Biology subjects

Guo, C.

Publications and source records attributed to Guo, C..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

In vivo proximity proteomics uncovers palmdelphin (PALMD) as a Z-line-associated mitigator of isoproterenol-induced cardiac injury

Z-lines are core ultrastructural organizers of cardiomyocytes that modulate many facets of cardiac pathogenesis. Yet a comprehensive proteomic atlas of Z-line-associated components remain incomplete. Here, we established an adeno-associated virus (AAV)-delivered, cardiomyocyte-specific, proximity-labeling approach to characterize the Z-line proteome in vivo. We found palmdelphin (PALMD) as a novel Z-line-associated protein in both adult murine cardiomyocytes and human pluripotent stem cell-derived cardiomyocytes. Germline and cardiomyocyte-specific palmd knockout mice were grossly normal at baseline but exhibited compromised cardiac hypertrophy and aggravated cardiac injury upon long-term isoproterenol treatment. By contrast, cardiomyocyte-specific PALMD overexpression was sufficient to mitigate isoproterenol-induced cardiac injury. PALMD ablation perturbed transverse tubules (T-tubules) and their association with sarcoplasmic reticulum, which formed the Z-line-associated junctional membrane complex (JMC) essential for calcium handling and cardiac function. These phenotypes were associated with disrupted localization of T-tubule markers caveolin-3 (CAV3) and junctophilin-2 (JPH2) and the reduction of nexilin (NEXN) protein, a crucial Z-line-associated protein that is essential for both Z-line and JMC structures and functions. PALMD was found to interact with NEXN and enhance its protein stability while the Nexn mRNA level was not affected. Together, this study discovered PALMD as a potential target for myocardial protection and highlighted in vivo proximity proteomics as a powerful approach to nominate novel players regulating cardiac pathogenesis. HighlightsO_LIIn vivo proximity proteomics uncover novel Z-line components that are undetected in in vitro proximity proteomics in cardiomyocytes. C_LIO_LIPALMD is a novel Z-line-associated protein that is dispensable for baseline cardiomyocyte function in vivo. C_LIO_LIPALMD mitigates cardiac dysfunction and myocardial injury after repeated isoproterenol insults. C_LIO_LIPALMD stabilizes NEXN, an essential Z-line-associated regulator of the junctional membrane complex and cardiac systolic function. C_LI

molecular biology↗

bioRxiv2023

Comparative analysis of methodologies for detecting extrachromosomal circular DNA

Extrachromosomal circular DNA (eccDNA) is crucial in oncogene amplification, gene transcription regulation, and intratumor heterogeneity. While various analysis pipelines and experimental methods have been developed for eccDNA identification, their detection efficiencies have not been systematically assessed. To address this, we evaluated the performance of 7 analysis pipelines using three simulated datasets, in terms of accuracy, similarity, duplication rate, and computational resource consumption. We also compared the eccDNA detection efficiency of 7 experimental methods through twenty-one real sequencing datasets. Our results identified Circle-Map and CReSIL as the most effective pipelines for eccDNA detection through short-read and long-read sequencing data, respectively. Moreover, third-generation sequencing-based Circle-Seq showed superior efficiency in detecting copy number-amplified eccDNA over 10 kb in length. These results offer valuable insights for researchers in choosing the most suitable methods for eccDNA research.

cancer biology↗

bioRxiv2023

Single-cell transcriptomic analysis of immune cell dynamics in the healthy human endometrium

The microenvironment of the endometrial immune system is crucial to the success of placental implantation and healthy pregnancy. However, the functionalities of immune cells across various stages of the reproductive cycle have yet to be fully comprehended. To address this, we conducted advanced bioinformatic analyses on 230,049 high-quality single-cell transcriptomes from healthy endometrial samples obtained during the proliferative, secretory, early pregnancy, and late pregnancy stages. Our investigation revealed that proliferative natural killer (NK) cells, a potential source of endometrial NK cells, exhibit the most robust proliferative and differentiation potential during non-pregnant stages. During early pregnancy, NK cells display high oxidative phosphorylation metabolism activity, and together with macrophages and T cells, exhibit a strong type II interferon response. Based on our cell-cell interaction analyses, we identify a large majority of interaction pairs to occur in late pregnancy. Finally, we explored the correlation between stage-specific alterations in transcriptomics and the risk genes of common reproductive diseases, unveiling that MHC class I/II molecules, along with TGFBR1, exhibited the potential to serve as biomarkers. Our study provides insights into the dynamics of the endometrial immune microenvironment during different reproductive cycle stages, thus serving as a reference for detecting pathological changes during pregnancy.

bioinformatics↗

bioRxiv2023

Adaptive immunity: from CRISPR to CRIHSP?

