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Multi-color live-cell super-resolution volume imaging with multi-angle interference microscopy
摘要： Imaging and tracking of near-surface three-dimensional volumetric nanoscale dynamic processes of live cells remains a challenging problem. In this paper, we propose a multi-color live-cell near-surface-volume super-resolution microscopy method that combines total internal reflection fluorescence structured illumination microscopy with multi-angle evanescent light illumination. We demonstrate that our approach of multi-angle interference microscopy is perfectly adapted to studying subcellular dynamics of mitochondria and microtubule architectures during cell migration.
关键词： super-resolution microscopy,live-cell imaging,multi-angle interference microscopy,mitochondria,microtubules
更新于2025-09-10 09:29:36
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A FRET sensor for live-cell imaging of MAP kinase activity in Arabidopsis
摘要： The catalytic activity of mitogen activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain, and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the F?rster resonance energy transfer (FRET) efficiency of the sensor. FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live-cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22, and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial FRET changes. We also expressed the sensor in a conditional loss-of function double-mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the sensor’s NaCl-induced FRET gain, while other MAPKs are likely contributing to the chitin and flg22-induced FRET increases. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.
关键词： Arabidopsis thaliana,FRET sensor,live-cell imaging,MAP kinase
更新于2025-09-09 09:28:46
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A novel colorimetric and ratiometric fluorescent probe for selective detection of bisulfite in real samples and living cells
摘要： An abnormal level of bisulfite can induce toxicological effects associated with lung cancer, cardiovascular and neurological disorders. Therefore, it is of significance to develop an effective fluorescent probe to detect bisulfite in living cells. Herein, a novel fluorescent probe QPCT, based on a 1,4-addition mechanism, was constructed for the colorimetric and ratiometric detection of bisulfite. QPCT displayed high selectivity and anti-interference ability to bisulfite over other anions and biothiols. The probe renders a sensitive ratiometric response to bisulfite with a remarkable fluorescence blue shift from 590 to 537 nm and the fluorescence ratio was linear with bisulfite concentration over the range of 0-120 μM. More importantly, QPCT has been successfully applied to detect bisulfite in sugars and living A549 cells, which indicated that QPCT had a great capability for monitoring bisulfite in complex systems.
关键词： Real samples,Fluorescent probe,Live-cell imaging,Bisulfite,Colorimetric and ratiometric
更新于2025-09-09 09:28:46
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Photochemically Active Dyes for Super-Resolution Microscopy
摘要： The development of super-resolved optical microscopies has revolutionized the way we visualize cell biology. These techniques strongly rely on the use of photochemically active fluorophores that display changes in their photophysical properties upon irradiation with light. Many reversible and irreversible photochemical transformations have been explored for this purpose, and different imaging techniques require specific mechanisms of photoconversion. In this review, we provide an overview of the most common strategies used for the development of fluorophores for super-resolution microscopy and give specific examples of state-of-the-art fluorogenic probes. Furthermore, we discuss their main field of application and possible directions for future developments.
关键词： Live-cell imaging,Photoswitchable,Super-resolution microscopy,Fluorescent probes,Photoactivatable
更新于2025-09-09 09:28:46
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A 1,8 Naphthalimide anchor Rhodamine B Based FRET Probe for Ratiometric Detection of Cr3+ion in Living Cells
摘要： A 2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)acetaldehydeanchored rhodamine B based probe, RDNAP detects Cr3+ ion by fluorescence resonance energy transfer (FRET) process in aqueous buffered acetonitrile media (7:3, v/v). Conversely, conjugation of 2-(1,3-dioxo-6-(piperidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)acetaldehyde with rhodamine B provides another probe, RDNAP-PY that undergoes Cr3+assisted ratiometric fluorescence and colorimetric change in the same media. RDNAP-PY provides higher FRET efficiency and detects as low as 1.81×10?6 M Cr3+with an association constant, 15.9 × 104 M-1. Other common ions do not interfere. RDNAP-PY efficiently images intracellular Cr3+ in live Hep3B, MCF-7, HeLa, SiHa and HEK 293T cells under fluorescence microscope in a ratiometric and time dependent manner. 1H NMR titration and DFT studies strongly support experimental findings.
