RESUMO
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.
Assuntos
Aterosclerose , Ácidos Graxos Ômega-3 , Humanos , Lipólise , Fluorescência , Ácidos Graxos Ômega-3/metabolismo , Óleos de Peixe/farmacologia , Ácidos Docosa-Hexaenoicos/metabolismo , Macrófagos/metabolismo , TriglicerídeosRESUMO
Superior photostability, minimal phototoxicity, red-shifted absorption/emission wavelengths, high brightness, and an enlarged Stokes shift are essential characteristics of top-tier organic fluorophores, particularly for long-lasting super-resolution imaging in live cells (e.g., via stimulated emission depletion (STED) nanoscopy). However, few existing fluorophores possess all of these properties. In this study, we demonstrate a general approach for simultaneously enhancing these parameters through the introduction of 9,9-dimethyl-9,10-dihydroacridine (DMA) as an electron-donating auxochrome. DMA not only induces red shifts in emission wavelengths but also suppresses photooxidative reactions and prevents the formation of triplet states in DMA-based fluorophores, greatly improving photostability and remarkably minimizing phototoxicity. Moreover, the DMA group enhances the fluorophores' brightness and enlarges the Stokes shift. Importantly, the "universal" benefits of attaching the DMA auxochrome have been exemplified in various fluorophores including rhodamines, difluoride-boron complexes, and coumarin derivatives. The resulting fluorophores successfully enabled the STED imaging of organelles and HaloTag-labeled membrane proteins.
Assuntos
Corantes Fluorescentes , Humanos , Rodaminas , Microscopia de Fluorescência/métodos , Células HeLa , IonóforosRESUMO
When using High-field asymmetric ion mobility spectrometry (FAIMS) to process complex mixtures for deep learning analysis, there is a problem of poor recognition performance due to the lack of high-quality data and low sample diversity. In this paper, a Generative Adversarial Network (GAN) method is introduced to simulate and generate highly realistic and diverse spectral for expanding the dataset using real mixture spectral data of 15 classes collected by FAIMS. The mixed datasets were put into VGG and ResNeXt for testing respectively, and the experimental results proved that the best recognition effect was achieved when the ratio of real data to generated data was 1:4: where accuracy improved by 24.19 % and 6.43 %; precision improved by 23.71 % and 6.97 %; recall improved by 21.08 % and 7.09 %; and F1-score improved by 24.50 % and 8.23 %. The above results strongly demonstrate that GAN can effectively expand the data volume and increase the sample diversity without increasing the additional experimental cost, which significantly enhances the experimental effect of FAIMS spectral for the analysis of complex mixtures.
Assuntos
Aprendizado Profundo , Espectrometria de Mobilidade Iônica , Espectrometria de Mobilidade Iônica/métodos , Redes Neurais de ComputaçãoRESUMO
In practical applications, analytical instruments are used for both qualitative and quantitative analysis. However, for high-field asymmetric-waveform ion mobility spectrometry (FAIMS), most studies to date have been focused on the qualitative analysis of substances, with limited research on quantitative analysis. Explored here is the feasibility of using deep learning in FAIMS for quantitative analysis, aided by redesigning the FAIMS upper computer. Integrating spectrum creation and deep learning analysis into the FAIMS upper computer boosts the processing and analysis of FAIMS data, laying a foundation for applying FAIMS practically. For analysis using image processing, multiple FAIMS spectral lines obtained under different conditions are converted into a three-dimensional thermodynamic map known as a FAIMS spectrum, and multiple FAIMS spectrum are preprocessed to obtain the data set of this experiment. The principles of partial-least-squares regression and the XGBoost and ResNeXt models are introduced in detail, and the data are analyzed using these models, while exploring the effects of different model parameters and determining their optimal values. The experimental results show that the pre-trained ResNeXt deep learning model performs the best on the test set, with a root mean square error of 0.86 mg/mL, indicating the potential of deep learning in realizing quantitative analysis of substances in FAIMS.
