Fluorescence Detection Research Articles

Evaluation and optimization of measurement conditions for heavy ions with fluorescent nuclear track detectors.

In applying fluorescent nuclear track detectors for heavy ion measurement, the fluorescence intensity directly influences linear energy transfer (LET) estimation. However, the conventional analysis highly depends on the analyzed depth and the reference frames from the optical readout. To improve the accuracy of measurements of heavy ions, the gradient of intensity and moving average fluorescence intensity were acquired and calculated to optimize the measurement conditions. The results indicate that the intensity gradient is an alternative quantity to discriminate ions with different LET, and the moving average fluorescence intensity is readily differentiated among individual ions without overlapping values in fluorescence intensity. The scanned range of 25-45μm is suggested to be the most suitable depth for measurement. To exclude the impact of the variation in fluorescence intensity on evaluation, it is recommended to use the quantities calculated in moving averages with depth in the future.

Genotype and Organ-Specific Variability in Antioxidant Capacities as Well as Polyamine and Osmolyte Levels in Eleven Lisianthus (Eustoma grandiflorum Raf.) Cultivars with Different Flowering Periods

Lisianthus (Eustoma grandiflorum Raf. Shinn.) is a valued plant known for its diverse flower colors and long vase life. Despite considerable research on the physiological roles of osmolytes, polyamines, and phenolic compounds, there is a lack of understanding regarding their specific accumulation patterns across various lisianthus cultivars and organs. This study aims to compare eleven lisianthus cultivars with varying flowering periods according to their accumulation of osmolytes, polyamines, phenolic content, and antioxidant capacities and measure their resistance to abiotic stress factors. High-performance liquid chromatography coupled with fluorescent detection was employed to quantify putrescine (PUT), spermidine (SPD), and spermine (SPM). In addition, proline (PRO), glycine betaine (GB), antioxidant capacities, phenolic content, and flavonoid contents were assessed spectrophotometrically. This comprehensive analysis allowed for a detailed understanding of the biochemical markers. The result indicates a significant genotype and organ-dependent variation in accumulation patterns of inspected metabolites and antioxidant activities. The petals of Rosita Green exhibited the highest levels of phenols and flavonoids, whereas the petals of Rosita Blue Picote demonstrated the highest concentrations of osmolytes. Polyamines were found to be more concentrated in the petals than in the leaves. The average values indicated high levels of polyamines in the Mariachi Carmine (PUT 186.72 nmol g−1 DW) and Mariachi Pink (SPD 227.4 nmol g−1 DW) cultivars. These findings underscore the significance of inspected compounds in stress tolerance among cultivars with different flowering periods, providing insights for optimizing cultivation strategies for lisianthus.

Detection of Ochratoxin A in Maize and Its Potential Impact on Avian Pathology in Romanian Farms

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that commonly contaminates maize, posing significant health risks to both poultry and humans. In this study, a rapid and sensitive method utilizing ultra-performance liquid chromatography coupled with fluorescence detection (UPLC-FLD) was developed for the quantification of OTA levels in maize. The method utilizes immunoaffinity column purification for improved specificity. Accuracy and precision were validated in line with European Union Reference Laboratory (EURL-MP) guidelines, meeting regulatory standards for linearity, trueness, detection and quantification limits, precision, and uncertainty, as per European Commission Regulation (EC) No. 401/2006 and its amendments. The method demonstrated an average recovery rate of 116.78% for maize, with RSDwR values (within-laboratory reproducibility) of 12.72%. Furthermore, OTA occurrence and its possible effects were investigated in several farms in South Romania, where necropsy and histopathological analyses of poultry revealed severe kidney damage, including renal tubular degeneration.

Pyrene Based Schiff Base for Cascade Fluorescent "On-Off" Detection of Aluminium(III) and Fluoride Ions and its Applications in Live Cells Imaging.