Clustered regularly interspaced short palindromic repeats (CRISPR) confer adaptive immunity in prokaryotes against viral infection and plasmid transformation. Here, we identified abundant clustered regularly interspaced homologous stem-loop pairs (CRIHSP) within human adaptive immunity-related genes, such as antigen receptor germline genes. By examining genomic stability under activation-induced cytidine deaminase (AID) overexpression, we found that CRIHSP structures are preferred targets for AID. Through collecting and analyzing cancer cell genomic mutation databases, we discovered that these mutation sites tend to cluster toward stem-loop structures. Importantly, the frequency of stem-loop sequences within immunoglobulin heavy chain variable (IGHV) gene segments was over 3-fold higher compared to that within other regions. We concurrently observed stem-loop structures frequently flanking RSS motifs. Conservative estimates indicate a 74.07% probability of immunoglobulin heavy chain diversity (IGHD) gene segment enclosure within stem-loop. These RSS-proximal stem-loops may assemble into homologous stem-loop pair (HSP)-like intermediates and subsequent CRIHSP arrays. Our results implicate CRIHSP architectures as critical enhancer of antigen receptor gene recombination and diversification. Further molecular dissection of these processes could potentially elucidate mechanisms underlying cancer cell genomic instability, chromatin folding and dynamic regulation, gene expression control, etc.

immunology↗

bioRxiv2023

Divide-and-conquer quantum algorithm for hybrid de novo genome assembly of short and long reads

Computational biology holds immense promise as a domain that can leverage quantum advantages due to its involvement in a wide range of challenging computational tasks. Researchers have recently explored the applications of quantum computing in genome assembly implementation. However, the issue of repetitive sequences remains unresolved. In this paper, we propose a hybrid assembly quantum algorithm using high-accuracy short reads and error-prone long reads to deal with sequencing errors and repetitive sequences. The proposed algorithm builds upon the variational quantum eigensolver and utilizes divide-and-conquer strategies to approximate the ground state of larger Hamiltonian while conserving quantum resources. Using simulations of 10-qubit quantum computers, we address problems as large as 140 qubits, yielding optimal assembly results. The convergence speed is significantly improved via the problem-inspired ansatz based on the known information about the assembly problem. Besides, entanglement within quantum circuits is qualitatively verified to notably accelerate the assembly path optimization.

bioinformatics↗

bioRxiv2023

A monoclonal antibody collection for C. difficile typing

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in adults. Various C. difficile strains circulate currently, associated with different outcomes and antibiotic resistance profiles. However, most studies still focus on the reference strain 630 that does not circulate anymore, partly due to the lack of immunological tools to study current clinically important C. difficile PCR ribotypes. Herein, we immunized mice expressing human variable antibody genes with the Low Molecular Weight (LMW) subunit of the surface layer protein SlpA from various C. difficile strains. Monoclonal antibodies purified from hybridomas bound LMW with high-affinity and whole bacteria from current C. difficile ribotypes with different cross-specificities. This first collection of anti-C. difficile mAbs represent valuable tools for basic and clinical research.

immunology↗

bioRxiv2023

Anti-S-layer monoclonal antibodies impact Clostridioides difficile physiology

Clostridioides difficile (C. difficile), a gram-positive anaerobic and spore-forming bacterium, is the leading cause of nosocomial antibiotic-associated diarrhea in adults and is characterized by high levels of recurrence and mortality. Surface-layer Protein A (SlpA), the most expressed protein on bacterial surface, plays a crucial role in the early stages of infection although its role in C. difficile physiology is yet to be fully understood. Anti-S-layer antibodies have been identified in the sera of convalescent patients and correlate with improved outcome of C. difficile infection (CDI). However, the precise mechanisms of how anti-S-layer antibodies can confer protection to the host remain unknown. In this study, we report the first monoclonal antibodies (mAbs) targeting S-layer of the reference strain 630. Characterization of these mAbs unravels important roles for S-layer protein in growth, toxin secretion, and biofilm formation with, surprisingly, opposite effects of different anti-SlpA mAbs on these functions. One anti-SlpA mAb impaired C. difficile growth and restored sensitivity to lysozyme-induced lysis. These findings suggest that anti-S-layer antibody responses may include protective and detrimental effects for the host and provide important insights for designing adequate S-layer-targeting therapeutics.