Assuntos
Aprendizado Profundo , Espectrometria de Mobilidade Iônica , Espectrometria de Mobilidade Iônica/métodos , AcetonaRESUMO
A precisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Limite de Detecção , RNA Mensageiro , Timidina Quinase , Timidina Quinase/genética , Humanos , Transferência Ressonante de Energia de Fluorescência/métodos , RNA Mensageiro/análise , RNA Mensageiro/genética , Corantes Fluorescentes/química , Técnicas Biossensoriais/métodos , Hibridização de Ácido Nucleico , Fluorometria/métodos , Biomarcadores/análiseRESUMO
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.
Assuntos
Corantes Fluorescentes , Fotodegradação , Espécies Reativas de Oxigênio , Microscopia de Fluorescência/métodosRESUMO
Herein, we report a mild, one-pot method for silver-catalyzed tandem cycloisomerization/[5 + 2] cycloaddition reactions between readily accessible cyclopropyl-tethered allenyl ketones and benzopyranone-derived oxidopyrylium ylides. The reactions proceed via a cyclobutene-fused furan intermediate generated in situ by a cycloisomerization/1,2-carbene transfer/ring-expansion cascade. This method, which features an unprecedented formal [5 + 2] cycloaddition, delivers good to excellent yields of structurally complex bibridged benzocycloheptanones bearing a strained cyclobutane ring and an O-bridged ring.
RESUMO
Endoplasmic reticulum (ER) is an important organelle of a membranous tubule network in cells for the synthesis, assembly, and modification of peptides, proteins, and enzymes. Autophagy and destruction of ER commonly occur during normal cellular activities. These processes have been studied extensively, but the spontaneous ER regeneration process is poorly understood because of the lack of molecular tools capable of distinguishing the intact, damaged, autophagic, and regenerative ER in live cells. Herein, we report a dual-localizing, environment-responsive, and lifetime-sensitive fluorescent probe for real-time monitoring ER autophagy and regeneration in live cells. Using this tool, the fluorescence lifetime imaging can quantitatively determine the degrees of ER destruction and spontaneous recovery. Significantly, we show that triglycerides supplied in lipid droplets can efficiently repair ER via the two critical pathways: (i) supplying materials for ER repair by converting triglycerides into fatty acids and diglycerides and (ii) partially inhibiting autophagy for stressed ER.
Assuntos
Retículo Endoplasmático , Imagem Óptica , Autofagia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Corantes Fluorescentes/metabolismo , Imagem Óptica/métodos , Triglicerídeos/metabolismoRESUMO
As a specific biological marker for the occurrence and progression of tumor cells, detection of telomerase activity is of great importance for the physiological research of tumors. However, in situ measurement of telomerase activity in living cells still remains a challenge. Herein, we report a precisely designed oligonucleotide-functionalized gold nanoparticle probe that has realized high-efficiency detection of telomerase activity for cellular imaging toward the identification of tumors. Our method has achieved intracellular imaging of telomerase activity and shows good performance towards the distinction of tumor cells from normal ones. Moreover, the method reported here for tracking tumor cells in blood has wide applications in cancer diagnosis. This strategy offers an opportunity for cancer diagnosis, guiding therapy and evaluating prognosis.
Assuntos
Nanopartículas Metálicas , Neoplasias , Telomerase , Ouro , Células HeLa , Humanos , Neoplasias/diagnóstico por imagem , Oligonucleotídeos , Imagem Óptica , Telomerase/metabolismoRESUMO
Understanding signaling molecules in regulating organelles dynamics and programmed cell death is critical for embryo development but is also challenging because current imaging probes are incapable of simultaneously imaging the signaling molecules and the intracellular organelles they interact with. Here, we report a chemically and environmentally dual-responsive imaging probe that can react with gasotransmitters and label cell nuclei in distinctive fluorescent colors, similar to the adaptive coloration of chameleons. Using this intracellular chameleon-like probe in three-dimensional (3D) super-resolution dynamic imaging of live cells, we discovered SO2 as a critical upstream signaling molecule that activates nucleophagy in programmed cell death. An elevated level of SO2 prompts kiss fusion between the lysosomal and nuclear membranes and nucleus shrinkage and rupture. Significantly, we revealed that the gasotransmitter SO2 is majorly generated in the yolk, induces autophagy there at the initial stage of embryo development, and is highly related to the development of the auditory nervous system.
