Atomic Pt Sites Anchored in the Interface between Grains on Vacancy-Enriched CeO2 Nanosheets: One-Step Precursor Combustion Synthesis.

Atomic metal catalysts have unique electronic, structural, and catalytic properties, which are widely used in the field of catalysis. However, designing new simple synthesis methods to fabricate atomic metal catalysts is a challenge in catalytic applications. Herein, a one-step precursor combustion strategy is presented that starts directly from precursors of metal salts, using a spontaneous combustion process convert platinum nitrate to atomic Pt sites. The atomic Pt sites with low valence are anchored in the formed interface between grains on vacancy-enriched CeO2 nanosheets. The obtained Pt/CeO2-2 catalyst exhibits much higher three-way catalytic activities at low temperatures than Pt/CeO2-C catalysts prepared using the traditional impregnation method. Density functional theory calculations show that the generated lower valent Pt atoms in the CeO2 interface promote catalytic activity through reducing the energy barrier, and lead to an overall improvement of three-way catalytic activities. This facile strategy provides new insights into the study of the properties and applications of atomic noble metal catalysts.

Substantial viral diversity in bats and rodents from East Africa: insights into evolution, recombination, and cocirculation.

BACKGROUND: Zoonotic viruses cause substantial public health and socioeconomic problems worldwide. Understanding how viruses evolve and spread within and among wildlife species is a critical step when aiming for proactive identification of viral threats to prevent future pandemics. Despite the many proposed factors influencing viral diversity, the genomic diversity and structure of viral communities in East Africa are largely unknown. RESULTS: Using 38.3 Tb of metatranscriptomic data obtained via ultradeep sequencing, we screened vertebrate-associated viromes from 844 bats and 250 rodents from Kenya and Uganda collected from the wild. The 251 vertebrate-associated viral genomes of bats (212) and rodents (39) revealed the vast diversity, host-related variability, and high geographic specificity of viruses in East Africa. Among the surveyed viral families, Coronaviridae and Circoviridae showed low host specificity, high conservation of replication-associated proteins, high divergence among viral entry proteins, and frequent recombination. Despite major dispersal limitations, recurrent mutations, cocirculation, and occasional gene flow contribute to the high local diversity of viral genomes. CONCLUSIONS: The present study not only shows the landscape of bat and rodent viromes in this zoonotic hotspot but also reveals genomic signatures driven by the evolution and dispersal of the viral community, laying solid groundwork for future proactive surveillance of emerging zoonotic pathogens in wildlife. Video Abstract.

A lipid/PLGA nanocomplex to reshape tumor immune microenvironment for colon cancer therapy.

Immune checkpoint blockade therapy provides a new strategy for tumor treatment; however, the insufficient infiltration of cytotoxic T cells and immunosuppression in tumor microenvironment lead to unsatisfied effects. Herein, we reported a lipid/PLGA nanocomplex (RDCM) co-loaded with the photosensitizer Ce6 and the indoleamine 2,3-dioxygenase (IDO) inhibitor 1MT to improve immunotherapy of colon cancer. Arginine-glycine-aspartic acid (RGD) as the targeting moiety was conjugated on 1,2-distearoyl-snglycero-3-phosphoethanolamine lipid via polyethylene glycol (PEG), and programmed cell death-ligand 1 (PD-L1) peptide inhibitor DPPA (sequence: CPLGVRGK-GGG-d(NYSKPTDRQYHF)) was immobilized on the terminal group of PEG via matrix metalloproteinase 2 sensitive peptide linker. The Ce6 and 1MT were encapsulated in PLGA nanoparticles. The drug loaded nanoparticles were composited with RGD and DPPA modified lipid and lecithin to form lipid/PLGA nanocomplexes. When the nanocomplexes were delivered to tumor, DPPA was released by the cleavage of a matrix metalloproteinase 2-sensitive peptide linker for PD-L1 binding. RGD facilitated the cellular internalization of nanocomplexes via avß3 integrin. Strong immunogenic cell death was induced by 1O2 generated from Ce6 irradiation under 660 nm laser. 1MT inhibited the activity of IDO and reduced the inhibition of cytotoxic T cells caused by kynurenine accumulation in the tumor microenvironment. The RDCM facilitated the maturation of dendritic cells, inhibited the activity of IDO, and markedly recruited the proportion of tumor-infiltrating cytotoxic T cells in CT26 tumor-bearing mice, triggering a robust immunological memory effect, thus effectively preventing tumor metastasis. The results indicated that the RDCM with dual IDO and PD-L1 inhibition effects is a promising platform for targeted photoimmunotherapy of colon cancer.

