Development of the CRISPR-Cas12a system for editing of Pseudomonas aeruginosa phages.

Pseudomonas aeruginosa is a common opportunistic pathogen. The potential efficacy of phage therapy has attracted the attention of researchers, but efficient gene-editing tools are lacking, limiting the study of their biological properties. Here, we designed a type V CRISPR-Cas12a system for the gene editing of P. aeruginosa phages. We first evaluated the active cutting function of the CRISPR-Cas12a system in vitro and discovered that it had a higher gene-cutting efficiency than the type II CRISPR-Cas9 system in three different P. aeruginosa phages. We also demonstrated the system's ability to precisely edit genes in Escherichia coli phages, Salmonella phages, and P. aeruginosa phages. Using the aforementioned strategies, non-essential P. aeruginosa phage genes can be efficiently deleted, resulting in a reduction of up to 5,215 bp (7.05%). Our study has provided a rapid, efficient, and time-saving tool that accelerates progress in phage engineering.

Au24Cd Nanoenzyme Coating for Enhancing Electrochemical Sensing Performance of Metal Wire Microelectrodes.

Dopamine (DA), ascorbic acid (AA), and uric acid (UA) are crucial neurochemicals, and their abnormal levels are involved in various neurological disorders. While electrodes for their detection have been developed, achieving the sensitivity required for in vivo applications remains a challenge. In this study, we proposed a synthetic Au24Cd nanoenzyme (ACNE) that significantly enhanced the electrochemical performance of metal electrodes. ACNE-modified electrodes demonstrated a remarkable 10-fold reduction in impedance compared to silver microelectrodes. Furthermore, we validated their excellent electrocatalytic activity and sensitivity using five electrochemical detection methods, including cyclic voltammetry, differential pulse voltammetry, square-wave pulse voltammetry, normal pulse voltammetry, and linear scanning voltammetry. Importantly, the stability of gold microelectrodes (Au MEs) modified with ACNEs was significantly improved, exhibiting a 30-fold enhancement compared to Au MEs. This improved performance suggests that ACNE functionalization holds great promise for developing micro-biosensors with enhanced sensitivity and stability for detecting small molecules.

Advances in Multifunctional Electronic Catheters for Precise and Intelligent Diagnosis and Therapy in Minimally Invasive Surgery.

The advent of catheter-based minimally invasive surgical instruments has provided an effective means of diagnosing and treating human disease. However, conventional medical catheter devices are limited in functionalities, hindering their ability to gather tissue information or perform precise treatment during surgery. Recently, electronic catheters have integrated various sensing and therapeutic technologies through micro/nanoelectronics, expanding their capabilities. As micro/nanoelectronic devices become more miniaturized, flexible, and stable, electronic surgical catheters are evolving from simple tools to multiplexed sensing and theranostics for surgical applications. The review on multifunctional electronic surgical catheters is lacking and thus is not conducive to the reader's comprehensive understanding of the development trend in this field. This review covers the advances in multifunctional electronic catheters for precise and intelligent diagnosis and therapy in minimally invasive surgery. It starts with the summary of clinical minimally invasive surgical instruments, followed by the background of current clinical catheter devices for sensing and therapeutic applications. Next, intelligent electronic catheters with integrated electronic components are reviewed in terms of electronic catheters for diagnosis, therapy, and multifunctional applications. It highlights the present status and development potential of catheter-based minimally invasive surgical devices, while also illustrating several significant challenges that remain to be overcome.

Multichannel microneedle dry electrode patches for minimally invasive transdermal recording of electrophysiological signals.

The collection of multiple-channel electrophysiological signals enables a comprehensive understanding of the spatial distribution and temporal features of electrophysiological activities. This approach can help to distinguish the traits and patterns of different ailments to enhance diagnostic accuracy. Microneedle array electrodes, which can penetrate skin without pain, can lessen the impedance between the electrodes and skin; however, current microneedle methods are limited to single channels and cannot achieve multichannel collection in small areas. Here, a multichannel (32 channels) microneedle dry electrode patch device was developed via a dimensionality reduction fabrication and integration approach and supported by a self-developed circuit system to record weak electrophysiological signals, including electroencephalography (EEG), electrocardiogram (ECG), and electromyography (EMG) signals. The microneedles reduced the electrode-skin contact impedance by penetrating the nonconducting stratum corneum in a painless way. The multichannel microneedle array (MMA) enabled painless transdermal recording of multichannel electrophysiological signals from the subcutaneous space, with high temporal and spatial resolution, reaching the level of a single microneedle in terms of signal precision. The MMA demonstrated the detection of the spatial distribution of ECG, EMG and EEG signals in live rabbit models, and the microneedle electrode (MNE) achieved better signal quality in the transcutaneous detection of EEG signals than did the conventional flat dry electrode array. This work offers a promising opportunity to develop advanced tools for neural interface technology and electrophysiological recording.