Assuntos
Corantes Fluorescentes , Dióxido de Enxofre , Autofagia , Núcleo Celular , Desenvolvimento Embrionário , Células HeLa , HumanosRESUMO
Intracellular lipid metabolism occurs in lipid droplets (LDs), which is critical to the survival of cells. Imaging LDs is an intuitive way to understand their physiology in live cells. However, this is limited by the availability of specific probes that can properly visualize LDs in vivo. Here, an LDs-specific red-emitting probe is proposed to address this need, which is not merely with an ultrahigh signal-to-noise (S/N) ratio and a large Stokes shift (up to 214 nm) but also with superior resistance to photobleaching. The probe has been successfully applied to real-time tracking of intracellular LDs behaviors, including fusion, migration, and lipophagy processes. We deem that the proposed probe here offers a new possibility for deeper understanding of LDs-associated behaviors, elucidation of their roles and mechanisms in cellular metabolism, and determination of the transition between adaptive lipid storage and lipotoxicity as well.
Assuntos
Corantes Fluorescentes/química , Luz , Gotículas Lipídicas/química , Animais , Transporte Biológico , Cor , Transporte de Elétrons , Corantes Fluorescentes/metabolismo , Células HeLa , Células Hep G2 , Humanos , Gotículas Lipídicas/metabolismo , Imagem Molecular , Peixe-ZebraRESUMO
Understanding the biomolecular interactions in a specific organelle has been a long-standing challenge because it requires super-resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super-resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H2 O2 ), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H2 O2 level leads to disassociation of nucleic acid-protein complexes.
Assuntos
Corantes Fluorescentes/química , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Hep G2 , Humanos , Peróxido de Hidrogênio/metabolismo , Ácidos Nucleicos/metabolismoRESUMO
Unveiling the synergism among multiple organelles for fully exploring the mysteries of the cell has drawn more and more attention. Herein, we developed two two-photon fluorescent bioprobes (Lyso-TA and Mito-QA), of which the conformational change triggered an "off-on" fluorescent response. Lyso-TA can real-time monitor the fusion and movement of lysosomes as well as unveil the mitophagy process with the engagement of lysosomes. Mito-QA was transformed from Lyso-TA by one-step ambient temperature reaction, visualizing the dysfunctional mitochondria through a shift from mitochondria to nucleoli. With superior two-photon absorption cross section, good biocompatibility, and greater penetration depth, two small bioprobes were both applied in in vivo bioimaging of brain tissues and zebrafish.
Assuntos
Acrilonitrila/síntese química , Corantes Fluorescentes/química , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Acrilonitrila/análogos & derivados , Acrilonitrila/efeitos da radiação , Acrilonitrila/toxicidade , Animais , Encéfalo/metabolismo , Nucléolo Celular/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/efeitos da radiação , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Mitofagia/fisiologia , Conformação Molecular , Fótons , Peixe-ZebraRESUMO
Visualizing and dynamic tracking lipid droplets (LDs) are of great importance to biological research. Herein, two-photon absorption fluorescent small bioprobes based on lipophilic coumarin were developed, which exhibited high selectivity toward LDs in HeLa cells. Because of good biocompatibility and excellent photostability, the probes were applied to realize specific super-resolution visualization of the intracellular LDs in HeLa cells, offering us the quantitative results of the amount and diameters of LDs as well. Furthermore, the bioprobes were capable of monitoring the movements of the LDs in real time. We believe that bioprobes would provide new avenues to designing bioimaging and biological diagnosis.
