Biology subjects

Guo, J.

Publications and source records attributed to Guo, J..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Prior epigenetic status predicts future susceptibility to seizures in mice

Wide variation of responses to identical stimuli presented to genetically inbred mice suggests the hypothesis that stochastic epigenetic variation during neurodevelopment can mediate such phenotypic differences. However , this hypothesis is largely untested since capturing pre-existing molecular states requires non-destructive, longitudinal recording. Therefore, we tested the potential of Calling Cards (CC) to record transient neuronal enhancer activity during postnatal development, and thereby associate epigenetic variation with a subsequent phenotypic presentation - degree of seizure response to the pro-convulsant pentylenetetrazol. We show that recorded differences in epigenetics at 243 loci predict a severe vs. mild response, and that these are enriched near genes associated with human epilepsy. We also validated pharmacologically a seizure -modifying role for two novel genes, Htr1f and Let7c. This proof-of-principle supports using CC broadly to discover predisposition loci for other neuropsychiatric traits and behaviors. Finally, as, human disease is also influenced by non-inherited factors, similar epigenetic predispositions are possible in humans.

genomics↗

bioRxiv2025

New Insights into Microbial Nitrogen Utilization in the Rumen Enabled by Genome-Resolved Multi-Omics

Optimizing nitrogen (N) utilization in ruminant production systems holds both economic and environmental significance. However, traditional paradigms of N metabolism, derived primarily from well-studied model rumen bacteria, cannot fully capture the diverse and complex N metabolic dynamics within the rumen ecosystem. To address this gap, we utilized comparative genomics and genome-resolved multi-omics analyses using a curated set of microbial genomes to investigate N assimilation and regulation in rumen microbes. We discovered that canonical mechanism of ammonia assimilation and regulation, such as the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathways and its regulatory proteins, are absent in the genomes of many key and predominant rumen microbes, which likely utilize alternative pathways for ammonia assimilation. These findings challenge the applicability of E. coli-based N regulation models to rumen bacteria. We further linked polysaccharide utilization and ammonia assimilation across hundreds of rumen microbial species. Furthermore, we identified specific microbial species involved in ureolysis and denitrification, as well as phages carrying auxiliary metabolic genes that perform N assimilation. Using an animal trial involving 11 pairs of lamb twins in a crossover design, we demonstrated that dietary crude protein (CP) concentrations had minimal impact on rumen microbiome composition and expression of N assimilation genes. Instead, shifts in concentrate levels triggered alterations in N assimilation, including increased expression of amino acid biosynthesis pathways. These findings indicate a nuanced, species-specific microbial response to dietary interventions, highlighting the limitations of traditional N metabolism models applied to rumen microbes and the need for more granular studies of rumen microbial ecosystems.

microbiology↗

bioRxiv2025

Commensal-derived Trehalose Monocorynomycolate Triggers γδ T Cell-driven Protective Ocular Barrier Immunity

Commensals shape host physiology through molecular crosstalk with host receptors. Identifying specific microbial factors that causally influence host immunity is key to understanding homeostasis at the host-microbe interface and advancing microbial-based therapeutics. Here, we identify trehalose monocorynomycolate (TMCM) from Corynebacterium mastitidis (C. mast) as a potent stimulator of IL-17 production by {gamma}{delta} T cells at the ocular surface. Mechanistically, TMCM-driven IL-17 responses require both IL-1 signals and {gamma}{delta} TCR signaling, which also supports endogenous {gamma}{delta} T cell IL-1R1 expression. Notably, synthetic TMCM alone is sufficient to mimic the effect of C. mast in inducing {gamma}{delta} T cell immunity and protect against pathogenic corneal infection. Our findings establish TMCM as a key mediator of commensal-driven immune defense, highlighting its potential as a {gamma}{delta} T cell adjuvant and a microbiome-informed therapeutic to enhance IL-17-driven protection at barrier sites such as the ocular surface. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/643820v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@63139forg.highwire.dtl.DTLVardef@9d6978org.highwire.dtl.DTLVardef@775e37org.highwire.dtl.DTLVardef@ca84b6_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LICorynomycolates enable ocular C. mast colonization and protective IL-17 immunity C_LIO_LITMCM drives IL-17 from {gamma}{delta} T cells through TCR and IL-1R signaling C_LIO_LI{gamma}{delta} TCR signaling maintains the expression of endogenous IL-1R1 C_LIO_LISynthetic TMCM mimics the ability of C. mast to induce {gamma}{delta} T cell immunity in the eye C_LIO_LITMCM protects against P. aeruginosa keratitis, highlighting its therapeutic potential C_LI

immunology↗

bioRxiv2025

Retrieval practice prevents stress-induced inference impairment by restoring rapid memory reactivation