Integrated metabolomic and metagenomic strategies shed light on interactions among planting environments, rhizosphere microbiota, and metabolites of tobacco in Yunnan, China.

Changes in climatic factors and rhizosphere microbiota led plants to adjust their metabolic strategies for survival under adverse environmental conditions. Changes in plant metabolites can mediate crop growth and development and interact with rhizosphere microbiota of the plant rhizosphere. To understand the interactions among environmental factors, rhizosphere microbiota, and metabolites of tobacco, a study was conducted by using integrated metagenomic and metabolomic strategies at four typical representative tobacco planting sites in Yunnan, China. The results showed that the agronomical and biochemical traits were significantly affected by temperature, precipitation (PREP), soil pH, and altitude. Correlation analyses revealed a significant positive correlation of temperature with length, width, and area of the leaf, while PREP correlated with plant height and effective leaf numbers. Furthermore, total sugar and reducing sugar contents of baked leaves were significantly higher, while the total nitrogen and total alkaloid levels were lower in tobacco leaves at site with low PREP. A total of 770 metabolites were detected with the highest number of different abundant metabolites (DMs) at Chuxiong (CX) with low PREP as compared to the other three sites, in which secondary metabolites were more abundant in both leaves and roots of tobacco. A total of 8,479 species, belonging to 2,094 genera with 420 individual bins (including 13 higher-quality bins) harboring 851,209 CDSs were detected. The phyla levels of microorganisms such as Euryarchaeota, Myxococcota, and Deinococcota were significantly enriched at the CX site, while Pseudomonadota was enriched at the high-temperature site with good PREP. The correlation analyses showed that the metabolic compounds in low-PREP site samples were positively correlated with Diaminobutyricimonas, Nissabacter, Alloactinosynnema, and Catellatospora and negatively correlated with Amniculibacterium, Nordella, Noviherbaspirillum, and Limnobacter, suggesting that the recruitment of Diaminobutyricimonas, Nissabacter, Alloactinosynnema, and Catellatospora in the rhizosphere induces the production and accumulation of secondary metabolites (SMs) (e.g., nitrogen compounds, terpenoids, and phenolics) for increasing drought tolerance with an unknown mechanism. The results of this study may promote the production and application of microbial fertilizers and agents such as Diaminobutyricimonas and Alloactinosynnema to assemble synthetic microbiota community or using their gene resources for better cultivation of tobacco as well as other crops in drought environments.

Unexpected omega-3 activities in intracellular lipolysis and macrophage foaming revealed by fluorescence lifetime imaging.

Omega-3 polyunsaturated fatty acids (PUFA) found primarily in fish oil have been a popular supplement for cardiovascular health because they can substantially reduce circulating triglyceride levels in the bloodstream to prevent atherosclerosis. Beyond this established extracellular activity, here, we report a mode of action of PUFA, regulating intracellular triglyceride metabolism and lipid droplet (LD) dynamics. Real-time imaging of the subtle and highly dynamic changes of intracellular lipid metabolism was enabled by a fluorescence lifetime probe that addressed the limitations of intensity-based fluorescence quantifications. Surprisingly, we found that among omega-3 PUFA, only docosahexaenoic acid (DHA) promoted the lipolysis in LDs and reduced the overall fat content by approximately 50%, and consequently helped suppress macrophage differentiation into foam cells, one of the early steps responsible for atherosclerosis. Eicosapentaenoic acid, another omega-3 FA in fish oil, however, counteracted the beneficial effects of DHA on lipolysis promotion and cell foaming prevention. These in vitro findings warrant future validation in vivo.

Technological Advances of Wearable Device for Continuous Monitoring of In Vivo Glucose.