microbiology↗

bioRxiv2023

Magnetic voluntary head-fixation in transgenic rats enables lifetime imaging of hippocampal neurons

The precise neural mechanisms within the brain that contribute to the remarkable lifetime persistence of memory remain unknown. Existing techniques to record neurons in animals are either unsuitable for longitudinal recording from the same cells or make it difficult for animals to express their full naturalistic behavioral repertoire. We present a magnetic voluntary head-fixation system that provides stable optical access to the brain during complex behavior. Compared to previous systems that used mechanical restraint, there are no moving parts and animals can engage and disengage entirely at will. This system is failsafe, easy for animals to use and reliable enough to allow long-term experiments to be routinely performed. Together with a novel two-photon fluorescence collection scheme that increases two-photon signal and a transgenic rat line that stably expresses the calcium sensor GCaMP6f in dorsal CA1, we are able to track and record activity from the same hippocampal neurons, during behavior, over a large fraction of animals lives.

neuroscience↗

bioRxiv2023

Large-scale population genomics of Malayan pangolins reveals deep diversification and a new species

BackgroundArchipelagos and oceanic islands often present high percentage of endemism due to rapid speciation. The Malayan pangolin is a species distributing at both mainland (southern Yunnan, China) and oceanic islands via Malayan peninsula, which may result in deep differentiation among populations. In-depth investigation of population structure and genetic consequences for such species is of vital importance for their protection and conservation, practically for the critically endangered Malayan pangolin that is suffering from poaching, illegal trade, and habitat loss. ResultsHere we carried out a large-scale population genomic analysis for Malayan pangolins, and revealed three highly distinct genetic populations in this species, two of which are now being reported for the first time. Based on multiple lines of genomic and morphological evidence, we postulate the existence of a new pangolin species (Manis_1). Genetic diversity and recent inbreeding were both at a moderate level for both Malayan pangolins and Manis_1, but mainland Malayan pangolins presented relatively lower genetic diversity, higher inbreeding and fitness cost than island populations. ConclusionsWe found extremely deep and graded differentiation in Malayan pangolins, with two newly discovered genetic populations and a new pangolin species that is closely related to the Philippine pangolin than the typical Malayan pangolin, but a distant relative of the Indian pangolin. Anthropogenic factors did not significantly weaken the basis of genetic sustainability for Malayan pangolins, but mainland Malayan pangolins should be paid more attention for conservation due to higher genetic risks than island populations.

genomics↗

bioRxiv2023

Dietary n-3 PUFA enhances DMI in transition cows by regulating taste transduction gene expression in liver associated with rumen microbial alteration

We hypothesised that the addition of n-3 polyunsaturated fatty acids (PUFAs) in the diet could affect gene expression in the liver and have beneficial effects on the recovery of cows in the transition phase. A total of 30 multiparous non-lactating Holstein dairy cows (35 days before expected calving) were randomly fed a diet with either 1% dry matter (DM) of hydrogenated fatty acid (C16:00 enriched; CON) or 3.5% DM of extruding flaxseed (n-3 enriched; HN3). Parity, body weight (BW), body condition score (BCS) and milk yield were 2.6 {+/-} 1.2, 757.5 {+/-} 65.8 kg, 3.3 {+/-} 0.2 and 10,286.5 {+/-} 1464.8 kg/d (mean {+/-} SD), respectively, at the beginning of the experiment. The relative abundance of Bacteroidota (P = 0.047) and Spirochaetota (P = 0.091) was higher and that of Patescibacteria (P = 0.076) was lower in the HN3 group than in the CON group on prepartum day 4. The DMI of cows was positively correlated with the abundance of bacteria in the rumen (Spirochaetota: r = 0.871, P < 0.001; Bacteroidota: r = 0.896, P < 0.001) and the differential expression of genes involved in taste transduction (ACSL1: r = 0.673, P < 0.001; PLIN4: r = 0.632, P < 0.01; CPT1A: r = 0.694, P < 0.001). These results suggest that dietary n-3 PUFA at an appropriate concentration can promote DMI recovery by upregulating the expression of these genes and maintaining the balance of the rumen microbiota.

