Fluorescent Labeling Technique Research Articles

Specificity determinants of pathogens in forest

Abstract Host‐specific pathogens have long been suggested to act as a major driver of species diversity in tropical forests. However, determining the degree of host specificity of potential pathogens coupled to key cellular characteristics of infection is difficult and time‐consuming. These challenges have delayed progress in relating the functional specificity of pathogenic fungi to ecological consequences. We tested the pathogenicity of 27 (of 215) fungi that were isolated from surface sterilized roots of seedlings from four common tree species in a diverse subtropical forest. Inoculation experiments showed that six fungi exhibited strong pathogenicity on the host seedlings. Five of these only infected their specific hosts (i.e. host‐specific pathogen species). Green fluorescent protein labelling revealed that in three host‐specific pathogens fungal hyphae were able to grow into the vascular tissues only in their specific host plant, in contrast to other fungal‐host combinations that exhibit non‐pathogenic interactions. This fluorescent labelling technique allows direct tracking of the progress of fungal infection in different host tissues. Synthesis. By coupling the green fluorescent protein technique with standard host inoculation experiments, we determined the developmental differences between infections by pathogenic and non‐pathogenic fungi in a relatively simple and straightforward way. Our work provides a useful tool for rapidly screening and categorizing host–fungal interactions, reflecting the functional basis of host specificity of pathogenic fungi. More broadly, this technique can contribute to understanding the roles of pathogens in species coexistence and biodiversity maintenance in forest communities.

Exploring the functionality of fluorescent liposomes in cancer: diagnosis and therapy

Abstract Fluorescent liposomes are pivotal in cancer research, serving as adaptable vehicles for imaging and therapeutics. These small lipid vesicles, capable of encapsulating fluorescent dyes, offer precise visualization and monitoring of their targeted delivery to cancer cells. This review delves into the critical role fluorescent liposomes play in enhancing both cancer diagnosis and treatment. It provides an in-depth analysis of their structural features, fluorescent labeling techniques, targeting strategies, and the challenges and opportunities they present. In the domain of cancer diagnosis, the article sheds light on various imaging modalities enabled by fluorescent liposomes, including fluorescence imaging and multimodal techniques. Emphasis is placed on early detection strategies, exhibiting the utility of targeted contrast agents and biomarker recognition for enhanced diagnostic precision. Moving on to cancer treatment, the review discusses the sophisticated drug delivery mechanisms facilitated by fluorescent liposomes, focusing on chemotherapy and photodynamic therapy. Moreover, the exploration extends to targeted therapy, explaining the applications of fluorescent liposomes in gene delivery and RNA interference. In a nutshell, his article comprehensively explores the multifaceted impact of fluorescent liposomes on advancing cancer diagnosis and treatment, combining existing knowledge with emerging trends.

A novel Bacillus aerolatus CX253 attenuates inflammation induced by Streptococcus pneumoniae in childhood and pregnant rats by regulating gut microbiome

Streptococcus pneumoniae (Spn) is the predominant pathogen responsible for community-acquired pneumonia (CAP) in children under five years old, and it can induce over 17% of pregnant women. However, no more effective measures exist to prevent infection induced by Spn in these two special populations. The beneficial microbes can antagonize Spn and provide new targets for preventing pneumococcal infections. This study used 16S rRNA gene sequencing and targeted metabolomics to evaluate the role of the Bacillus aerolatus CX253 (CX253) in alleviating Spn infection. Additionally, the colonization of CX253 was observed in nose, trachea, and lung by using confocal laser scanning microscopy and fluorescent labeling techniques. Compared with the model group, the expression level of interleukin-1β was dropped 1.81-fold and 2.22-fold, and interleukin-6 was decreased 2.39-fold and 1.84-fold. The express of tumor necrosis factor-α was down 2.30-fold and 3.84-fold in prevention group of childhood and pregnant rats, respectively. The 16S rRNA sequencing results showed that CX253 administration alone significantly increased the abundance of Lactobacillus, Limosilactobacillus, and Prevotella in the gut of childhood and pregnant rats. Furthermore, the CX253 increased propionate in the gut of childhood rats and increased propionate and butyrate in the gut of pregnant rats to inhibit pulmonary inflammation. In summary, CX253 attenuated Spn-induced inflammation by regulating the gut microbiota and SCFAs. The research provides valuable information for the prevention of pneumonia.Graphical