An easy-to-prepare pyrene-based Schiff base PNZ was synthesized by condensing equimolar amount of 1-pyrenebutyric hydrazide with 2-hydroxy-naphthaldehyde, and employed as a fluorescent chemosensor for in-situ cascade detection of aluminium (Al3+) and fluoride (F-) ions. In DMSO:H2O (1 : 1, v/v), the weakly emissive PNZ showed a significant fluorescence enhancement at 455 nm selectively upon the addition of Al3+ due to the complexation-induced formation of a pyrene excimer. Schiff base PNZ and Al3+ formed a complex in 2 : 1 binding ratio with the estimated binding constants of K1:1=9826.01 M-1 and K2:1=3188.49 M-1. The sensing mechanism was explored by performing quantum mechanical calculations and 1H-NMR titration of PNZ with Al3+. The in-situ formed PNZ-Al3+ complex species enabled the fluorescent turn-off detection of F-. Using PNZ and PNZ-Al3+, the concentrations of Al3+ and F- ions can be detected down to 2.89×10-7 M and 1.88×10-7 M, respectively. The cytotoxicity of the PNZ and its ability to bioimage Al3+ and F- ions was examined in the human cervical cancer cell line. Finally, the receptor PNZ was applied for the quantification of Al3+ and F- ions in various real samples, such as tap water, river water, rainwater, mouthwash, and toothpaste.

Fluorescent Sensing for the Detection and Quantification of Sulfur-Containing Gases.

Sulfur-containing gases, such as H2S and SO2, play significant roles in a multitude of biological processes affecting human life and health. Precise and efficient detection of these gases is therefore crucial for advancing one's understanding of their biological roles and developing effective diagnostic strategies. Fluorescent sensing offers a highly sensitive and versatile approach for detecting these gases. This Review examines the recent advances in the fluorescent detection of H2S and SO2, highlighting the key mechanisms involved in fluorescence signal transduction, including changes in intensity and wavelength shifts. The diverse array of probe molecules employed for this purpose, including those utilizing mechanisms such as nucleophilic reactions, Förster resonance energy transfer (FRET), and sulfur affinity interactions are explored. In additional to organic sensors, the focus of the Review is particularly directed toward quantum dot (QD) systems, emphasizing their tunable optical properties that hold immense potential for fluorescence sensing. Beyond the traditional III-V QDs, we delve into the emerging fluorescence sensors based on halide perovskite QDs, upconversion nanocrystals, and other novel materials. These advanced QD systems hold promise for the development of highly sensitive and cost-effective gas detectors, paving the way for significant advances in biomedical and environmental monitoring. This Review provides a comprehensive overview of the current state-of-the-art in QD-based fluorescence sensing of sulfur-containing gases and provides a multifaceted discussion comparing organic fluorescent sensors with QD sensors, highlighting the key challenges and opportunities for the integration of fluorescence sensing as it evolves. The Review aims to facilitate further research and development of innovative sensing platforms to enable more accurate and sensitive detection of these important gases.

Activation of Hedgehog pathway by circEEF2/miR-625-5p/TRPM2 axis promotes prostate cancer cell proliferation through mitochondrial stress.