IL-6/STAT3 axis is hijacked by GCRV to facilitate viral replication via suppressing type â IFN signaling.

Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type Ⅰ IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.

Tuning Pd species via electronic metal-support interaction for methane combustion.

Utilizing catalytic combustion to convert methane (CH4) into CO2 and H2O stands as one of the most effective approaches for mitigating unburnt CH4 emissions from natural gas engines. Supported Pd catalysts have been extensively researched for their role in low-temperature CH4 combustion, with their catalytic activity greatly influenced by metal-support interactions. Surface interaction Pd phases, as a special type of Pd species originating from metal-support interactions on supported Pd catalysts, show controversial catalytic performance in CH4 combustion. Moreover, the impact of electronic metal-support interactions (EMSI, which refers to metal-support interactions associated with electron transfer) remains unclear. Hence, we opted for Ce-Zr solid solutions with different Ce:Zr molar ratios as supports and synthesized a range of supported Pd catalysts with varying EMSI intensities. Characterization revealed that as the oxygen vacancy concentration on the support increased, electron transfer weakened, leading to a higher Pd-O-Ce content, resulting in a lower CH4 activation barrier and better catalytic performance. This study offers a promising approach for regulating EMSI and active Pd species on supported catalysts in practical applications.

Unveiling the Pivotal Role of dx2-y2 Electronic States in Nickel-based Hydroxide Electrocatalysts for Methanol Oxidation.

The anodic methanol oxidation reaction (MOR) plays a crucial role in coupling with the cathodic hydrogen evolution reaction (HER) and enables the sustainable production of the high-valued formate. Nickel-based hydroxide (Ni(OH)2) as MOR electrocatalyst has attracted enormous attention. However, the key factor determining the intrinsic catalytic activity remains unknown, which significantly hinders the further development of Ni(OH)2 electrocatalyst. Here, we found that the dx2-y2 electronic state within antibonding bands plays a decisive role in the whole MOR process. The onset potential depends on the deprotonation ability (Ni2+ to Ni3+), which was closely related to the band center of dx2-y2 orbital. The closer of dx2-y2 orbital to the Fermi level showed the stronger the deprotonation ability. Meanwhile, in the high potential region, the broadening of dx2-y2 orbital would facilitate the electron transfer from methanol to catalysts (Ni3+ to Ni2+), further enhancing the catalytic properties. Our work for the first time clarifies the intrinsic relationship between dx2-y2 electronic state and the MOR activities, which adds a new layer of understanding to the methanol electrooxidation research scene.

Palladium-catalyzed alkynylation of allylic gem-difluorides.

Herein, a palladium-catalyzed regioselective alkynylation, esterification, and amination of allylic gem-difluorides via C-F bond activation/transmetallation/ß-C elimination or nucleophilic attack has been achieved. This innovative protocol showcases an extensive substrate range and operates efficiently under mild reaction conditions, resulting in high product yields and Z-selectivity. Particularly noteworthy is its exceptional tolerance towards a wide array of functional groups. This developed methodology provides effective and convenient routes to access a diverse array of essential fluorinated enynes, esters and amines.

Targeting SLC22A5 fosters mitophagy inhibition-mediated macrophage immunity against septic acute kidney injury upon CD47-SIRPα axis blockade.