Exploration of Anticancer Activity of Portieria Hornemanni Against Lung Cancer Cellline (A549)

Cancer remains a major global health issue for which there are now no entirely viable treatments. As a result, natural product-based alternative therapies have been suggested. Studies have demonstrated the biological activity of seaweed, particularly its anticancer properties. The review’s main subject is lung cancer. It also lists some substances that have been taken out of a variety of seaweeds and demonstrated to either completely prevent cancer or significantly delay its spread. The main objectives of the study are to find the cytotoxicity effect of Porteria hornimanni in human lung cancer cell line A549 adopting MTT assay and utilizing fluorescent labelling techniques to detect morphological changes in the cells following P. hornimanni therapy.The present study indicates that P. hornemanni’s ethanolic extract exhibits anti-cancer properties in the lung cancer cell line A549.This study shows how cancer cells can employ their anticancer properties. Additional research is necessary to identify the specific chemical in P. hornemanni that has cytotoxic effects on cancer.

Parascedosporium putredinis NO1 tailors its secretome for different lignocellulosic substrates.

Parascedosporium putredinis NO1 is a plant biomass-degrading ascomycete with a propensity to target the most recalcitrant components of lignocellulose. Here we applied proteomics and activity-based protein profiling (ABPP) to investigate the ability of P. putredinis NO1 to tailor its secretome for growth on different lignocellulosic substrates. Proteomic analysis of soluble and insoluble culture fractions following the growth of P. putredinis NO1 on six lignocellulosic substrates highlights the adaptability of the response of the P. putredinis NO1 secretome to different substrates. Differences in protein abundance profiles were maintained and observed across substrates after bioinformatic filtering of the data to remove intracellular protein contamination to identify the components of the secretome more accurately. These differences across substrates extended to carbohydrate-active enzymes (CAZymes) at both class and family levels. Investigation of abundant activities in the secretomes for each substrate revealed similar variation but also a high abundance of "unknown" proteins in all conditions investigated. Fluorescence-based and chemical proteomic ABPP of secreted cellulases, xylanases, and β-glucosidases applied to secretomes from multiple growth substrates for the first time confirmed highly adaptive time- and substrate-dependent glycoside hydrolase production by this fungus. P. putredinis NO1 is a promising new candidate for the identification of enzymes suited to the degradation of recalcitrant lignocellulosic feedstocks. The investigation of proteomes from the biomass bound and culture supernatant fractions provides a more complete picture of a fungal lignocellulose-degrading response. An in-depth understanding of this varied response will enhance efforts toward the development of tailored enzyme systems for use in biorefining.IMPORTANCEThe ability of the lignocellulose-degrading fungus Parascedosporium putredinis NO1 to tailor its secreted enzymes to different sources of plant biomass was revealed here. Through a combination of proteomic, bioinformatic, and fluorescent labeling techniques, remarkable variation was demonstrated in the secreted enzyme response for this ascomycete when grown on multiple lignocellulosic substrates. The maintenance of this variation over time when exploring hydrolytic polysaccharide-active enzymes through fluorescent labeling, suggests that this variation results from an actively tailored secretome response based on substrate. Understanding the tailored secretomes of wood-degrading fungi, especially from underexplored and poorly represented families, will be important for the development of effective substrate-tailored treatments for the conversion and valorization of lignocellulose.

Comparative Analysis of Fluorescent Labeling Techniques for Tracking Canine Amniotic Stem Cells.