Assuntos
Cumarínicos/química , Corantes Fluorescentes/química , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Células HeLa , Humanos , Estrutura Molecular , Tamanho da Partícula , Fótons , Propriedades de SuperfícieRESUMO
Enzyme activity in live cells is dynamically regulated by small-molecule transmitters for maintaining normal physiological functions. A few probes have been devised to measure intracellular enzyme activities by fluorescent imaging, but the study of the regulation of enzyme activity via gasotransmitters in situ remains a long-standing challenge. Herein, we report a three-channel imaging correlation by a single dual-reactive fluorescent probe to measure the dependence of phosphatase activity on the H2 S level in cells. The two sites of the probe reactive to H2 S and phosphatase individually produce blue and green fluorescent responses, respectively, and resonance energy transfer can be triggered by their coexistence. Fluorescent analysis based on the three-channel imaging correlation shows that cells have an ideal level of H2 S to promote phosphatase activity up to its maximum. Significantly, a slight deviation from this H2 S level leads to a sharp decrease of phosphatase activity. The discovery further strengthens our understanding of the importance of H2 S in cellular signaling and in various human diseases.
Assuntos
Corantes Fluorescentes/metabolismo , Gasotransmissores/metabolismo , Imagem Óptica , Monoéster Fosfórico Hidrolases/metabolismo , Corantes Fluorescentes/química , Gasotransmissores/química , Células HeLa , Humanos , Sulfeto de Hidrogênio/metabolismo , Estrutura MolecularRESUMO
The dynamics of DNA and RNA structures in live cells are important for understanding cell behaviors, such as transcription activity, protein expression, cell apoptosis, and hereditary disease, but are challenging to monitor in live organisms in real time. The difficulty is largely due to the lack of photostable imaging probes that can distinguish between DNA and RNA, and more importantly, are capable of crossing multiple membrane barriers ranging from the cell/organelle to the tissue/organ level. We report the discovery of a cationic carbon quantum dot (cQD) probe that emits spectrally distinguishable fluorescence upon binding with double-stranded DNA and single-stranded RNA in live cells, thereby enabling real-time monitoring of DNA and RNA localization and motion. A surprising finding is that the probe can penetrate through various types of biological barriers inâ vitro and inâ vivo. Combined with standard and super-resolution microscopy, photostable cQDs allow time-lapse imaging of chromatin and nucleoli during cell division and Caenorhabditis elegans (C. elegans) growth.
Assuntos
Caenorhabditis elegans/metabolismo , Carbono/química , Permeabilidade da Membrana Celular , DNA/química , Imagem Óptica/métodos , Pontos Quânticos/química , RNA/química , Animais , Caenorhabditis elegans/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , DNA/análise , Fluorescência , Células HeLa , Humanos , Conformação de Ácido Nucleico , RNA/análiseRESUMO
Hydrogen sulfide (H2S) is a key signaling molecule in the cytoprotection, vascular mediation and neurotransmission of living organisms. In-depth understanding of its production, trafficking, and transformation in cells is very important in the way H2S mediates cellular signal transductions and organism functions; it also motivates the development of H2S probes and imaging technologies. A fundamental challenge, however, is how to engineer probes with sensitivity and cellular penetrability that allow detection of spontaneous production of H2S in the entire cell space and live animals. Here, we report a rationally designed molecular switch capable of accessing all intracellular compartments, including the nucleus, lysosomes and mitochondria, for H2S detection. Our probe comprised three functional domains (H2S sensing, fluorescence, and biomembrane penetration), could enter almost all cell types readily, and exhibit a rapid and ultrasensitive response to H2S (≤120-fold fluorescence enhancement) for the dynamic mapping of spontaneously produced H2S as well as its distribution in the whole cell. In particular, the probe traversed blood/tissue/cell barriers to achieve mapping of endogenous H2S in metabolic organs of a live Danio rerio (zebrafish). These results open-up exciting opportunities to investigate H2S physiology and H2S-related diseases.