zoology↗

bioRxiv2023

Non-cell autonomous cardiomyocyte regulation complicates gene supplementation therapy for LMNA cardiomyopathy

AimsRecombinant adeno-associated viruses (rAAVs) are federally approved gene delivery vectors for in vivo gene supplementation therapy. Loss-of-function truncating variants of LMNA, the coding gene for Lamin-A/C, are one of the primary causes of inherited dilate cardiomyopathy (DCM). Here we aim to study whether AAV-based LMNA supplementation could treat LMNA deficiency-triggered cardiac defects. Methods and ResultsWe compared whole-body, cardiomyocyte-specific and genetic-mosaic mouse models that carry Lmna truncating variants at the same genetic loci and uncovered primarily a non-cell autonomous impact of Lmna on cardiomyocyte maturation. Whole-body lamin-A supplementation by rAAVs moderately rescued the cardiac defects in Lmna germline mutants. By contrast, cardiomyocyte-specific lamin-A addback failed to restore the cardiomyocyte growth defects. A Cre-loxP-based AAV vector that expresses lamin-A throughout the body but excluding the heart was able to restore cardiomyocyte growth in Lmna germline mutants. ConclusionsLmna regulates cardiomyocyte growth non-cell autonomously. Non-myocytes are the key cell targets for a successful gene therapy for LMNA-associated cardiac defects. Translational perspectiveLMNA truncating mutations are among the major causes of inherited DCM. AAV gene supplementation therapy is emerging as a promising strategy to treat genetic cardiomyopathy, but whether this strategy is suitable for LMNA cardiomyopathy remained unclear. Our study counterintuitively showed that the cardiomyocytes are not necessarily the correct therapeutic cell targets for AAV-based treatment of LMNA cardiomyopathy. By contrast, careful elucidation of cell-autonomous versus non-cell-autonomous gene functions is essential for the proper design of a gene supplementation therapy for cardiomyopathy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/549413v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@37cfdaorg.highwire.dtl.DTLVardef@2f2cd1org.highwire.dtl.DTLVardef@1278d08org.highwire.dtl.DTLVardef@19c4869_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

MicroRNA-122-mediated liver detargeting enhances the tissue specificity of cardiac genome editing

BackgroundThe cardiac troponin T (Tnnt2) promoter is broadly utilized for cardiac specific gene expression, particularly via adeno-associated virus (AAV)-based gene transfer. However, these vectors drive lower-level ectopic gene expression in other tissues, most notably in the liver. Whether the AAV-Tnnt2 vectors remain tissue-specific in applications sensitive to low or transient gene expression, such as gene editing, remains unclear. MethodsThe tissue specificity of AAV9-Tnnt2 vectors was evaluated in mice using Cre-LoxP-based fluorescence reporters and CRISPR/Cas9-mediated somatic mutagenesis. CRISPR/Cas9-triggered AAV integration into host genome was further assessed by quantitative PCR. ResultsIn mice treated with AAV-Tnnt2-GFP, GFP signal was specifically observed in the heart by confocal imaging. However, when AAV-Tnnt2-Cre was administered to mice carrying LoxP-STOP-LoxP fluorescence reporters, the reporter signals were observed in up to 50% hepatic cells. Similarly, the AAV-Tnnt2-SaCas9 vector extensively edited the hepatic genome as measured by targeted amplicon-sequencing. Cas9-triggered AAV integration into the host genome was also validated in the liver. Inclusion of target sequences for microRNA-122, a highly expressed, liver-specific microRNA, in the AAV transgenes 3 untranslated region (3 UTR) markedly reduced ectopic transgene expression, genome editing and AAV integration in the liver. ConclusionsThe heavily used AAV-Tnnt2 system exhibits liver leakiness that severely impairs the cardiac specificity of AAV-based genetic manipulation. This problem can be mitigated via miR122-mediated liver detargeting.