The utmost aim of regenerative medicine is to promote the regeneration of injured tissues using stem cells. Amniotic mesenchymal stem cells (AmMSCs) have been used in several studies mainly because of their easy isolation from amniotic tissue postpartum and immunomodulatory and angiogenic properties and the low level of rejection. These cells share characteristics of both embryonic/fetal and adult stem cells and are particularly advantageous because they do not trigger tumorigenic activity when injected into immunocompromised animals. The large-scale use of AmMSCs for cellular therapies would greatly benefit from fluorescence labeling studies to validate their tracking in future therapies. This study evaluated the fluorophore positivity, fluorescence intensity, and longevity of canine AmMSCs. For this purpose, canine AmMSCs from the GDTI/USP biobank were submitted to three labeling conditions, two commercial fluorophores [CellTrace CFSE Cell Proliferation kit - CTrace, and CellTracker Green CMFDA - CTracker (CellTracker Green CMFDA, CT, #C2925, Molecular Probes®; Life Technologies)] and green fluorescent protein (GFP) expression after lentiviral transduction, to select the most suitable tracer in terms of adequate persistence and easy handling and analysis that could be used in studies of domestic animals. Fluorescence was detected in all groups; however, the patterns were different. Specifically, CTrace and CTracker fluorescence was detected 6 h after labeling, while GFP was visualized no earlier than 48 h after transduction. Flow cytometry analysis revealed more than 70% of positive cells on day 7 in the CTrace and CTracker groups, while fluorescence decreased significantly to 10% or less on day 20. Variations between repetitions were observed in the GFP group under the present conditions. Our results showed earlier fluorescence detection and more uniform results across repetitions for the commercial fluorophores. In contrast, fluorescence persisted for more extended periods in the GFP group. These results indicate a promising direction for assessing the roles of canine AmMSCs in regenerative medicine without genomic integration.

Theoretical calculation of NMDA receptor desensitization and intracellular calcium determination through simulation

One of the crucial processes in preventing cellular damage due to excitotoxicity involves the calcium-mediated desensitization of the N-methyl-D-aspartate (NMDA) receptor. The opening of NMDA channels results in the influx of Na+ and Ca2+ ions and efflux of K+ ions. This condition leads to an increase in intracellular calcium ([Ca2+]i), thereby diminishing the NMDA current and preventing damage induced by constant stimulation of the receptor by glutamate. In this study, a simulator based on a phenomenological mathematical model was developed, employing the Visual Basic 6.0 language for the Windows® environment. Input variables encompass intracellular calcium, peak currents (Ip), and steady-state currents (Iss). Input currents were derived from previously published electrophysiological recordings. The simulator adeptly reproduces the NMDA receptor desensitization phenomenon under varying experimental conditions and accurately calculates internal calcium with a 10% margin of error. It proves valuable in scenarios where simultaneous recordings of NMDA current and [Ca2+]i via fluorescence labeling techniques are impractical. In the realm of education, it enables students to conduct virtual experiments, providing an immersive introduction to the topic.

Modularity impacts cellulose surface oxidation by a lytic polysaccharide monooxygenase from Streptomyces coelicolor

AbstractLytic polysaccharide monooxygenases (LPMOs) catalyze the oxidation of β-(1,4)-linked polysaccharides, such as cellulose, in a reaction that requires an electron donor and H2O2 as co-substrate. Several LPMOs include a carbohydrate-binding module (CBM), which promotes action on insoluble substrates. Herein, a fluorescent labeling technique was used to track LPMO action on microcrystalline cellulose and evaluate the impact of CBMs on the distribution of LPMO activity across the fiber surface. Confocal microscopic images revealed that the distribution of oxidized positions on the cellulose surface was CBM-dependent: fluorescent spots were concentrated in reactions with a CBM-containing LPMO whereas they were more dispersed for a CBM-deficient LPMO variant. The more dispersed oxidation pattern for the CBM-free LPMO coincided with the release of fewer soluble reaction products.

Compartmentalization of androgen receptors at endogenous genes in living cells.

A wide range of nuclear proteins are involved in the spatio-temporal organization of the genome through diverse biological processes such as gene transcription and DNA replication. Upon stimulation by testosterone and translocation to the nucleus, multiple androgen receptors (ARs) accumulate in microscopically discernable foci which are irregularly distributed in the nucleus. Here, we investigated the formation and physical nature of these foci, by combining novel fluorescent labeling techniques to visualize a defined chromatin locus of AR-regulated genes-PTPRN2 or BANP-simultaneously with either AR foci or individual AR molecules. Quantitative colocalization analysis showed evidence of AR foci formation induced by R1881 at both PTPRN2 and BANP loci. Furthermore, single-particle tracking (SPT) revealed three distinct subdiffusive fractional Brownian motion (fBm) states: immobilized ARs were observed near the labeled genes likely as a consequence of DNA-binding, while the intermediate confined state showed a similar spatial behavior but with larger displacements, suggesting compartmentalization by liquid-liquid phase separation (LLPS), while freely mobile ARs were diffusing in the nuclear environment. All together, we show for the first time in living cells the presence of AR-regulated genes in AR foci.

Diffracted X-ray Tracking for Observing the Internal Motions of Individual Protein Molecules and Its Extended Methodologies.