Assuntos
Corantes Fluorescentes , Sulfeto de Hidrogênio/análise , Animais , Linhagem Celular , Núcleo Celular/química , Fluorescência , Humanos , Lisossomos/química , Mitocôndrias/química , Peixe-ZebraRESUMO
A triphenylamine-pyridine derivative, viz., a fluorescence probe was designed and synthesized. The probe displayed an "on-off-on" fluorescence switch, upon the sequential addition of Cu2+ and ethylenediaminetetraacetic acid (EDTA). The detection mechanism was confirmed by using UV-vis, fluorescence and 1H NMR titration spectra. Interestingly, the probe can accumulate in mitochondria, and thus detect Cu2+ in real-time there, which indicated that probe L can be used to monitor intracellular mitochondrial Cu2+ levels. Further understanding of the biological functions of Cu2+ in mitochondria has important implications for treating cancer and other diseases.
Assuntos
Cobre/análise , Corantes Fluorescentes/química , Mitocôndrias/química , Imagem Óptica , Sobrevivência Celular/efeitos dos fármacos , Cobre/metabolismo , Ácido Edético/química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Mitocôndrias/metabolismo , Estrutura Molecular , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Fatores de TempoRESUMO
Fluorescent probes are powerful tools for the investigations of reactive oxygen species (ROS) in living organisms by visualization and imaging. However, the multiparallel assays of several ROS with multiple probes are often limited by the available number of spectrally nonoverlapping chromophores together with large invasive effects and discrepant biological locations. Meanwhile, the spontaneous ROS profilings in various living organs/tissues are also limited by the penetration capability of probes across different biological barriers and the stability in reactive in vivo environments. Here, we report a single fluorescent probe to achieve the effective discrimination and profiling of hydroxyl radicals (â¢OH) and hypochlorous acid (HClO) in living organisms. The probe is constructed by chemically grafting an additional five-membered heterocyclic ring and a lateral triethylene glycol chain to a fluorescein mother, which does not only turn off the fluorescence of fluorescein, but also create the dual reactive sites to ROS and the penetration capability in passing through various biological barriers. The reactions of probe with â¢OH and HClO simultaneously result in cyan and green emissions, respectively, providing the real-time discrimination and quantitative analysis of the two ROS in cellular mitochondria. Surprisingly, the accumulation of probes in the intestine and liver of a normal-state zebrafish and the transfer pathway from intestine-to-blood-to-organ/tissue-to-kidney-to-excretion clearly present the profiling of spontaneous â¢OH and HClO in these metabolic organs. In particular, the stress generation of â¢OH at the fresh wound of zebrafish is successfully visualized for the first time, in spite of its extremely short lifetime.
Assuntos
Corantes Fluorescentes/química , Espécies Reativas de Oxigênio/análise , Animais , Sistemas Computacionais , Fluoresceínas/química , Células HeLa , Humanos , Radical Hidroxila/análise , Radical Hidroxila/metabolismo , Ácido Hipocloroso/análise , Ácido Hipocloroso/metabolismo , Camundongos , Polietilenoglicóis/química , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Fluorescência/métodos , Ferimentos e Lesões/metabolismo , Peixe-ZebraRESUMO
Fluorescent colorimetry test papers are promising for the assays of environments, medicines, and foods by the observation of the naked eye on the variations of fluorescence brightness and color. Unlike dye-absorption-based pH test paper, however, the fluorescent test papers with wide color-emissive variations with target dosages for accurate quantification remain unsuccessful even if the multicolorful fluorescent probes are used. Here, we report the dosage-sensitive fluorescent colorimetry test paper with a very wide/consecutive "from red to cyan" response to the presence and amount of arsenic ions, As(III). Red quantum dots (QDs) were modified with glutathione and dithiothreitol to obtain the supersensitivity to As(III) by the quenching of red fluorescence through the formation of dispersive QDs aggregates. A small amount of cyan carbon dots (CDs) with spectral blue-green components as the photostable internal standard were mixed into the QDs solution to produce a composited red fluorescence. Upon the addition of As(III) into the sensory solution, the fluorescence color could gradually be reversed from red to cyan with a detection limit of 1.7 ppb As(III). When the sensory solution was printed onto a piece of filter paper, surprisingly a serial of color evolution from peach to pink to orange to khaki to yellowish to yellow-green to final cyan with the addition of As(III) was displayed and clearly discerned the dosage scale as low as 5 ppb. The methodology reported here opens a novel pathway toward the real applications of fluorescent test papers.