A hallmark of human memory is its ability to form novel inferences by linking discrete but related events. We examined whether acute stress impairs memory inference and if retrieval practice can buffer this effect. Participants were trained on image pairs, AB and BC, to establish interconnected triads (ABC) with a shared bridge element B. Twenty-four hours later, we induced acute stress in half of the participants and then tested their capacity to infer the indirect AC associations. Behavioral results indicated that acute stress-induced memory inference accuracy and speed, and retrieval practice was associated with attenuation of stress-induced impairment in A-C inference accuracy and speed. Using multivariate decoding analysis of human electroencephalogram (EEG) recordings, we found neural evidence that bridge element B is rapidly reactivated during the inferential process, a neural signature predictive of subsequent successful inference. Importantly, stress disrupts this rapid neural reactivation of the bridge element, but retrieval practice buffers the stress effect and enhances the strength of reactivation signals beyond the non-stress condition. Time-frequency analyses of theta oscillations mirrored these effects of stress and retrieval practice on inference performance and neural reactivation. Collectively, our findings pinpoint rapid reactivation of the bridge element as an essential neural mechanism for memory-based inference. Although susceptible to stress, this mechanism can be enhanced through retrieval practice, suggesting that building robust memory traces renders subsequent inferences resilient to stress.

neuroscience↗

bioRxiv2025

Developing Foundation Models for Predicting Viral Animal Host Range in Intelligent Surveillance

Emerging human infectious viruses originating from animals continue to pose a persistent threat to global public health. Understanding the host range of animal viruses is crucial for identifying potential spillover pathways and mitigating the risk of future pandemics. Here, we present VirHRanger, a prediction method that integrates foundation models trained on viral genome and protein sequences, alongside genomic and protein compositional traits, viral phylogeny, and protein-protein interactions. To systematically predict the animal host range, VirHRanger incorporates host taxonomy-aware neural networks trained on a comprehensive collection of animal-virus associations spanning mammals, birds, and arthropods. Within a dataset of 4,006 virus species spanning 99 viral families, our model achieved robust performance with a micro-averaged AUROC of 0.938 across all host categories, demonstrating its effectiveness in capturing generalizable host signals from viral genetic data. On a dataset of 315 novel viruses, which are associated with key reservoir animal hosts and insect vectors, VirHRanger notably outperformed the homology-based method, exhibiting a strong generalizability to novel viruses. Furthermore, VirHRanger identified host range variations among closely related viruses within the Coronaviridae family and successfully predicted the ability of SARS-CoV-2 to infect humans and other animal hosts. These findings highlight the potential of VirHRanger to transform sequencing data into timely insights for disease control during the early stages of zoonotic outbreaks.

bioinformatics↗

bioRxiv2025

Mobile genetic elements that shape microbial diversity and functions inthawing permafrost soils

The worlds ecosystems are shaped by microbiota. Their niches and their impacts depend on functional profiles influenced by gene gains and losses. While culture-based experiments demonstrate that mobile genetic elements (MGEs) can mediate gene flux, quantitative field data on the rates and impacts of MGE activity remains scarce. Here we leverage large-scale soil meta-omic data to develop and apply analytics for studying MGEs in complex natural systems. In our model permafrost-thaw ecosystem, Stordalen Mire, we identify [~]2.1 million MGE recombinases across 89 microbial phyla to assess ecological distributions, affected functions, past mobility, and current activity. This revealed MGEs shaping natural genetic diversity via differential impacts on major phyla; affecting a wide range of functions, including diverse regulatory and metabolic genes affecting carbon flux and nutrient cycling; and moving at rates that should significantly influence the realized functional profiles of natural microbiomes. These findings and this systematic meta-omic framework open new avenues to better investigate MGE diversity, activity, mobility, and impacts in nature.