In 1998, the diffracted X-ray tracking (DXT) method pioneered the attainment of molecular dynamics measurements within individual molecules. This breakthrough revolutionized the field by enabling unprecedented insights into the complex workings of molecular systems. Similar to the single-molecule fluorescence labeling technique used in the visible range, DXT uses a labeling method and a pink beam to closely track the diffraction pattern emitted from the labeled gold nanocrystals. Moreover, by utilizing X-rays with extremely short wavelengths, DXT has achieved unparalleled accuracy and sensitivity, exceeding initial expectations. As a result, this remarkable advance has facilitated the search for internal dynamics within many protein molecules. DXT has recently achieved remarkable success in elucidating the internal dynamics of membrane proteins in living cell membranes. This breakthrough has not only expanded our knowledge of these important biomolecules but also has immense potential to advance our understanding of cellular processes in their native environment.

Deubiquitination Detection of p53 Protein in Living Cells by Fluorescence Cross-Correlation Spectroscopy.

Deubiquitination is a reverse post-translational modification of ubiquitination and plays significant roles in various signal transduction cascades and protein stability. The p53 is a very important tumor-suppressor protein and closely implicates more than 50% of human cancers. Although extracellular studies on the deubiquitination of p53 were reported, the process of p53 deubiquitination in living cells due to the shortage of an efficient in situ method for single living cells is still not clear. In this study, we described an in situ method for studying p53 deubiquitination in living cells by combining fluorescence cross-correlation spectroscopy with a fluorescent protein labeling technique. We first constructed the stable cell line expressing EGFP-Ub-p53-mCherry as the substrate of p53 deubiquitination. Then, we established a method for in situ monitoring of the deubiquitination of p53 in living cells. Based on the amplitudes of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy curves from living cells, we obtained the deubiquitination percentage for evaluating the level of p53 protein deubiquitination. Furthermore, we studied the effects of ubiquitin structures on p53 deubiquitination in living cells and found that the C-terminal Gly75-Gly76 motif of ubiquitin is a key location for p53 deubiquitination and the deubiquitination cannot occur when ubiquitin lacks the C-terminal Gly75-Gly76 motif. Our results documented that the developed strategy is an efficient method for in situ study of deubiquitination of proteins in living cells.

Heptamethine cyanines in bioorthogonal chemistry

Due to their excellent fluorescence properties and biological function, cyanine dyes have been widely applied in biological imaging. Heptamethine cyanine (Cy7) dyes, as a type of classic near-infrared (NIR) fluorescent dyes, are considered as one of the effective fluorescent tools in the living organisms due to their good biocompatibility and very low background interference. Bioorthogonal reactions performed in living cells and tissues have developed by leaps and bounds in recent years. The NIR fluorescent labeling technique involving cyanine has attracted widespread attention. This review summarizes their recent application in the field of bioorthogonal imaging, mainly concluding Cy7-type dyes, labeling strategy, bioimaging application, etc. We expect this work can provide some helps for the studies of NIR bioorthogonal reaction in vivo.

Cerebrovascular segmentation from mesoscopic optical images using Swin Transformer

Vascular segmentation is a crucial task in biomedical image processing, which is significant for analyzing and modeling vascular networks under physiological and pathological states. With advances in fluorescent labeling and mesoscopic optical techniques, it has become possible to map the whole-mouse-brain vascular networks at capillary resolution. However, segmenting vessels from mesoscopic optical images is a challenging task. The problems, such as vascular signal discontinuities, vessel lumens, and background fluorescence signals in mesoscopic optical images, belong to global semantic information during vascular segmentation. Traditional vascular segmentation methods based on convolutional neural networks (CNNs) have been limited by their insufficient receptive fields, making it challenging to capture global semantic information of vessels and resulting in inaccurate segmentation results. Here, we propose SegVesseler, a vascular segmentation method based on Swin Transformer. SegVesseler adopts 3D Swin Transformer blocks to extract global contextual information in 3D images. This approach is able to maintain the connectivity and topology of blood vessels during segmentation. We evaluated the performance of our method on mouse cerebrovascular datasets generated from three different labeling and imaging modalities. The experimental results demonstrate that the segmentation effect of our method is significantly better than traditional CNNs and achieves state-of-the-art performance.