Managing diabetes is a chronic challenge today, requiring monitoring and timely insulin injections to maintain stable blood glucose levels. Traditional clinical testing relies on fingertip or venous blood collection, which has facilitated the emergence of continuous glucose monitoring (CGM) technology to address data limitations. Continuous glucose monitoring technology is recognized for tracking long-term blood glucose fluctuations, and its development, particularly in wearable devices, has given rise to compact and portable continuous glucose monitoring devices, which facilitates the measurement of blood glucose and adjustment of medication. This review introduces the development of wearable CGM-based technologies, including noninvasive methods using body fluids and invasive methods using implantable electrodes. The advantages and disadvantages of these approaches are discussed as well as the use of microneedle arrays in minimally invasive CGM. Microneedle arrays allow for painless transdermal puncture and are expected to facilitate the development of wearable CGM devices. Finally, we discuss the challenges and opportunities and look forward to the biomedical applications and future directions of wearable CGM-based technologies in biological research.

Systematic review and meta-analysis of current evidence in endograft therapy for spontaneous isolated superior mesenteric artery dissection.

The role of endovascular stent therapy (EST) in the treatment of spontaneous isolated superior mesenteric artery dissection (SISMAD) has gained momentum in recent years but remains controversial. We gathered research examining the advantages and disadvantages of EST for SISMAD patients. Primary outcomes involved both immediate and long-term results. Random or fixed effect models were used for effect size (ES) calculation with 95% confidence interval (CI) based on 50% heterogeneity threshold. Our analysis incorporated data from 21 studies including 611 SISMAD cases treated by EST. Our findings show a complication rate of approximately 1% following EST (95%CI 0.01-0.02, I2 = 0%, P = 0.97), with a bare minimum mortality rate of < 1% (95%CI 0.00-0.01, I2 = 0%, P > 0.05) and a reintervention rate of < 1% (95%CI 0.00-0.01, I2 = 0%, P = 0.89). We also found technique success and symptom resolution approaching 94% and 99%, respectively, in the immediate postoperative phase. In the long run, we observed a recurrence of symptoms at 3% (95%CI 0.00-0.06, I2 = 58.6%, P < 0.01), creation of new dissections at 1% (95%CI 0.00-0.02, I2 = 0%, P = 0.73), aneurysm progression at 2% (95%CI 0.00-0.03, I2 = 42.7%, P = 0.12), reintervention due to complications at 3% (95%CI 0.00-0.05, I2 = 0%, P = 0.43) and stenotic stents at 12% (95%CI 0.04-0.23, I2 = 77.5%, P < 0.01). Nevertheless, high levels of stent patency 98% (95% CI 0.97-1.00, I2 = 0%, P = 0.51) and complete remodeling 88% (95% CI 0.82-0.94, I2 = 65.5%, P < 0.05) were observed postoperatively. Overall, EST presents minimal complications and promising long-term outcomes for SISMAD, although the prevalence of stent stenosis requires further attention.

Coupling of nanostraws with diverse physicochemical perforation strategies for intracellular DNA delivery.

Effective intracellular DNA transfection is imperative for cell-based therapy and gene therapy. Conventional gene transfection methods, including biochemical carriers, physical electroporation and microinjection, face challenges such as cell type dependency, low efficiency, safety concerns, and technical complexity. Nanoneedle arrays have emerged as a promising avenue for improving cellular nucleic acid delivery through direct penetration of the cell membrane, bypassing endocytosis and endosome escape processes. Nanostraws (NS), characterized by their hollow tubular structure, offer the advantage of flexible solution delivery compared to solid nanoneedles. However, NS struggle to stably self-penetrate the cell membrane, resulting in limited delivery efficiency. Coupling with extra physiochemical perforation strategies is a viable approach to improve their performance. This study systematically compared the efficiency of NS coupled with polyethylenimine (PEI) chemical modification, mechanical force, photothermal effect, and electric field on cell membrane perforation and DNA transfection. The results indicate that coupling NS with PEI modification, mechanical force, photothermal effects provide limited enhancement effects. In contrast, NS-electric field coupling significantly improves intracellular DNA transfection efficiency. This work demonstrates that NS serve as a versatile platform capable of integrating various physicochemical strategies, while electric field coupling stands out as a form worthy of primary consideration for efficient DNA transfection.

Young evolutionary origins of dioecy in the genus Asparagus.