microbiology↗

bioRxiv2025

RNA virus ecogenomics along a subarctic permafrost thaw gradient

Climate change thaws permafrost, which releases greenhouse gases partly from dormant microorganisms awakening and metabolizing organic matter. Though DNA viruses that infect these soil microbes have been studied, little is known on soil RNA viruses, which typically infect microeukaryotes. Here we identify and characterize 2,651 RNA viruses from a 4-year time series of bulk soil metatranscriptomes derived from the climatically fragile Stordalen Mire ecosystem --a long studied permafrost peatland. RNA virus diversity was structured by habitat (palsa, bog, and fen), and these patterns correlated with pH and carbon dioxide and methane emissions. Further, host prediction, virus-encoded metabolite-transforming and information-processing functions suggested roles in ecosystem-scale carbon fluxes and contribute to greenhouse gases emissions. Together, these RNA virus ecogenomic data in permafrost provide essential baseline information for integration into predictive models to support hypothesis testing.

microbiology↗

bioRxiv2025

SMTdb: A comprehensive spatial meta-transcriptome resource in cancer

Microbes have been found in various tumors and the research of tumor microbiome has been garnering increased attention. However, it remains challenge to investigate the microbiome in cancer at spatial resolution. The rapid advent of spatially resolved transcriptomics techniques has given rise to map transcripts at single-cell resolution in various types of cancer. Here, we constructed a comprehensive spatial meta-transcriptome resource by manually curating 203 fresh frozen (FF) slices from 20 cancers encompassing 334,253 spots and 1,908,646 cells. SMTdb (http://bio-bigdata.hrbmu.edu.cn/SMTdb/) was constructed to provide comprehensive insights into the abundance, distribution and enriched TME regions of 1218 microbiota in spatial tissue slices. SMTdb enables vast interactive data exploration of spatial distribution and expression of microbiota, host gene modules associated with certain microbiota and co-occurrence between microbiota and immune cells within tumor microenvironment. The atlas resource serves as a one-stop and time-effective platform to investigate the interactions among microbial ecosystems and hosts in cancer.

bioinformatics↗

bioRxiv2025

Predicting the structures of cyclic peptides containing unnatural amino acids by HighFold2

Cyclic peptides containing unnatural amino acids possess many excellent properties and have become promising candidates in drug discovery. Therefore, accurately predicting the three-dimensional structures of cyclic peptides containing unnatural residues will significantly advance the development of cyclic peptide-based therapeutics. Although deep learning-based structural prediction models have made tremendous progress, these models still cannot predict the structures of cyclic peptides containing unnatural amino acids. To address this gap, we introduce a novel model, HighFold2, built upon the AlphaFold-Multimer framework. HighFold2 first extends the pre-defined rigid groups and their initial atomic coordinates from natural amino acids to unnatural amino acids, thus enabling structural prediction for these residues. Then, it incorporates an additional neural network to characterize the atom-level features of peptides, allowing for multi-scale modeling of peptide molecules while enabling the distinction between various unnatural amino acids. Besides, HighFold2 constructs a relative position encoding matrix for cyclic peptides based on different cyclization constraints. Except for training using spatial structures with unnatural amino acids, HighFold2 also parameterizes the unnatural amino acids to relax the predicted structure by energy minimization for clash elimination. Extensive empirical experiments demonstrate that HighFold2 can accurately predict the three-dimensional structures of cyclic peptide monomers containing unnatural amino acids and their complexes with proteins, with the median RMSD for C reaching 1.891 [A]. All these results indicate the effectiveness of HighFold2, representing a significant advancement in cyclic peptide-based drug discovery.

bioinformatics↗

bioRxiv2025

Multi-Region Brain Organoid: Fusion Organoid with Cerebral, Endothelial and Mid-Hindbrain Components

Brain organoid technology has revolutionized our ability to model human neurodevelopment in vitro. However, current techniques remain limited by their reliance on simplified endothelial cell populations rather than a complete endothelial system. We engineered Multi-Region Brain Organoids (MRBOs) that integrate cerebral, mid/hindbrain, and complex endothelial organoids into one structure. Different from the earlier approaches based on isolated HUVECs, our endothelial organoids contain diverse vascular cell types, including vascular progenitors, mature endothelial cells, pericytes, proliferating angiogenic cells and stromal cells. Our strategy employs a sequential modulation of key developmental pathways to generate individual organoids, followed by optimized fusion conditions that maintain regional identities while supporting cellular integration. Single-nucleus RNA sequencing shows that MRBOs develop discrete neural populations specific to each brain region alongside specialized endothelial populations that establish paracrine signalling networks. Integration analysis with human fetal brain data shows that MRBOs contribute to 80% of cellular clusters found in human fetal brain tissue (Carnegie stages 12-16), whereas CellChat analysis identifies 13 previously uncharacterized endothelial-neural signalling interactions. Notably, we uncover endothelial-derived factors that support the persistence of intermediate progenitor populations during hindbrain development, but not during the cerebral one thereby revealing a new role for complex endothelial populations in regional brain patterning. This platform enables matching multiple developmental regions, fully incorporating the endothelial nature at the same time providing opportunities for studying neurodevelopmental disorders in which neural-endothelial interactions are disrupted. Our engineered MRBO system establishes a foundation for investigating complex neurodevelopmental processes, providing an enabling context closer to physiological relevance.