Simultaneous Detection of Two Extracellular Vesicle Subpopulations in Saliva Assisting Tumor T Staging of Oral Squamous Cell Carcinoma.

Extracellular vesicles (EVs), acting as important mediators of intercellular communication, play an essential role in physiological processes, which have unique potential in the medical field. However, the heterogeneity of EVs limits their development for disease diagnosis and therapy, making the EV subpopulation analysis extremely valuable. In this article, a simple microfluidic approach was presented for the on-chip specific isolation and detection of two phenotypes of EVs (Annexin V+ EGFR+ EVs and Annexin V- EGFR+ EVs) based on different biomolecule-modified magnetic nanospheres and a fluorescence labeling technique. Combined with the control of the magnetic field in the microzone and fluid flow, it was easy to form two separate functional regions in the chip to capture different EV subpopulations. This method was successfully applied to the tests of clinical saliva samples in 75 oral squamous cell carcinoma (OSCC) patients and 10 healthy people. The results showed that the total level of EGFR+ EVs was much higher in OSCC patients that in healthy people. Meantime, the ratio of Annexin V+ EGFR+ EVs to Annexin V- EGFR+ EVs was found to be negatively correlated with tumor T stage of OSCC patients with a statistical difference, which suggested the ratio as a clinical index for monitoring the progression of OSCC in real time based on a noninvasive method. The approach provided a novel idea for evaluating the tumor T stage of OSCC and a powerful tool for clinical application.

Key mechanism of enzymatic dehairing technology for leather-making: permeation behaviors of protease into animal hide and the mechanism of charge regulation

Enzymes have been widely used as alternatives to conventional chemicals in cleaner leather processes due to their advantages of meeting increasing environmental demands. Especially, enzymatic unhairing based on protease has been applied to leather-making for a long time, however, it still has the key problem of slow permeation in the animal hide, resulting in slow unhairing rate, poor hair removal effect, excessive proteolysis of hide collagen, and decreased leather quality. Aiming at the key problem of bio-unhairing technology, fluorescent labeling technique and confocal laser scanning microscopy were used to investigate the protease permeation behaviors into the animal hide based on well-prepared labeled proteases, as well as the quantitative analysis of the protease amount in different hide layers. The results show that the protease mainly permeates the bovine hide through transfollicular routes from the hair side, and although the intercellular-interfibrillar pathway also exists, it does not play an important role. Additionally, the protease permeation behaviors into the hide are greatly impacted by the charge states of the hide and protease proteins, depending on the isoelectric points (pI) of the proteins and solution pH values. When the solution pH is not between the pI values of the two proteins, the similarly charged protease can quickly and deeply penetrate the hide because of the electrostatic repulsion. The established mechanism provides a theoretical basis for developing an efficient enzymatic unhairing technology for leather-making, and this can also be applied to other processes involving the enzyme permeation into the hide or leather.Graphical

Radiolabeled Biodistribution of Expansile Nanoparticles: Intraperitoneal Administration Results in Tumor Specific Accumulation.

Nanoparticle biodistribution in vivo is an essential component to the success of nanoparticle-based drug delivery systems. Previous studies with fluorescently labeled expansile nanoparticles, or "eNPs", demonstrated a high specificity of eNPs to tumors that is achieved through a materials-based targeting strategy. However, fluorescent labeling techniques are primarily qualitative in nature and the gold-standard for quantitative evaluation of biodistribution is through radiolabeling. In this manuscript, we synthesize 14C-labeled eNPs to quantitatively evaluate the biodistribution of these particles in a murine model of intraperitoneal mesothelioma via liquid scintillation counting. The results demonstrate a strong specificity of eNPs for tumors that lasts one to 2 weeks postinjection with an overall delivery efficiency to the tumor tissue of 30% of the injected dose which is congruent with prior reports of preclinical efficacy of the technology. Importantly, the route of administration is essential to the eNP's material-based targeting strategy with intraperitoneal administration leading to tumoral accumulation while, in contrast, intravenous administration leads to rapid clearance via the reticuloendothelial system and low tumoral accumulation. A comparison against nanoparticle delivery systems published over the past decade shows that the 30% tumoral delivery efficiency of the eNP is significantly higher than the 0.7% median delivery efficiency of other systems with sufficient quantitative data to define this metric. These results lay a foundation for targeting intraperitoneal tumors and encourage efforts to explore alternative, nonintravenous routes, of delivery to accelerate the translation of nanoparticle therapies to the clinic.