PREMISE: Dioecy (separate sexes) has independently evolved numerous times across the angiosperm phylogeny and is recently derived in many lineages. However, our understanding is limited regarding the evolutionary mechanisms that drive the origins of dioecy in plants. The recent and repeated evolution of dioecy across angiosperms offers an opportunity to make strong inferences about the ecological, developmental, and molecular factors influencing the evolution of dioecy, and thus sex chromosomes. The genus Asparagus (Asparagaceae) is an emerging model taxon for studying dioecy and sex chromosome evolution, yet estimates for the age and origin of dioecy in the genus are lacking. METHODS: We use plastome sequences and fossil time calibrations in phylogenetic analyses to investigate the age and origin of dioecy in the genus Asparagus. We also review the diversity of sexual systems present across the genus to address contradicting reports in the literature. RESULTS: We estimate that dioecy evolved once or twice approximately 2.78-3.78 million years ago in Asparagus, of which roughly 27% of the species are dioecious and the remaining are hermaphroditic with monoclinous flowers. CONCLUSIONS: Our findings support previous work implicating a young age and the possibility of two origins of dioecy in Asparagus, which appear to be associated with rapid radiations and range expansion out of Africa. Lastly, we speculate that paleoclimatic oscillations throughout northern Africa may have helped set the stage for the origin(s) of dioecy in Asparagus approximately 2.78-3.78 million years ago.

Genomic analysis and experimental pathogenic characterization of Riemerella anatipestifer isolates from chickens in China.

Waterfowl have a high likelihood of being infected with Riemerella anatipestifer. Although the pathogen is found in domestic ducks, turkeys, geese, and wild birds, there is little information available about the consequences of infection during egg laying and hatching in chickens. Here, we present the first report of a novel sequence type of R. anatipestifer S63 isolated from chickens in China. On the basis of pan-genome analysis, we showed S63's genome occupies a distinct branch with other R. anatipestifer isolates from other hosts. Galleria mellonella larval tests indicated that S63 is less virulent than R. anatipestifer Ra36 isolated from ducks. Ducks and hens are susceptible to S63 infection. There is no mortality rate for chickens or ducks, but adult chickens experience neurological symptoms that reduce egg production and hatching rates. In chickens, S63 might be passed vertically from parents to offspring, resulting in "jelly-like" lifeless embryos. Using quantitative PCR, S63 was detected in the brain, liver, reproductive organs, and embryos. As far as we know, this is the first report of R. anatipestifer in hens, a disease that can reduce egg productivity, lower hatching rates, and produce jelly-like lifeless embryos, and the first report to raise the possibility that hens can be infected by roosters via semen.

Integrated electronic/fluidic microneedle system for glucose sensing and insulin delivery.

Background: Precise and dynamic blood glucose regulation is paramount for both diagnosing and managing diabetes. Continuous glucose monitoring (CGM) coupled with insulin pumps forms an artificial pancreas, enabling closed-loop control of blood glucose levels. Indeed, this integration necessitates advanced micro-nano fabrication techniques to miniaturize and combine sensing and delivery modules on a single electrode. While microneedle technology can mitigate discomfort, concerns remain regarding infection risk and potential sensitivity limitations due to their short needle length. Methods: This study presents the development of an integrated electronic/fluidic microneedle patch (IEFMN) designed for both glucose sensing and insulin delivery. The use of minimally invasive microneedles mitigates nerve contact and reduces infection risks. The incorporation of wired enzymes addresses the issue of "oxygen deprivation" during glucose detection by decreasing the reliance on oxygen. The glucose-sensing electrodes employ wired enzyme functionalization to achieve lower operating voltages and enhanced resilience to sensor interference. The hollow microneedles' inner channel facilitates precise drug delivery for blood glucose regulation. Results: Our IEFMN-based system demonstrated high sensitivity, selectivity, and a wide response range in glucose detection at relatively low voltages. This effectively reduced interference from both external and internal active substances. The microneedle array ensured painless and minimally invasive skin penetration, while wired enzyme functionalization not only lowered sensing potential but also improved glucose detection accuracy. In vivo, experiments conducted in rats showed that the device could track subcutaneous glucose fluctuations in real-time and deliver insulin to regulate blood glucose levels. Conclusions: Our work suggests that the IEFMN-based system, developed for glucose sensing and insulin delivery, exhibits good performance during in vivo glucose detection and drug delivery. It holds the potential to contribute to real-time, intelligent, and controllable diabetes management.

Genome sequence of Shiga toxin-producing Escherichia coli jumbo bacteriophage vB_EcoM_JNE01.

Bacteriophage vB_EcoM_JNE01 was isolated from chicken farm sewage using Escherichia coli O157:H7 as the host bacteria. The total length of the vB_EcoM_JNE01 genome is 355,583 bp, with 584 open reading frames and 36% G+C content. It shares an 80% nucleotide identity with 59% query coverage with the bacteriophage PBECO4 (NC_027364).