Efferocytosis of apoptotic neutrophils (PMNs) by macrophages is helpful for inflammation resolution and injury repair, but the role of efferocytosis in intrinsic nature of macrophages during septic acute kidney injury (AKI) remains unknown. Here we report that CD47 and signal regulatory protein alpha (SIRPα)-the anti-efferocytotic 'don't eat me' signals-are highly expressed in peripheral blood mononuclear cells (PBMCs) from patients with septic AKI and kidney samples from mice with polymicrobial sepsis and endotoxin shock. Conditional knockout (CKO) of SIRPA in macrophages ameliorates AKI and systemic inflammation response in septic mice, accompanied by an escalation in mitophagy inhibition of macrophages. Ablation of SIRPA transcriptionally downregulates solute carrier family 22 member 5 (SLC22A5) in the lipopolysaccharide (LPS)-stimulated macrophages that efferocytose apoptotic neutrophils (PMNs). Targeting SLC22A5 renders mitophagy inhibition of macrophages in response to LPS stimuli, improves survival and deters development of septic AKI. Our study supports further clinical investigation of CD47-SIRPα signalling in sepsis and proposes that SLC22A5 might be a promising immunotherapeutic target for septic AKI.

Metagenomic analysis of individual mosquito viromes reveals the geographical patterns and drivers of viral diversity.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Here, using a meta-transcriptomic approach, we determined the viromes of 2,438 individual mosquitoes (81 species), spanning ~4,000 km along latitudes and longitudes in China. From these data we identified 393 viral species associated with mosquitoes, including 7 (putative) species of arthropod-borne viruses (that is, arboviruses). We identified potential mosquito species and geographic hotspots of viral diversity and arbovirus occurrence, and demonstrated that the composition of individual mosquito viromes was strongly associated with host phylogeny. Our data revealed a large number of viruses shared among mosquito species or genera, enhancing our understanding of the host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, perhaps reflecting long-distance mosquito dispersal. Together, these results greatly expand the known mosquito virome, linked viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the biogeography and diversity of viruses in insect vectors.

Circulating soluble CD30 is associated with renal tertiary lymphoid structures and the progression of IgA nephropathy.

BACKGROUND: Renal tertiary lymphoid structures (TLSs) are involved in renal pathology and prognosis of IgA nephropathy (IgAN). CD30 and its ligands participate in the formation of renal TLSs. However, the relationship between circulating CD30 and renal prognosis is unclear. The objective of this study was to evaluate the relationship between circulating CD30 and prognosis in patients with IgAN. METHODS: We conducted a retrospective study including 351 patients with biopsy proved IgAN. We collected clinical and pathologic features at the time of biopsy and recorded renal follow-up outcomes. Circulating CD30 levels in IgAN patients at the time of biopsy were measured via enzyme-linked immunosorbent assay (ELISA). The association between elevated CD30 levels and the composite endpoint (defined as a ≥ 50 % decline in eGFR from baseline, end-stage renal disease, or death) was investigated using Cox regression analysis. RESULTS: During a median follow-up period of 5.12 years, 44 (12.5 %) patients in the cohort reached the composite endpoint. Kaplan-Meier survival curve analysis revealed a significant association between higher circulating CD30 levels and a poorer renal prognosis (log-rank P < 0.001). Cox regression analysis showed that high CD30 was an independent factor for the composite endpoints in multivariable-adjusted models (HR 3.397, 95 % CI: 1.230-9.384, P = 0.018). These associations were also observed in a subgroup of patients with concomitant renal TLSs formation (10.443, 95 % CI: 1.680-65.545, P = 0.012), proteinuria > 1 g/d (HR 12.287, 95 % CI: 1.499-100.711, P = 0.019), and female patients (HR 22.372, 95 % CI: 1.797-278.520, P = 0.016). CONCLUSION: Elevated level of circulating CD30 is an independent risk factor for renal disease progression in patients with IgAN.

Rapid and sensitive detection of SARS-CoV-2 IgM through luciferase luminescence on an automatic platform.

SARS-CoV-2 has brought a global health crisis worldwide. IgM is an early marker in sera after the infections, and the detection of IgM is crucial to assist diagnosis and evaluate the vaccination clinically. Herein, we developed an automated platform to identify IgM against SARS-CoV-2 in sera. Streptavidin-magnetic beads were utilized to bind to a biotinylated anti-IgM antibody, which was employed to capture IgM in sera. RBD fused luciferase hGluc was employed to label the trapped IgM against RBD and the signal of luminescence of hGluc with the substrate of coelenterazine corresponded to the amount of SARS-CoV-2 IgM conjugated to the magnetic beads. An appropriate cut-off value of the designed method was defined by a set of negative samples and positive samples with 100 % sensitivity and 100 % specificity. Through serial dilution of a positive sample, it was found that the method has a better sensitivity than ELISA. The application to determine IgM against SARS-CoV-2 demonstrated a good performance of the method. The developed system can complete the analysis of SARS-CoV-2 IgM within 25 min. Through the substitution of RBD antigen with antigens of other pathogens in this platform, the automated detection of IgM against the corresponding pathogens can be realized.