Direct Observation of Archaellar Motor Rotation by Single-Molecular Imaging Techniques.

Single-molecular techniques have characterized dynamics of molecular motors such as flagellum in bacteria and myosin, kinesin, and dynein in eukaryotes. We can apply these techniques to a motility machine of archaea, namely, the archaellum, composed of a thin helical filament and a rotary motor. Although the size of the motor hinders the characterization of its motor function under a conventional optical microscope, fluorescence-labeling techniques allow us to visualize the architecture and function of the archaellar filaments in real time. Furthermore, a tiny polystyrene bead attached to the filament enables the visualization of motor rotation through the bead rotation and quantification of biophysical properties such as speed and torque produced by the rotary motor imbedded in the cell membrane. In this chapter, I describe the details of the above biophysical method based on an optical microscope.

Morphological transitions of flexible fibers in viscous flows

<h2>Abstract</h2> Here we present the individual dynamics of flexible and Brownian filaments under shear and compression. We use actin filaments as a model system and observe their dynamics in microfluidic flow geometries using fluorescent labeling techniques and microscopic tracking methods. Under shear we characterize successive transitions from tumbling to buckling and finally snake turns as a function of an elasto-viscous number. Under compression we reveal the formation of three dimensional helicoidal structures and characterize their formation. Finally, we attempt at linking the microscopic observations to the macroscopic suspension properties with preliminary measurements of the shear viscosity of dilute suspensions of actin filaments in microfluidic rheometers.

Bioaccumulation of differently-sized polystyrene nanoplastics by human lung and intestine cells

The bioaccumulation of nanoplastics (NPs) has been intensively examined using the fluorescence-labeling technique. As the fluorescence intensity per particle is different for NPs with different physicochemical properties, it’s hard to directly compare their bioaccumulation based on fluorescence. Therefore, how physicochemical properties may affect NPs’ bioaccumulation remains unclear. In the present study, we chose polystyrene NPs (PSNPs) with the primary particle size of 70 nm (PS70), 200 nm (PS200), and 500 nm (PS500), and examined their uptake by human lung and intestine cells. We found that PSNPs had low cytotoxicity, but could be taken up by both cell lines. The particle-mass-, particle-number-, and particle-surface-area-based accumulation of the differently-sized PSNPs were then compared. Smaller PSNPs showed lower particle-mass-based but higher particle-number-based uptake rate than the larger ones. Nevertheless, much less difference was observed when the unit of uptake rate was based on particle surface area, suggesting the critical role of surface area during PSNPs’ interaction with the cell membrane. Additionally, all three PSNPs could enter the cells by phagocytosis and PS70 could also be internalized by clathrin- and caveolae-mediated endocytosis. Overall, the effects of size on the bioaccumulation of NPs need to be considered when evaluating their environmental and health risks.

Multi-perspective label based deep learning framework for cerebral vasculature segmentation in whole-brain fluorescence images.

The popularity of fluorescent labelling and mesoscopic optical imaging techniques enable the acquisition of whole mammalian brain vasculature images at capillary resolution. Segmentation of the cerebrovascular network is essential for analyzing the cerebrovascular structure and revealing the pathogenesis of brain diseases. Existing deep learning methods use a single type of annotated labels with the same pixel weight to train the neural network and segment vessels. Due to the variation in the shape, density and brightness of vessels in whole-brain fluorescence images, it is difficult for a neural network trained with a single type of label to segment all vessels accurately. To address this problem, we proposed a deep learning cerebral vasculature segmentation framework based on multi-perspective labels. First, the pixels in the central region of thick vessels and the skeleton region of vessels were extracted separately using morphological operations based on the binary annotated labels to generate two different labels. Then, we designed a three-stage 3D convolutional neural network containing three sub-networks, namely thick-vessel enhancement network, vessel skeleton enhancement network and multi-channel fusion segmentation network. The first two sub-networks were trained by the two labels generated in the previous step, respectively, and pre-segmented the vessels. The third sub-network was responsible for fusing the pre-segmented results to precisely segment the vessels. We validated our method on two mouse cerebral vascular datasets generated by different fluorescence imaging modalities. The results showed that our method outperforms the state-of-the-art methods, and the proposed method can be applied to segment the vasculature on large-scale volumes.