关键词： 1,8 Naphthalimide,Live cell imaging,Ratiometric probe,fluorescence resonance energy transfer,DFT calculation
更新于2025-09-09 09:28:46
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Media Dependent Switching of Selectivity and Continuous Near Infrared Turn-on Fluorescence Response through Cascade Interactions from Noncovalent to Covalent Binding for Detection of Serum Albumin in Living Cells
摘要： Abnormal level of proteins is proved to be associated with diseases. Thus, protein sensing is helpful for clinical diagnosis and therapy. However, there is a great variety of protein species and relatively low concentration of each protein in complicated biological systems including other non-protein biomolecules. Therefore, it remains challenging to develop an effective method for detecting protein with high selectivity and sensitivity. Herein, a new self-assembly method based on a robust dye SQSS of which two squaraine molecules were conjugated through disulfide bond was developed for highly selective and sensitive detection of serum albumin (SA) in aqueous solution and live cells. SQSS can self-assemble into “compact” aggregates, offering “inert” disulfide group and very low background fluorescence through the combination of aggregation quenching and homogeneous fluorescence resonance energy transfer (homoFRET) quenching. The response of SQSS to SA undergoes two cascade stages. At the first stage, SA drives the compact assemblies of SQSS to form loose ones with fast speed (30 s) through noncovalent interaction, resulting in the enhancement of fluorescence to some extent. In this loose assembly state, the disulfide bond in SQSS is reactive. At the second stage, the Cys34 in SA slowly induced further disassembly through covalent binding with reactive disulfide bond, resulting in fluorescence further increasing and SQSS labeling to SA that cannot be displaced by site binding ligands of SA. The self-assemblies of SQSS can selectively detect SA with continuous near infrared (NIR) turn-on fluorescence response in 100% aqueous buffer solution. In addition, SQSS showed the potential application of imaging SA in living cells. On the other hand, the loose assembly state of SQSS was also achieved in aqueous solution with 20% CH3CN. In this media, thiol-containing glutathione (GSH) caused the disassembly of SQSS with turn-on fluorescence response through interaction with disulfide bond. SQSS can selectively recognize GSH over other amino acids even in the presence of other sulfhydryl amino acids. As a proof-of-concept method, the molecular self-assembly through multi-steps interactions would provide an ideal strategy for detection and live-cell imaging of bio-related molecules with high selectivity and signal-to-noise ratio.
关键词： squaraine dyes,disulfide linkage,glutathione,live-cell imaging,serum albumin,self-assemblies,noncovalent and covalent interactions
更新于2025-09-09 09:28:46
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A machine learning approach for online automated optimization of super-resolution optical microscopy
摘要： Traditional approaches for finding well-performing parameterizations of complex imaging systems, such as super-resolution microscopes rely on an extensive exploration phase over the illumination and acquisition settings, prior to the imaging task. This strategy suffers from several issues: it requires a large amount of parameter configurations to be evaluated, it leads to discrepancies between well-performing parameters in the exploration phase and imaging task, and it results in a waste of time and resources given that optimization and final imaging tasks are conducted separately. Here we show that a fully automated, machine learning-based system can conduct imaging parameter optimization toward a trade-off between several objectives, simultaneously to the imaging task. Its potential is highlighted on various imaging tasks, such as live-cell and multicolor imaging and multimodal optimization. This online optimization routine can be integrated to various imaging systems to increase accessibility, optimize performance and improve overall imaging quality.
关键词： machine learning,multicolor imaging,online automated optimization,live-cell imaging,super-resolution optical microscopy,multimodal optimization
更新于2025-09-04 15:30:14
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Deep-red fluorogenic probe for rapid detection of nitric oxide in Parkinson’s disease models
摘要： Nitric oxide (NO), as one of important gaseous signaling molecule in human body, has been associated with a variety of physiologic processes. However, excessive production of NO has been profoundly implicated in the pathogenesis of neurodegenerative disorders in particular, Parkinson's disease (PD). Therefore, accurate and facile detection of NO is of great significance for investigating its functions in PD, and the subsequent diagnosis and/or treatment. Herein, we developed a deep-red fluorogenic probe (BT-NH), with high sensitivity and good selectivity to detect NO, which was successfully used to visualize exogenous/endogenous NO level in living cells, and further applied to in vitro and in vivo PD models.
关键词： Parkinson’s disease,Drosophila,live cell imaging,nitric oxide,fluorogenic probe
更新于2025-09-04 15:30:14