The purpose of this study was to identify the role played by circEEF2 (has-circ-0048559) in prostate cancer (PCa) development and to determine the potential mechanism involved. circEEF2, miR-625-5p, and the transient receptor potential M2 channel protein (TRPM2) were determined using RT-qPCR in PCa. Cell proliferation was determined by CCK-8 assay and colony formation assay, whereas migration and invasion were assessed by Transwell assay, and apoptosis was evaluated by flow cytometry after annexin V-FITC and propidium iodide staining. The interactions between circEEF2 and miRNAs were investigated through the Circular RNA Interactome database, and the downstream targets of miR-625-5p were forecasted using TargetScan. The interaction was confirmed using both the dual luciferase reporter gene assay and RNA pull-down assay. TRPM2, Hedgehog signaling pathway proteins (GLI1 and GLI2), ubiquinone oxidase subunit B8, and cytochrome C oxidase subunit IV (COX4) were analyzed by protein blotting. JC-1 fluorescence detection was applied for mitochondrial membrane potential changes, fluorescent probe assay for intracellular ROS levels, and immunofluorescence staining for γ-H2AX expression. The role of circEEF2 in PCa tumor growth was tested by xenograft experiments. CircEEF2 expression was upregulated in PCa (p<0.05). Cells of PCa were inhibited in proliferation, migration, invasion, and enhanced in apoptosis by depleting circEEF2 (p<0.05). circEEF2 directly targeted adsorbed miR-625-5p. TRPM2 bound to miR-625-5p. Upregulating TRPM2 likewise reversed the therapeutic effect of depleting circEEF2 on cancer development in PCa cells. circEEF2 activated the Hedgehog pathway through the miR-625-5p/TRPM2 axis, promotes mitochondrial stress, and promotes PCa development in vivo. circEEF2 upregulates mitochondrial stress to promote PCa by activating the Hedgehog pathway through the miR-625-5p/TRPM2 axis.

A novel, rapid, ultrasensitive diagnosis platform for detecting Candida albicans using restriction endonuclease‐mediated real-time loop-mediated isothermal amplification

IntroductionCandida albicans (C. albicans, CA) is an essential invasive fungus in clinical diagnosis. Although several detection methods exist, none meet the need for early diagnosis. A rapid, sensitive, and specific diagnostic tool is crucial for effective prevention and control of C. albicans infections.MethodsThis study aimed to develop a new, rapid, and ultrasensitive diagnostic tool for C. albicans detection based on restriction endonuclease-mediated real-time loop-mediated isothermal amplification (ERT-LAMP-CA). The ERT-LAMP-CA technology combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence detection in a single reaction tube, which can complete a diagnosis of C. albicans in a short time (approximately 1 h).ResultsHerein, we developed the primer sequences required for ERT-LAMP-CA based on the ITS2 gene of C. albicans and found that ERT-LAMP-CA limit of detection was approximately 500 ag/μL genomic DNA and can present negative results for non-C. albicans templates. We tested sputum samples from 64 patients with suspected C. albicans infections to validate ERT-LAMP-CA applicability in clinical sample testing and found that ERT-LAMP-CA was consistent with multiplex PCR-capillary electrophoresis.DiscussionIn conclusion, ERT-LAMP-CA is a rapid, accurate, and sensitive assay with excellent potential for clinical and basic laboratory diagnosis and an efficient screening strategy.

Real-time fluorescent detection of food spoilage with doped quantum dots-anchored hydrogel sensor

Real-time fluorescent detection of food spoilage with doped quantum dots-anchored hydrogel sensor

Reversible addition-fragmentation chain transfer enhanced aggregation signal-on fluorescence detection of alkaline phosphatase.

The instability of the signal intensity of fluorescent biosensors and the false signals have been significant factors affecting the performance of biosensors. Herein, a novel signaling system is devised through the application of reversible addition-fragmentation chain transfer (RAFT) polymerization with monomers containing the tetraphenylethylene (TPE) groups. TPE exhibits an aggregation-induced emission (AIE) phenomenon in certain solvents, mainly due to the blockage of the rotation of its four benzene rings, which also exist in the aggregated state. With this property, a series of molecules are modified based on click chemistry for RAFT polymerization using Fe3O4 magnetic beads as the carriers, and stable aggregated luminescent TPE polymers are formed on the surface of magnetic beads to realize the transformation of fluorescence signal from "0" to "1". In addition, the fluorescence signal demonstrates a positive correlation with alkaline phosphatase (ALP) activity, which can be quantified by measuring the fluorescence intensity. The biosensor exhibits high sensitivity and good linearity in the range of 0.1-5 U/L, with a LOD of 0.079 U/L. Furthermore, the designed strategy demonstrated satisfactory performance in the quantitative determination of ALP activity in serum samples, indicating that the signaling system developed by combining RAFT polymerization and AIE molecules has an important application in the field of fluorescent biosensors.