Effect of salting and dehydration treatments on the physicochemical and gel properties of hen and duck egg yolks, plasma and granules.

BACKGROUND: Salted hen egg yolks are less oily and less flavorful than salted duck egg yolks. However, hen eggs have a more adequate market supply and have a broader application prospect than duck eggs. In the present study, egg yolks, plasma, and granules were dehydrated by adding 1% NaCl to simulate traditional curing process of salted egg yolk. The changes in the pickling process of hen egg yolks (HEY) and duck egg yolks (DEY) plasma and granules were compared to reveal the gelation mechanism and the underlying causes of quality differences in salted HEY and DEY. Salted HEY can be compared with the changes in DEY during the pickling process to provide a theoretical basis for the quality improvement of salted HEY to salted DEY. RESULTS: The results showed that both plasma and granules were involved in gel formation, but exhibited different aggregation behaviors. Based on the intermolecular forces, the HEY proteins achieved aggregation mainly through hydrophobic interactions and DEY proteins mainly through covalent binding. According to spin-spin relaxation time, HEY gels immobilized a large amount of lipid and interacted strongly with lipids. DEY gels showed much free lipid and had weak interaction with lipid. The microstructure showed that HEY proteins were easily unfolded to form a homogeneous three-dimensional gel network structure after salting, whereas heterogeneous aggregates were formed to hinder the gel development in DEY. Changes in protein secondary structure content showed that pickling can promote the transformation of the α-helices to ß-sheets structure in HEY gels, whereas more α-helices structure was formed in DEY gels. CONCLUSION: The present study has demonstrated that different gelation behaviors of hen and duck egg yolk proteins (especially in plasma) through salting treatment led to the difference in the quality of salted HEY and DEY. © 2024 Society of Chemical Industry.

A novel variable neighborhood search approach for cell clustering for spatial transcriptomics.

This paper introduces a new approach to cell clustering using the Variable Neighborhood Search (VNS) metaheuristic. The purpose of this method is to cluster cells based on both gene expression and spatial coordinates. Initially, we confronted this clustering challenge as an Integer Linear Programming minimization problem. Our approach introduced a novel model based on the VNS technique, demonstrating the efficacy in navigating the complexities of cell clustering. Notably, our method extends beyond conventional cell-type clustering to spatial domain clustering. This adaptability enables our algorithm to orchestrate clusters based on information gleaned from gene expression matrices and spatial coordinates. Our validation showed the superior performance of our method when compared to existing techniques. Our approach advances current clustering methodologies and can potentially be applied to several fields, from biomedical research to spatial data analysis.

Egg Yolk Selenopeptides: Preparation, Characterization, and Immunomodulatory Activity.

In this study, egg yolk selenium peptides (Se-EYP) were prepared using double-enzyme hydrolysis combined with a shearing pretreatment. The properties of the selenopeptides formed were then characterized, including their yield, composition, molecular weight distribution, antioxidant activity, in vitro digestion, and immunomodulatory activity. The peptide yield obtained after enzymatic hydrolysis using a combination of alkaline protease and neutral protease was 74.5%, of which 82.6% had a molecular weight <1000 Da. The selenium content of the lyophilized solid product was 4.01 µg/g. Chromatography-mass spectrometry analysis showed that 88.6% of selenium in Se-EYP was in the organic form, of which SeMet accounted for 60.3%, SeCys2 for 21.8%, and MeSeCys for 17.9%. After being exposed to in vitro simulated digestion, Se-EYP still had 65.1% of oligopeptides present, and the in vitro antioxidant activity was enhanced. Moreover, Se-EYP exhibited superior immune detection indices, including immune organ index, level of immune factors in the serum, histopathological changes in the spleen, and selenium content in the liver. Our results suggest that Se-EYP may be used as selenium-enriched ingredients in functional food products.

Sulfur Migration Enhanced Proton-Coupled Electron Transfer for Efficient CO2 Desorption with Core-Shelled C@Mn3O4.