Production of chitosan from shrimp shell using ultrasound followed by subcritical water hydrolysis.

The interest in shrimp shell valorization has been growing in line with sustainability goals. Therefore, the main objective of this study was to obtain chitosan from shrimp shell using ultrasound followed by subcritical water treatment. Ultrasonication of shells was performed at 600 and 1200 W for 5 min. Then, shells were hydrolyzed at 140-260 °C and 50 bar for 10-60 min followed by demineralization using citric acid, bleaching using hydrogen peroxide and deacetylation using sodium hydroxide solution. The highest deproteination (80.93 %) was obtained by ultrasonication at 1200 W/5 min followed by subcritical water hydrolysis at 260 °C/50 bar/60 min, where the residue with a yield of 10.56 %, whiteness index of 60.42, degree of deacetylation of 64.27 %, relative crystallinity of 32.66 % and similar functional groups to the commercial sample was obtained. These results indicated that the combination of ultrasound with subcritical water is promising to valorize shrimp shell towards production of value-added compounds.

Profiles of phenolics and their synthetic pathways in Asparagus officinalis L.

The synthetic pathways of some phenolics compounds in asparagus have been reported, however, the diversified phenolics compounds including their modification and transcription regulation remains unknown. Thus, multi-omics strategies were applied to detect the phenolics profiles, contents, and screen the key genes for phenolics biosynthesis and regulation in asparagus. A total of 437 compounds, among which 204 phenolics including 105 flavonoids and 82 phenolic acids were detected with fluctuated concentrations in roots (Rs), spears (Ss) and flowering twigs (Fs) of the both green and purple cultivars. Based on the detected phenolics profiles and contents correlated to the gene expressions of screened synthetic enzymes and regulatory TFs, a full phenolics synthetic pathway of asparagus was proposed for the first time, essential for future breeding of asparagus and scaled healthy phenolics production using synthetic biological strategies.

Phage Tail Fiber Protein as a Specific Probe for Recognition of Shiga Toxin-Producing Escherichia coli O91, O103, and O111.

The ability to quickly identify specific serotypes of Shiga toxin-producing Escherichia coli (STEC) could facilitate the monitoring and control of STEC pathogens. In this study, we identified the receptors and receptor-binding proteins (RBPs) of three novel phages (pO91, pO103, and pO111) isolated from hospital wastewater. Recombinant versions of these RBPs (pO91-ORF43, pO103-ORF42, and pO111-ORF8) fused to a fluorescent reporter protein were then constructed. Both fluorescence microscopy and transmission electron microscopy showed that all three recombinant RBPs were bound to the bacterial surface. Indirect enzyme-linked immunosorbent assay was used to verify that each recombinant RBP bound specifically to E. coli O91, O103, or O111, but not to any of the 83 strains of E. coli with different O-antigens, nor to 10 other bacterial species that were tested. The recombinant RBPs adsorbed to their respective host bacteria within 10 min of incubation. The minimum concentration of bacteria required for detection by the recombinant RBPs was 33 colony-forming units (CFU)/mL (range: 3.3 × 10 to 3.3 × 108 CFU/mL). Furthermore, each recombinant RBP was also able to detect bacteria in lettuce, chicken breast meat, and infected mice, indicating that their usage will facilitate the detection of STEC and may help to reduce the spread of STEC-related infections and diseases.

Metabolic syndrome prediction model using Bayesian optimization and XGBoost based on traditional Chinese medicine features.

Metabolic syndrome (MetS) has a high prevalence and is prone to many complications. However, current MetS diagnostic methods require blood tests that are not conducive to self-testing, so a user-friendly and accurate method for predicting MetS is needed to facilitate early detection and treatment. In this study, a MetS prediction model based on a simple, small number of Traditional Chinese Medicine (TCM) clinical indicators and biological indicators combined with machine learning algorithms is investigated. Electronic medical record data from 2040 patients who visited outpatient clinics at Guangdong Chinese medicine hospitals from 2020 to 2021 were used to investigate the fusion of Bayesian optimization (BO) and eXtreme gradient boosting (XGBoost) in order to create a BO-XGBoost model for screening nineteen key features in three categories: individual bio-information, TCM indicators, and TCM habits that influence MetS prediction. Subsequently, the predictive diagnostic model for MetS was developed. The experimental results revealed that the model proposed in this paper achieved values of 93.35 %, 90.67 %, 80.40 %, and 0.920 for the F1, sensitivity, FRS, and AUC metrics, respectively. These values outperformed those of the seven other tested machine learning models. Finally, this study developed an intelligent prediction application for MetS based on the proposed model, which can be utilized by ordinary users to perform self-diagnosis through a web-based questionnaire, thereby accomplishing the objective of early detection and intervention for MetS.