Nanowire-like phosphorus modulated carbon-based iron nanozyme with oxidase-like activity for sensitive detection of choline and dye degradation

Choline is mainly supplemented through food intake, lack of choline would result in diseases like liver cirrhosis, hardening of the arteries, or neurodegenerative disorders. The accurate detection of choline is important for human health. Herein, a novel nanowire-like phosphorus/nitrogen co-doped carbon-based iron nanozyme (Fe-NPC) with outstanding oxidase-like activity was synthesized, and the influence of phosphorus doping on oxidase-like activity was explored. Proper phosphorus doping was found to enhance the oxidase-like activity of Fe-NPC by increasing surface defects, promoting iron loading, and modulating chemical environment of central site. The oxidase-like activity of Fe-NPC was successfully applied to colorimetric-fluorescent dual mode detection of choline, and good linear relationship in the ranges of 3–200 μM were achieved with LODs of 1.95 and 1.50 μM, respectively. The fluorescent detection was constructed based on the fluorescence quenching of silicon quantum dots (Si QDs) by quinone-imine. The quenching mechanism was attributed to FRET due to the large spectra overlap, reduced fluorescence lifetime and proper donor−acceptor distance. The successful application to the detection of choline in milk proved the practicability of the proposed sensing method. The oxidase-like properties of Fe-NPC was further used in the degradation of cationic dye methyl blue, high degradation efficiency of 74 % was achieved within 5 min under optimal conditions, proving the enormous potential of Fe-NPC for application in environment protection.

High‐Throughput Absorbance‐Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases

AbstractRecent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy‐intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet‐based microfluidic systems have emerged as a powerful high‐throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet‐based microfluidic systems, we present an absorbance‐activated droplet sorter that allows droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105‐member aldehyde dehydrogenase library towards D‐glyceraldehyde using a NADH mediated coupled assay that generates WST‐1 formazan as the colorimetric product. We successfully identify a variant with a 51 % improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

Standardization of FTIR-Based Methodologies for Microplastics Detection in Drinking Water: A Meta-Analysis Indeed and Practical Approach

The detection of microplastics (MPs) in drinking water presents significant environmental and public health challenges. This study comprises two stages: a meta-analysis aimed at standardizing Fourier Transform Infrared Spectroscopy (FTIR) methods for MP detection, followed by the practical implementation of these findings in the laboratory. The review of studies conducted from 2019 to 2023 identifies 0.45 μm cellulose nitrate filters and Nile red staining as the most effective techniques for fluorescent detection. Experimental results demonstrate the superior retention capabilities of cellulose nitrate filters and the uniformity of Nile red staining. This dual approach not only optimizes water treatment processes but also enhances the accuracy of MP detection. The findings contribute to improved water quality management and public health protection by establishing robust protocols for MP analysis.

Dual-Modality Accurate Visualization of Drug Synergy Based on Mass Spectrometry and Fluorescence Imaging.

There is a potential synergistic effect between nonsteroidal anti-inflammatory drugs and hydrogen sulfide (H2S), but direct evidence for the study is lacking. With a single fluorescence detection method, it is difficult to accurately confirm the effectiveness of the synergistic effect. In this study, the fluorescent probe and the nonsteroidal anti-inflammatory drug naproxen were combined via different self-immolative spacer groups to obtain a diagnostic and therapeutic integrated fluorescent probe Nap-NP-NSB, which can release H2S. The quantitative release of H2S by Nap-NP-NSB was evaluated in vitro and in cells, and the synergistic effect of H2S and naproxen was confirmed by monitoring the treatment process of cellular inflammation and oxidative damage of gastric mucosa cells. Finally, in vivo fluorescence imaging and mass spectrometry imaging of the liver and stomach tissues and their sections were performed in the mouse model of acute hepatitis. The dual-modal detection method not only confirmed that Nap-NP-NSB had better anti-inflammatory activity and less gastric mucosal damage, but also enabled a more accurate visualization of the drug synergistic effect of naproxen and H2S. This work provides a dual visualization imaging method combining fluorescence and mass spectrometry imaging and develops a new idea for studying drug synergies based on self-immolative structures.