bioengineering↗

bioRxiv2025

Ancient gene clusters initiate monoterpene indole alkaloid biosynthesis and C-3 stereochemistry inversion

The inversion of C3 stereochemistry in monoterpenoid indole alkaloids (MIAs), derived from the central precursor strictosidine (3S), is a critical step for the biosynthesis of numerous 3R MIAs and spirooxindoles, including the antihypertensive drug reserpine. While early MIA biosynthesis preserves the 3S configuration, the mechanism underlying C3 inversion has remained unresolved. Here, we identify and biochemically characterize a conserved oxidase-reductase pair in the Gentianales order: the heteroyohimbine/yohimbine/corynanthe C3-oxidase (HYC3O) and C3-reductase (HYC3R), which together invert the 3S stereochemistry to 3R across diverse substrates. Notably, HYC3O and HYC3R reside in gene clusters in Rauvolfia tetraphylla and Catharanthus roseus, homologous to an elusive geissoschizine synthase (GS) cluster we also uncovered. In R. tetraphylla, these clusters are in tandem on a single chromosome, likely derived from segmental duplication, whereas in C. roseus they reside on separate chromosomes due to translocation. Comparative genomics indicate the GS cluster originated at the base of Gentianales ([~]135 Mya), coinciding with the evolution of the strictosidine synthase cluster, while the reserpine cluster arose later in rauvolfioid Apocynaceae. Together, these findings uncover the genomic and biochemical basis for key events in MIA evolution and diversification, providing insights beyond the canonical vinblastine and ajmaline biosynthetic pathways.

biochemistry↗

bioRxiv2024

Neurodevelopmental deviations in schizophrenia: Evidences from multimodal connectome-based brain ages

BackgroundPathologic schizophrenia process originate early in brain development, leading to detectable brain alterations via structural and functional magnetic resonance imaging (MRI). Recent MRI studies have sought to characterize disease effects from a brain age perspective, but developmental deviations from the typical brain age trajectory in youths with schizophrenia remain unestablished. AimThis study investigated brain development deviations in early-onset schizophrenia (EOS) patients by applying machine learning algorithms to structural and functional MRI data. MethodsMultimodal MRI data, including T1-weighted MRI (T1w-MRI), diffusion MRI, and resting-state functional MRI (rs-fMRI) data, were collected from 80 antipsychotic-naive first-episode EOS patients and 91 typically developing (TD) controls. The morphometric similarity connectome (MSC), structural connectome (SC), and functional connectome (FC) were separately constructed by using these three modalities. According to these connectivity features, eight brain age estimation models were first trained with the TD group, the best of which was then used to predict brain ages in patients. Individual brain age gaps (BAGs) were assessed as brain ages minus chronological ages. ResultsBoth the SC and MSC features performed well in brain age estimation, whereas the FC features did not. Compared with the TD controls, the EOS patients presented widened structural BAGs, with opposite trends between childhood and adolescence. These increased absolute BAG scores for EOS patients were positively correlated with the severity of their clinical symptoms. ConclusionThese findings from a multimodal brain age perspective suggest that advanced BAGs exist early in youths with schizophrenia.

neuroscience↗

bioRxiv2024

Atypical Visual Selective Attention in Children with Dyslexia: Evidence from N2pc and PD

