pleiomorphism of both the bacteria and CCVs in three dimensions. We show that this technology can be leveraged to characterize CCV biogenesis defects displayed by C. burnetii mutants that are unable to generate the large CCV displayed by wild-type C. burnetii. Analysis of HeLa cells infected with a cig2::Tn mutant confirmed that this mutant creates vacuoles that have a defect in homotypic fusion but that the proportions of SCV to LCV in vacuoles in which this mutant resides are nearly identical to CCVs containing wild-type C. burnetii, which indicates the vacuole biogenesis defect displayed by the cig2::Tn mutant did not impact the developmental cycle intracellularly. Collectively, this study provides unprecedented three-dimensional images of the complex intracellular lifestyle of C. burnetii. This imaging technology also provides unique insights into the biphasic developmental cycle and will be a powerful approach to dissect CCV biogenesis defects displayed by mutant C. burnetii.

microbiology↗

bioRxiv2025

Hydrogen-bonded organic framework nanotransducers enabled sono-optogenetics for Parkinsonian rats

Cell-type-specific activation of parvalbumin (PV)-expressing neurons in the external globus pallidus (GPe) through optogenetics has shown promise in facilitating long-lasting movement dysfunction recovery in mice with Parkinsons disease. However, its translational potential is hindered by adverse effects stemming from the invasive implantation of optical fibers into the brain. In this study, we have developed a non-invasive optogenetics approach, utilizing focused ultrasound-triggered mechanoluminescent nanotransducers to enable remote photon delivery deep in the brain for genetically targeted neuromodulation. These mechanoluminescent nanotransducers consist of sonosensitized hydrogen-bonded frameworks and chemiluminescent L012, serving as a nanoscale light source through ultrasound-induced cascade reactions. This system offers high ultrasound-triggered brightness and long-lasting light emission, facilitating repeatable deep brain stimulation. Our sono-optogenetics technology demonstrated effective modulation in the mouse motor cortex for limb motion control and activation of PV-GPe neurons for rescuing movement dysfunction over time in dopamine-depleted Parkinsons disease rats. This approach demonstrates the pathway for achieving genetically targeted and non-invasive neuromodulation for long-lasting treatment of Parkinsons disease, towards non-human primate models and clinical applications.

bioengineering↗

bioRxiv2025

Ether Rhodamines with Enhanced Hydrophilicity, Fluorogenicity, and Brightness for Super-Resolution Imaging

Rhodamine dyes are widely used fluorophores in super-resolution fluorescence imaging due to their exceptional optical properties and "aggregation-disaggregation" induced fluorogenic activation. However, their excessive lipophilicity often reduces brightness in aqueous environments and causes off-target staining, limiting their effectiveness in high-resolution imaging. To address these challenges, we introduce an ether-decorated N-terminal modification strategy for rhodamine and silicon-rhodamine (Si-rhodamine), replacing conventional N-alkyl groups. The ether chains enhance water solubility, decrease aggregate size, and improve fluorogenicity across a wide concentration range. Their flexible, hydrophilic structure forms a protective shield around the xanthene core, minimizing dye-water interactions and reducing fluorescence quenching. Additionally, the inductive effect of the ether chains decreases the electron-donating strength of the amino groups, suppressing quenching caused by twisted intramolecular charge transfer (TICT). These modifications collectively increase the quantum yields of ER and ESiR in water from 0.35 and 0.19 (for tetraethyl-substituted analogs) to 0.70 and 0.41, respectively. Probes derived from ER and ESiR exhibit outstanding fluorogenicity, enhanced signal-to-noise ratios, and improved resolution in complex aqueous environments, demonstrating superior performance in advanced super-resolution imaging techniques such as structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single-molecule localization microscopy (SMLM). This work introduces an innovative strategy for fluorophore design, offering significant advancements for super-resolution imaging applications.

biochemistry↗