Occurrence and exposure to polycyclic aromatic hydrocarbons (PAHs) in traditional dry-cured or smoked meat products from Brazil

Polycyclic aromatic hydrocarbons (PAHs) are classified as environmental and food contaminants, with several adverse health effects, especially genotoxic and carcinogenic actions. In processed meats, they are the major contaminants, especially those subjected to smoking processes. Considering that the population is exposed to PAHs through several routes, and that the ingestion of contaminated food is considered the main one, evaluating the concentrations of these contaminants in food becomes essential, as well as the exposure and risk to the population at different ages through ingestion of the evaluated products. In the current study, the levels of nine PAHs (PAH9) were measured in 205 processed meat products commercially available and with high consumption in Brazil. The methodology involved saponification, extraction with n-hexane, purification with solid-phase extraction (SPE) silica cartridges, and quantification by liquid chromatography with fluorescence detection. In general, 83% of all samples were contaminated with at least one of the studied PAHs, and the measured PAH9 levels ranged between < LOQ-108.24 µg/kg. The highest mean of total PAHs was found in smoked sausage (108.24 μg/kg), while the lowest content was found in ham (1.83 μg/kg). Benzo[a]pyrene (BaP) and PAH4 (benz[a]anthracene, chrysene, benzo[b]fluoranthene, and BaP) exceeded the maximum permissible limits of the European Union (EU) in three (1.5%) and 18 samples (8.7%), respectively. The results of margin of exposure results (MOE ≥ 10,000) and incremental lifetime cancer risk (ILCR) values (10 − 6 < ILCR < 10 − 4) in all ten types of meat indicated there were low significant potential health problems related to meat products consumption for the Brazilian population.Graphical

Fluorescence Detection and Inhibition Mechanisms of DNTPH on Aβ42 Oligomers Characterized as Products in the Four Stages of Aggregation.

Aβ42 aggregation was implicated in the pathogenesis of Alzheimer's disease (AD) without effective treatment available currently. Future efforts in clinical trials should instead focus on applying those antiamyloid treatment strategies to the preclinical stage and "the earlier, the better". How to identify and inhibit Aβ42 oligomers in the different stages of aggregation is therefore becoming the key to controlling primary aggregation and consequent AD development. Aggregation-induced emission probe DNTPH was demonstrated recently, enabling detection of amyloid at wavelengths up to 710 nm and exhibiting strong inhibitory effects on Aβ fibrosis at low dose. However, the detection and inhibition mechanisms of Aβ oligomers at various early stages of aggregation remain unknown. To this end, we built four different morphologies of Aβ42 pentamers characterized by products in monomeric aggregate (PM), primary nucleation (PP), secondary nucleation (PS), and fibril stages (PF) to explore the distinguishable ability and inhibition mechanisms of DNTPH with different concentrations upon binding. The results showcased that DNTPH does detect the four different Aβ42 oligomers with conspicuous fluorescence (λPM = 657 nm, λPP = 639 nm, λPS = 630 nm, and λPF = 648 nm) but fails to distinguish them, indicating that additional improvements are required further for the probe to achieve it. The inhibition mechanisms of DNTPH on the four Aβ42 aggregation are however of amazing differences. For PM and PP, aggregation was inhibited by altering the secondary structural composition, i.e., by decreasing the β-sheet and toxic turn (residues 22-23) probabilities, respectively. For PS, inhibition was achieved by segregating and keeping the two disordered monomeric species (PSM) away from the ordered secondary seed species (PSF) and consequently blocking further growth of the PSF seed. The inhibition mechanism for PS is first probed and proposed so far, as far as we know, and the corresponding aggregation stage of PS is the most important one among the four stages. The inhibition of PF was triggered by distorting the fibril chains, disrupting the ordered fibril surface for the contact of monomers. In addition, the optimal inhibitory concentrations of DNTPH for PM, PP, and PF were determined to be 1:3, while for PS, it was 1:5. This outcome offers a novel perspective for designing drugs targeting Aβ42 oligomers at different aggregation stages.