Transforming hazardous species into active sites by ingenious material design was a promising and positive strategy to improve catalytic reactions in industrial applications. To synergistically address the issue of sluggish CO2 desorption kinetics and SO2-poisoning solvent of amine scrubbing, we propose a novel method for preparing a high-performance core-shell C@Mn3O4 catalyst for heterogeneous sulfur migration and in situ reconstruction to active -SO3H groups, and thus inducing an enhanced proton-coupled electron transfer (PCET) effect for CO2 desorption. As anticipated, the rate of CO2 desorption increases significantly, by 255%, when SO2 is introduced. On a bench scale, dynamic CO2 capture experiments reveal that the catalytic regeneration heat duty of SO2-poisoned solvent experiences a 32% reduction compared to the blank case, while the durability of the catalyst is confirmed. Thus, the enhanced PCET of C@Mn3O4, facilitated by sulfur migration and simultaneous transformation, effectively improves the SO2 resistance and regeneration efficiency of amine solvents, providing a novel route for pursuing cost-effective CO2 capture with an amine solvent.

Enhancing CO catalytic oxidation performance over Cu-doping manganese oxide octahedral molecular sieves catalyst.

The CO oxidation catalytic activity of catalysts is strongly influenced by the oxygen vacancy defects (OVDs) concentration and the valence state of active metal. Herein, a defect engineering approach was implemented to enhance the oxygen vacancy defects and to modify the valence of metal ions in manganese oxide octahedral molecular sieves (OMS-2) by the introduction of copper (Cu). The characterization and theoretical calculation results reveal that the incorporation of Cu2+ ion into the OMS-2 structure led to a rise in specific surface area and pore volume, weakening of Mn-O bonds, higher proportion of the low-coordinated oxygen species adsorbed in oxygen vacancies (Oads) and an increase in the average oxidation state of manganese. These structural modifications were discovered to considerably reduce the apparent activation energy (Ea), thus ultimately significantly enhancing the CO oxidation activity (T99 at 148 ℃at GHSV = 13,200 h-1) than the original OMS-2 (T99 = 215 ℃ at GHSV = 13,200 h-1). Furthermore, In-situ diffuse reflectance infrared Fourier transform (DRIFT) and In-situ near-ambient pressure X-ray photoelectron spectroscopy (in situ NAP-XPS) results indicate that the bimetallic synergy enhanced by doping strategy accelerates the conversion of oxygen to chemisorbed oxygen species and the reaction rate of CO oxidation through Mn3++Cu2+↔Mn4++Cu+ redox cycle. The findings of this study offer novel perspectives on the design of catalysts with exceptional performance in CO oxidation.

The Effect of Feeding Sequence on the Structure and Properties of the Ethylene/1-Octene Copolymer in the Semi-Continuous Polymerization Reaction System.

The performance of ethylene/1-octene copolymer primarily depends on the microstructure of the polymer chain. This study employed a new method to control the inter-distribution of hexyl chain branches directly on the backbone of the ethylene/1-octene copolymer. Three ethylene/1-octene copolymers with different inter-distributions of hexyl chain branches were synthesized using [Me2Si(C5Me4) (NtBu)] TiCl2 (Ti-CGC) by different feeding sequences in the semi-continuous polymerization reaction system. The three copolymers were named according to the feeding sequence of the materials: ethylene/1-octene/Ti-CGC (EOC), 1-octene/Ti-CGC/ethylene (OCE), and ethylene/Ti-CGC/1-octene (ECO), respectively. The structure and properties of the copolymers were characterized using HT-GPC, 13C-NMR, DSC, WAXD, DMA, MI, and Uniaxial Tension Test. The results showed that the feeding sequence greatly affected the comonomer distribution of the molecular chains, molecular weight, molecular weight distribution, and chemical composition of the copolymers, consequently influencing their thermal performance and mechanical properties. Thus, it is probable that one could obtain an ethylene/1-octene copolymer with designed properties by controlling the feeding sequence during the ethylene/1-octene semi-continuous copolymerization process.

Satellite Cell-Derived Exosomes: A Novel Approach to Alleviate Skeletal Muscle Atrophy and Fibrosis.