Efficient visual attention is fundamental to the development of reading abilities. Previous studies have identified visual attention impairments in individuals with dyslexia, particularly in visual attention span and sluggish attention shifting. However, findings regarding basic visual selective attention remain controversial. To address this issue, the present study provides event-related potential (ERP) evidence to verify whether children with developmental dyslexia (DD) suffered from deficits in visual selective attention. A pop-out visual search task was used to examine visual attentional patterns to a lateral target in 66 children (33 children with DD and 33 typically developing (TD) children) using electroencephalography. Compared to TD children, children with DD showed a larger and prolonged P1 component, as well as a larger target-evoked N2pc component. The larger P1 amplitude was associated with lower reading fluency while the larger N2pc amplitude was associated with poor reading comprehension performance. Interestingly, the target-elicited N2pc was followed by positivity (PD component) in TD children but not in children with DD; however, the N2pc amplitude was correlated with the PD amplitude in children with DD. Children with DD show immature early attentional processing and may have an imbalance in the regulation of visual selective attention between attentional selection and suppression. Our findings provide ERP evidence for interpreting the underlying neural mechanism of visual selective attention in children with DD and may offer a new perspective for exploring the mechanism of visual attention deficits in dyslexia. Research HighlightsO_LIIn a pop-out visual search task, children with dyslexia exhibited a larger N2pc and an absent PD component. C_LIO_LIThe absence of the PD component in children with dyslexia may be associated with their larger N2pc amplitudes. C_LIO_LIN2pc amplitude is associated with reading ability in children with dyslexia. C_LIO_LIChildren with dyslexia need to allocate more resources towards the target but also struggle to disengage from the attended location. C_LI

neuroscience↗

bioRxiv2024

Deciphering the thiolactonization mechanism in thiolactomycin biosynthesis

Thiolactomycin (1), which features a unique{gamma} -thiolactone ring, is a promising antibiotic candidate that specifically targets bacterial type II fatty acid synthase. Despite extensive studies on its pharmacological activities, modes of action, and chemical synthesis, the enzymatic processes responsible for forming the activity-determining{gamma} -thiolactone ring have remained largely unknown. Here, we resolve this problem by revealing that the condensation and heterocyclization (Cy) domain of the nonribosomal peptide synthetase (NRPS) TlnC (TlnCCy), along with the cytochrome P450 enzyme TlnA, cooperatively enable the{gamma} -thiolactone assembly. TlnCCy mediates an unusual sulfurtransfer reaction to sulfurate the polyketide intermediate, generating a thiocarboxylate intermediate. Subsequently, TlnA acts as a{gamma} -thiolactone synthase, converting the linear thiocarboxylate intermediate into 1 via a distal radical-based cyclization mechanism. These findings not only expand the functional and catalytic repertoires of NRPS Cy domains and P450 enzymes, but also highlight a special enzymatic strategy for{gamma} -thiolactone biosynthesis in nature. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/629141v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@7e0ad2org.highwire.dtl.DTLVardef@f86b80org.highwire.dtl.DTLVardef@159d136org.highwire.dtl.DTLVardef@1b633ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Stereochemical Insights into Sarpagan and Akuammiline Alkaloid Biosynthesis

The Apocynaceae family produces a diverse array of monoterpenoid indole alkaloids (MIAs) with significant pharmaceutical value. Among these, sarpagan and akuammiline alkaloids stand out for their complex stereochemistry, derived from the enzymatic cyclization and rearrangement of geissoschizine. This study investigates the stereochemical outcomes of sarpagan bridge enzymes (SBEs) and rhazimal synthases (RHS), key players in geissoschizine cyclization and MIA diversification. Using two known and five newly identified enzymes from six plant species, we show that RHS enzymes from Alstonia scholaris, Vinca minor, and Amsonia tabernaemontana exclusively produce the 16R rhazimal stereoisomer. Meanwhile, SBEs from Catharanthus roseus, Tabernaemontana elegans, Vinca minor, and Rauvolfia serpentina likely generate 16R polyneuridine aldehyde; however, downstream aldehyde reductase, deformylase, and esterase activities further epimerize and alter the C16 stereochemistry, yielding naturally occurring alkaloids with distinct C16 stereochemistry across species. These findings, supported by in vitro assays and in planta silencing of C. roseus CrSBE after we reroute biosynthetic flux toward mutated sarpagan MIAs, further reveal enzymatic control over C16 stereochemistry in sarpagan MIA biosynthesis. By elucidating the transformation of diastereomeric intermediates, this work provides key insights into the stereochemical and enzymatic diversification of MIAs in nature.

biochemistry↗

bioRxiv2024

Multimodal single-cell analyses reveal molecular markers of neuronal senescence in human drug-resistant epilepsy