physiology↗

bioRxiv2023

Syntenin and CD63 Promote Exosome Biogenesis from the Plasma Membrane by Blocking Cargo Endocytosis

Exosomes are small extracellular vesicles important in health and disease. Syntenin is thought to drive the biogenesis of CD63 exosomes by recruiting Alix and the ESCRT machinery to endosomes, initiating an endosome-mediated pathway of exosome biogenesis. Contrary to this model, we show here that syntenin drives the biogenesis of CD63 exosomes by blocking CD63 endocytosis, thereby allowing CD63 to accumulate at the plasma membrane, the primary site of exosome biogenesis. Consistent with these results, we find that inhibitors of endocytosis induce the exosomal secretion of CD63, that endocytosis inhibits the vesicular secretion of exosome cargo proteins, and that high-level expression of CD63 itself also inhibits endocytosis. These and other results indicate that exosomes bud primarily from the plasma membrane, that endocytosis inhibits their loading into exosomes, that syntenin and CD63 are expression-dependent regulators of exosome biogenesis, and that syntenin drives the biogenesis of CD63 exosomes even in Alix knockout cells.

cell biology↗

bioRxiv2023

SUMOylation of MFF is required for stress-induced mitochondrial fission

Mitochondrial fission regulates mitochondrial morphology, function, mitophagy and apoptosis. Fission is mediated by the GTPase dynamin related protein-1 (DRP1) and its recruitment to the outer mitochondrial membrane by DRP1 receptors. Mitochondrial fission factor (MFF) is considered the major pro-fission receptor, whereas the mitochondrial dynamics proteins (MiD49/51) sequester inactive DRP1 and facilitate the MFF-DRP1 interaction by forming a trimeric DRP1-MiD-MFF complex. Here, we identify MFF as a target of poly-SUMOylation at a single residue (Lys151). Following bioenergetic stress, AMPK phosphorylates MFF to promote its SUMOylation, a critical step in stress-induced fragmentation. MFF SUMOylation is not required for DRP1 recruitment from the cytosol but causes a rearrangement of the trimeric fission complex to displace MiD proteins. This alleviates MiD inhibition of DRP1 to facilitate formation of a fission-competent complex. Thus, our data demonstrate that MFF SUMOylation fine-tunes the ratio of MiD to DRP1 for the dynamic control of stress-induced mitochondrial fragmentation.

cell biology↗

bioRxiv2023

Contribution of the IGCR1 regulatory element and the 3'Igh CBEs to Regulation of Igh V(D)J Recombination

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJH-RC. Igh has a provocative number and organization of CTCF-binding-elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Down-regulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJH-RC-bound RAG to scan the VH domain and perform VH-to-DJH rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the "ordered" transition from D-to-JH to VH-to-DJH recombination, we tested effects of deleting or inverting IGCR1 or 3Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL down-regulation mechanism in progenitor B cells as opposed to a strict developmental switch. SIGNIFICANCE STATEMENTTo counteract diverse pathogens, vertebrates evolved adaptive immunity to generate diverse antibody repertoires through a B lymphocyte-specific somatic gene rearrangement process termed V(D)J recombination. Tight regulation of the V(D)J recombination process is vital to generating antibody diversity and preventing off-target activities that can predispose the oncogenic translocations. Recent studies have demonstrated V(D)J rearrangement is driven by cohesin-mediated chromatin loop extrusion, a process that establishes genomic loop domains by extruding chromatin, predominantly, between convergently-oriented CTCF looping factor-binding elements (CBEs). By deleting and inverting CBEs within a critical antibody heavy chain gene locus developmental control region and a loop extrusion chromatin-anchor at the downstream end of this locus, we reveal how these elements developmentally contribute to generation of diverse antibody repertoires.

immunology↗

bioRxiv2023

The "replacing surgery" of cpDNA: de novo chemical synthesis and in vivo functional testing of Chlamydomonas chloroplast genome