Personalized Machine Learning-Coupled Nanopillar Triboelectric Pulse Sensor for Cuffless Blood Pressure Continuous Monitoring.

A wearable system that can continuously track the fluctuation of blood pressure (BP) based on pulse signals is highly desirable for the treatments of cardiovascular diseases, yet the sensitivity, reliability, and accuracy remain challenging. Since the correlations of pulse waveforms to BP are highly individualized due to the diversity of the patients' physiological characteristics, wearable sensors based on universal designs and algorithms often fail to derive BP accurately when applied on individual patients. Herein, a wearable triboelectric pulse sensor based on a biomimetic nanopillar layer was developed and coupled with Personalized Machine Learning (ML) to provide accurate and continuous monitoring of BP. Flexible conductive nanopillars as the triboelectric layer were fabricated through soft lithography replication of a cicada wing, which could effectively enhance the sensor's output performance to detect weak signal characteristics of pulse waveform for BP derivation. The sensors were coupled with a personalized Partial Least-Squares Regression (PLSR) ML to derive unknown BP based on individual pulse characteristics with reasonable accuracy, avoiding the issue of individual variability that was encountered by General PLSR ML or formula algorithms. The cuffless and intelligent design endow this ML-sensor as a highly promising platform for the care and treatments of hypertensive patients.

Tumor-Targeted Fluorescent/Photoacoustic Imaging of Legumain Activity In Vivo.

Legumain has been identified as a target for diagnosis and treatment of associated cancers. Therefore, real-time imaging of legumain activity in vivo is helpful in diagnosing and evaluating therapeutic efficacy of associated cancers. Fluorescent/photoacoustic (FL/PA) dual-modal imaging developed rapidly because of its good sensitivity and spatial resolution. As far as we know, a tumor-targeted probe for FL/PA imaging of legumain activity in vivo has not been reported. Hence, we intended to develop a tumor-targeted hemicyanine (HCy) probe (HCy-AAN-Bio) for FL/PA imaging of legumain in vivo. The control probe HCy-AAN does not have tumor-targeting ability. Legumain can specifically cleave HCy-AAN-Bio or HCy-AAN with the generation of FL/PA signal while more HCy-AAN-Bio could be recognized by legumain than HCy-AAN with higher sensitivity in vitro. Due to the tumor-targeting ability, HCy-AAN-Bio could image 4T1 cells with an additional 1.3-fold FL enhancement and 1.9-fold PA enhancement than HCy-AAN. In addition, HCy-AAN-Bio could image legumain activity in vivo with an additional 1.5-fold FL enhancement and 1.9-fold PA enhancement than HCy-AAN. We expected that HCy-AAN-Bio will be a powerful tool for early diagnosis of associated cancer.

Synchronous Photoactivation-Imaging Fluorophores Break Limitations of Photobleaching and Phototoxicity in Live-cell Microscopy.

Fluorescence microscopy is one of the most important tools in the studies of cell biology and many other fields, but two fundamental issues, photobleaching and phototoxicity, associated with the fluorophores have still limited its use for long-term and strong-illumination imaging of live cells. Here, we report a new concept of fluorophore engineering chemistry, synchronous photoactivation-imaging (SPI) fluorophores, activating and exciting fluorophores by a single light source to thus avoid the repeated switches between activation and excitation lights. The chemically reconstructed, nonemissive fluorophores can be photolyzed to allow continuous replenishing of "bright-state" probes detectable by standard fluorescent microscopes in the imaging process so as to bypass the photobleaching barrier to greatly extend the imaging period. Equally importantly, SPI fluorophores substantially reduce photocytotoxicity due to the scavenging of reactive oxygen species (ROS) by a photoactivable group and the slow release of "bright-state" probes to minimize ROS generation. Using SPI fluorophores, the time-lapsed confocal (>16 h) and super-resolution (>3 h) imaging of subcellular organelles under intensive illumination (50 MW/cm2) were achieved in live cells.