LY354740, an agonist of glutamatergic metabotropic receptor mGlu2/3 increases the cytochrome P450 2D (CYP2D) activity in the frontal cortical area of rat brain.

Our previous studies indicated that changes in the functioning of the brain glutamatergic system involving the NMDA receptor may affect cytochrome P450 2D (CYP2D) in the brain. Since CYP2D may contribute to the metabolism of neurotransmitters and neurosteroids engaged in the pathology and pharmacology of neuropsychiatric diseases, in the present work we have investigated the effect of compound LY354740, an agonist of glutamatergic metabotropic receptor mGlu2/3, on brain and liver CYP2D. The activity (high performance liquid chromatography with fluorescence detection) and protein levels (Western blotting) of CYP2D were measured in the microsomes from the liver and different brain areas of male Wistar rats after 5day-treatment with LY354740 (10mg/kg ip). The results were analyzed statistically using Student's t-test. Among the investigated brain areas, the highest CYP2D activity was found in the cerebellum and brainstem, which exceeded that in the thalamus, cortex, hippocampus and frontal cortex. The mGlu2/3 receptor agonist LY354740 administered for five consecutive days significantly increased the protein level and activity of CYP2D in the frontal cortex. Such a tendency was also observed in the other brain areas. LY354740 did not affect the CYP2D activity in the liver. Repeated administration of the mGlu2/3 receptor agonist, the compound LY354740 specifically increases the protein level and activity of CYP2D in the frontal cortex, which may accelerate dopamine synthesis via an alternative CYP2D-mediated route in the mesocortical dopaminergic pathway, and thus may contribute to the beneficial pharmacological effect on negative symptoms of schizophrenia.

Divergent Synthesis of (CF3)‐Indazolo[3,2‐a]Isoquinolines with Potent Photophysical Property and Anticancer Activity from 3‐Aryl‐1H‐Indazoles and Sulfoxonium Ylides

AbstractTransition‐metal‐catalyzed inert C−H bond functionalization using sulfoxonium ylides as metal carbene surrogates to replace the explosive and labile diazo compounds is synthetically attractive. Presented herein is a divergent synthesis of (CF3)‐indazolo[3,2‐a]isoquinoline derivatives based on C−H bond activation‐initiated cascade reactions of 3‐aryl‐1H‐indazoles with sulfoxonium ylides. The formation of products involves Rh(III)‐catalyzed C(sp2)−H bond metalation, carbene formation, migratory insertion, proto‐demetalation and intramolecular condensation. In general, this newly developed protocol features simple substrates, structurally diverse products, step‐economy and scalability. In addition, evaluation of the photophysical properties and anti‐proliferative activity against three human cancer cell lines of the products thus obtained showed that some of them possess potent anticancer activity with potential self‐tracking capability through fluorescence detection.

Tetraphenylethene-Containing Nanofilm via Interfacial Confinement Self-Assembly for Fluorescent Monitoring of SOCl2 Leakage in a Li-SOCl2 Battery Electrolyte.