We have successfully designed and synthesized the 221,372-bp cpDNA SynCpV1.0 with the native cpDNA of Chlamydomonas reinhardtii as the template. Homoplasmic SynCpv1.0-harboring algal strains were obtained by biolistic transformation and selected with an ascending gradient of antibiotic pressure. Meanwhile, we were pleasantly surprised to find that SynCpV1.0 was able to re-introduce and replicate normally after the total DNA of transplastomic algal strains were transformed to Escherichia coli, it indicated that SynCpV1.0 was able to shuttle between C. reinhardtii and E. coli. Finally, we analyzed the photosynthetic properties of SynCpV1.0-harboring transplastomic strains, the results showed that they exhibited the same photosynthetic efficiency as the wild strain of C. reinhardtii CC125, and could rescue the photosynthetic defect in mutant strain of C. reinhardtii CC5168. Herein, we have performed the "replacing surgery" of cpDNA and established an ideal platform to complete multiple cycles of "Design-Build-Test" for optimizing the cpDNA of photosynthetic organisms. HighlightO_LIAn artificial cpDNA SynCpV1.0 is constructed by de novo chemical synthesis. C_LIO_LIThe "replacing surgery" of cpDNA was performed in the chloroplast of C. reinhardtii C_LIO_LIIt is found that artificial cpDNA was able to shuttle between Chlamydomonas chloroplast and E. coli. C_LIO_LIEstablish an ideal platform to complete multiple cycles of "Design-Build-Test" for optimizing the cpDNA. C_LI One-Sentence SummaryThe chloroplast genome can be replaced by a complete synthesized genome and performs the designed biological function in C. reinhardtii.

molecular biology↗

bioRxiv2022

ClpP/ClpX deficiency impairs mitochondrial functions and mTORC1 signaling during spermatogenesis and meiosis

Caseinolytic protease proteolytic subunit (ClpP) and caseinolytic protease X (ClpX) are mitochondrial matrix peptidases that activate mitochondrial unfolded protein response to maintain protein homeostasis in the mitochondria. However, the role of ClpP and ClpX in spermatogenesis remains largely unknown. In this study, we demonstrated the importance of ClpP/ClpX for meiosis and spermatogenesis with two conditional knockout (cKO) mouse models. We found that ClpP/ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply during meiosis and attenuated zygotene-pachytene transformation of the male germ cells. The dysregulated spermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within the seminiferous tubules. We found mTORC1 pathway was over-activated after deletion of ClpP/ClpX in spermatocytes. Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis. The data reveal the novel roles of ClpP and ClpX in regulating meiosis and spermatogenesis. One-Sentence SummaryClpP/ClpX is required for maintaining mitochondrial functions in spermatocytes during meiosis and spermatogenesis.

developmental biology↗

bioRxiv2022

Enhancing detectable fluorescence fluctuation for high-throughput and four-dimensional live-cell super-resolution imaging

Super-resolution (SR) imaging with high-throughput is invaluable to fast and high-precision profiling in a wide range of biomedical applications. However, prevalent SR methods require sophisticated acquisition devices and specific imaging control, and may cost a fairly long time on a single field-of-view. These essentially increase the construction difficulty, including challenges in imaging throughput, system establishment, and automation. Using the natural photophysics of fluorescence, fluctuation-based microscopy techniques can routinely break the diffraction limit with no need for additional optical components, but its long acquisition time still poses a challenge for high-throughput imaging or visualizing transient organelle dynamics. Here, we propose an SR method based on the Auto-Correlation with two-step Deconvolution (SACD) that reduces the number of frames required by maximizing the detectable fluorescence fluctuation behavior in each measurement, with further removal of tunable parameters by a Fourier ring correlation analysis. It only needs 20 frames for twofold lateral and axial resolution improvements, while the SR optical fluctuation imaging (SOFI) needs more than 1000 frames. By capturing raw images for [~]10 minutes, we record an SR image with [~]128 nm resolution that contains 2.4 gigapixels covering an area of [~]2.0 mm x 1.4 mm, including more than 2,000 cells. Beyond that, by applying continuity and sparsity joint constraint, the Sparse deconvolution-assisted SACD enables 4D live-cell SR imaging of events such as mitochondrial fission and fusion. Overall, as an open-sourced module, we anticipate SACD can offer direct access to SR, which may facilitate the biology studies of cells and organisms with high-throughput and low-cost.

biophysics↗