The histopathological neurons in the brain tissue of drug-resistant epilepsy exhibit aberrant cytoarchitecture and imbalanced synaptic circuit function. However, the gene expression changes of these neurons remain unknown, making it difficult for the diagnosis or to dissect the mechanism of drug-resistant epilepsy. By integrating whole-cell patch clamp recording and single-cell RNA sequencing approaches, we identified a transcriptionally distinct subset of cortical pyramidal neurons. These neurons highly expressed genes CDKN1A (P21), CCL2 and NFKBIA that associated with mTOR pathway, inflammatory response and cellular senescence. We confirmed the expression of senescent marker genes in a subpopulation of cortical pyramidal neurons with enlarged soma size in the brain tissue of drug-resistant epilepsy. We further revealed the expression of senescent cell markers P21, P53, COX2, {gamma}-H2AX, {beta}-Gal and reduction of nuclear integrity marker Lamin B1 in histopathological neurons in the brain tissue of drug-resistant epilepsy patients with different pathologies, but not in control brain tissue with no history of epilepsy. Additionally, chronic, but not acute, epileptic seizures induced senescent markers expression in cortical neurons in mouse models of drug-resistant epilepsy. These results provide important molecular markers for histopathological neurons and new insights into the pathophysiological mechanisms of drug-resistant epilepsy.

neuroscience↗

bioRxiv2024

Reduced expression of PER2 protein contrbutes to β1-AA induced cardiac autophagy rhythm disorder

It has been confirmed that heart failure may be linked to fluctuations in autophagy rhythm of cardiomyocytes throughout the day. It is known that circadian rhythms depend on the regulation of core biological clock proteins, with PER2 playing a crucial role. Our previous research has confirmed the presence of {beta}1-Adrenergic receptor autoantibodies ({beta}1-AA) could induce inhibition of myocardial autophagy, leading to cell death and heart failure. However, it remains unclear whether {beta}1-AA induces cardiac autophagy rhythm disorder by affecting PER2 expression. This study find that {beta}1-AA disrupts the autophagy rhythm in cardiomyocytes, primarily indicates by the decreased expression of the autophagy marker protein LC3; {beta}1-AA induces disruption of the rhythmic expression of PER2 protein in myocardial cells, mainly manifests by a decrease in PER2 protein expression; Metoprolol is employed to verify that the {beta}1-adrenergic receptor contributes to the reduction of Per2 protein caused by {beta}1-AA. Knocking down Per2 with lentivirus reduces the inhibition of LC3 expression caused by {beta}1-AA, while overexpressing Per2 in cardiomyocytes using lentivirus significantly restores {beta}1-AA-induced decline in LC3 expression. At the same time, mTORC1 activation is found to participate in {beta}1-AA-induced autophagy inhibition of cardiomyocytes after pretreatment with the mTORC1 inhibitor rapamycin. Furthermore, it is confirmed that the decreased expression of PER2 protein caused by {beta}1-AA disrupts the myocardial autophagy rhythm by promoting mTORC1 activation through lentiviruses that knock down or overexpress the Per2 gene. This study provides experimental basis for the precision treatment of cardiovascular diseases from the perspective of biological rhythm.

molecular biology↗

bioRxiv2024

Fast Photostable Expansion Microscopy Using QDots andDeconvolution

Expansion microscopy (ExM) enables sub-diffraction imaging by physically expanding labeled tissue samples. This increases the tissue volume relative to the instrument point spread function (PSF), thereby improving the effective resolution by reported factors of 4 - 20X [1, 2]. However, this volume increase dilutes the fluorescence signal, reducing both signal-to noise ratio (SNR) and acquisition speed. This paper proposes and validates a method for mitigating these challenges. We overcame the limitations of ExM by developing a fast photo-stable protocol to enable scalable widefield three-dimensional imaging with ExM. We combined widefield imaging with quantum dots (QDots). Widefield imaging provides a significantly faster acquisition of a single field-of-view (FOV). However, the uncontrolled incoherent illumination induces photobleaching. We mitigated this challenge using QDots, which exhibit a long fluorescence lifetime and improved photostability. First, we developed a protocol for QDot labeling. Next, we utilized widefield imaging to obtain 3D image stacks and applied deconvolution, which is feasible due to reduced scattering in ExM samples. We show that tissue clearing, which is a side-effect of ExM, enables widefield deconvolution, dramatically reducing the acquisition time for three-dimensional images compared to laser scanning microscopy. The proposed QDot labeling protocol is compatible with ExM and provides enhanced photostability compared to traditional fluorescent dyes. Widefield imaging significantly improves SNR and acquisition speed compared to conventional confocal microscopy. Combining widefield imaging with QDot labeling and deconvolution has the potential to be applied to ExM for faster imaging of large three-dimensional samples with improved SNR.

neuroscience↗