Skeletal muscle atrophy coincides with extensive fibrous tissue hyperplasia in muscle-atrophied patients, and fibrous tissue plays a vital role in skeletal muscle function and hinders muscle fiber regeneration. However, effective drugs to manage skeletal muscle atrophy and fibrosis remain elusive. This study isolated and characterized exosomes derived from skeletal muscle satellite cells (MuSC-Exo). The study investigated their effects on denervated skeletal muscle atrophy and fibrosis in Sprague Dawley (SD) rats via intramuscular injection. MuSC-Exo demonstrated the potential to alleviate skeletal muscle atrophy and fibrosis. The underlying mechanism using single-cell RNA sequencing data and functional analysis are analyzed. Mechanistic studies reveal close associations between fibroblasts and myoblasts, with the transforming growth factor ß1 (TGF-ß1)-Smad3-Pax7 axis governing fibroblast activation in atrophic skeletal muscle. MuSC-Exo intervention inhibited the TGF-ß1/Smad3 pathway and improved muscle atrophy and fibrosis. In conclusion, MuSC-Exo-based therapy may represent a novel strategy to alleviate skeletal muscle atrophy and reduce excessive fibrotic tissue by targeting Pax7 through the TGF-ß1/Smad3 pathway.

Astrocyte-derived exosomes-transported miRNA-26a-5p ameliorates sevoflurane-induced cognitive dysfunction in aged mice.

Prolonged sevoflurane anesthesia is the primary factor contributing to the development of perioperative neurocognitive disorders (PND). Recent studies have highlighted neuronal apoptosis and abnormal dendritic structures as crucial features of PND. Astrocytes-derived exosomes (ADEs) have been identified as carriers of microRNAs (miRNAs), playing a vital role in cell-to-cell communication through transmitting genetic material. Nevertheless, the specific mechanisms by which miRNAs in ADEs contribute to sevoflurane-induced cognitive deficit are currently unknown. Through a series of in vivo and in vitro experiments, we demonstrated that ADEs contributed to improved neurocognitive outcomes by reducing neuronal apoptosis and promoting dendritic development. Our miRNA microarray analysis revealed a significant increase in the expression level of miR-26a-5p within ADEs. Furthermore, we identified NCAM as the downstream target gene of miR-26a-5p. Subsequent gain- and loss-of-function experiments were conducted to validate the role of the miR-26a-5p/NCAM axis. Finally, we found that the AKT/GSK3-ß/CRMP2 signaling pathway was involved in regulating neurons through exosomal miR-26a-5p. Taken together, our findings suggest that the treatment with miR-26a-5p in ADEs can improve neurocognitive outcomes induced by long-term sevoflurane anesthesia, suggesting a promising approach for retarding the progress of PND.

An optimum study on the laser scanning confocal microscopy techniques for BiFC assay using plant protoplast.

BACKGROUND: The bimolecular fluorescence complementation (BiFC) assay is commonly used for investigating protein-protein interactions. While several BiFC detection systems have been developed, there is a limited amount of research focused on using laser scanning confocal microscope (LSCM) techniques to observe protoplasts. Protoplasts are more susceptible to damage and instability compared to their original cell state due to the preparation treatments they undergo, which makes it challenging for researchers to manipulate them during observation under LSCMs. Therefore, it is crucial to utilize microscope techniques properly and efficiently in BiFC assays. RESULTS: When the target fluorescence is weak, the autofluorescence of chloroplast particles in protoplasts can interfere with the detection of BiFC signals localized in the nuclear region. Spectrum analysis revealed that chloroplast autofluorescence can be excited by lasers of various types, with the highest fluorescence signal observed at around 660 nm. Furthermore, our investigation into the impact of different pipette tips on the integrity of protoplast samples indicated that the utilization of cut tips with larger openings can mitigate cell breakage. We presented a workflow of LSCM techniques for investigating protoplast BiFC and discussed the microscopic manipulation involved in sample preparation and image capturing. CONCLUSION: When the BiFC signals are weak, they may be affected by chloroplast autofluorescence. However, when used properly, the autofluorescence of chloroplasts can serve as an excellent internal marker for effectively distinguishing other signals. In combination with other findings, this study can provide valuable reference for researchers conducting BiFC assays and related studies.