Lithium thionyl chloride (Li-SOCl2) batteries are widely used due to their high energy density and long shelf life. However, the corrosive nature of SOCl2 poses a safety hazard, necessitating effective leak detection methods. We report an approach for real-time fluorescent detection of SOCl2 leakage in Li-SOCl2 batteries using a tetraphenylethene-based nanofilm. The nanofilms were prepared through the interfacial confined self-assembly method and exhibit excellent flexibility, homogeneity, tunable size and thickness, and adaptability to various substrates, enabling easy integration into sensor devices. They possess high photochemical stability and a photoluminescence quantum yield exceeding 25%, demonstrating their potential as high-performance fluorescent sensing material. The nanofilms also exhibit high sensitivity, good reproducibility, and selectivity toward SOCl2 detection. Upon exposure to SOCl2 vapor, the nanofilm shows a red-shifted and fluorescence quenching response, attributed to the protonation of the acylhydrazone bond in the nanofilm by the hydrolysis product of SOCl2, which disrupts the electronic structure of the nanofilm and leads to a decrease in the fluorescence intensity and a shift in the emission wavelength. A detection limit of ∼1.31 ppt and excellent repeatability over 300 cycles are demonstrated, highlighting their high sensitivity and reliability. This work paves the way for a highly sensitive and reliable leak detection system for Li-SOCl2 batteries, enhancing their safety and reliability in various applications.

Advanced Imaging Integration: Multi-Modal Raman Light Sheet Microscopy Combined with Zero-Shot Learning for Denoising and Super-Resolution.

This study presents an advanced integration of Multi-modal Raman Light Sheet Microscopy with zero-shot learning-based computational methods to significantly enhance the resolution and analysis of complex three-dimensional biological structures, such as 3D cell cultures and spheroids. The Multi-modal Raman Light Sheet Microscopy system incorporates Rayleigh scattering, Raman scattering, and fluorescence detection, enabling comprehensive, marker-free imaging of cellular architecture. These diverse modalities offer detailed spatial and molecular insights into cellular organization and interactions, critical for applications in biomedical research, drug discovery, and histological studies. To improve image quality without altering or introducing new biological information, we apply Zero-Shot Deconvolution Networks (ZS-DeconvNet), a deep-learning-based method that enhances resolution in an unsupervised manner. ZS-DeconvNet significantly refines image clarity and sharpness across multiple microscopy modalities without requiring large, labeled datasets, or introducing artifacts. By combining the strengths of multi-modal light sheet microscopy and ZS-DeconvNet, we achieve improved visualization of subcellular structures, offering clearer and more detailed representations of existing data. This approach holds significant potential for advancing high-resolution imaging in biomedical research and other related fields.

Highly Sensitive One-Pot Isothermal Assay Combining Rolling Circle Amplification and CRISPR/Cas12a for Aflatoxin B1 Detection.

Occurrences of mycotoxins in cereals are widespread throughout the world. However, the lack of efficient and ultrasensitive tests has largely impeded the identification of these substances in actual samples. Herein, a novel one-pot isothermal assay that integrates rolling-circle amplification (RCA) and CRISPR/Cas12a to detect aflatoxin B1 (AFB1) is reported. Upon addition of AFB1 to the magnetic bead functionalized with a duplex of the AFB1 aptamer and its complementary DNA (cDNA), the specific recognition of AFB1 by the aptamer causes the release of cDNA to activate the RCA reaction. Subsequently, the RCA amplicon initiates both trans-cleavage and cis-cleavage activities of the endonuclease Cas12a. The synergistic coupling of RCA and CRISPR/Cas12a enables exponential amplification of cDNA, which further promotes CRISPR/Cas12a to nonspecifically cleave the single-stranded DNA reporters with enhanced detection signals. Remarkably, the CRISPR/Cas12a-assisted one-pot isothermal assay can not only achieve ultrasensitive quantitative detection through fluorescence detection, but also achieve visual detection through a lateral flow strip, which improves accessibility to mycotoxin detection in resource-limited regions. The limit of detection was 0.016 and 0.408 ng/mL, respectively. The proposed assay successfully applies in real samples with satisfactory recoveries from 90 to 114%. This study presents a powerful and versatile method for reliable and ultrasensitive detection of mycotoxins in various applications.