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Fluorescence microscopy is a powerful tool used in scientific and medical research, but it is inextricably linked to phototoxicity. Neglecting phototoxicity can lead to erroneous or inconclusive results. Recently, several reports have addressed this issue, but it is still underestimated by many researchers, even though it can lead to cell death. Phototoxicity can be reduced by appropriate microscopic techniques and carefully designed experiments. This review focuses on recent strategies to reduce phototoxicity in microscopic imaging of living cells and tissues. We describe digital image processing and new hardware solutions. We point out new modifications of microscopy methods and hope that this review will interest microscopy hardware engineers. Our aim is to underscore the challenges and potential solutions integral to the design of microscopy systems. Simultaneously, we intend to engage biologists, offering insight into the latest technological advancements in imaging that can enhance their understanding and practice.
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Microscopía Fluorescente , Humanos , Microscopía Fluorescente/métodos , Animales , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
In this paper, we discuss how tetrahydrodibenzo[a,j]acridine (4-HA) loses its hydrogen, which makes dibenzo[a,j]acridine (ARM) and also how 4-HA can be synthesized effectively using 2-tetralone in high yield. Dehydrogenative condensation and dehydrogenation are the two processes that make up the overall reaction of this synthetic approach. In addition, the presence of BF3 caused a remarkable fluorescence shift in ARM. Test paper analysis was used for examining the practical usefulness of ARM, which can be seen under UV light, resulting in this unique phenomenon. The fluorescent bio imaging experiment demonstrates that the sensor ARM has the capability to detect BF3 in living HeLa cells.
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Acridinas , Colorantes Fluorescentes , Humanos , Células HeLa , FluorescenciaRESUMEN
Atomic force microscope enables ultra-precision imaging of living cells. However, atomic force microscope imaging is a complex and time-consuming process. The obtained images of living cells usually have low resolution and are easily influenced by noise leading to unsatisfactory imaging quality, obstructing the research and analysis based on cell images. Herein, an adaptive attention image reconstruction network based on residual encoder-decoder was proposed, through the combination of deep learning technology and atomic force microscope imaging supporting high-quality cell image acquisition. Compared with other learning-based methods, the proposed network showed higher peak signal-to-noise ratio, higher structural similarity and better image reconstruction performances. In addition, the cell images reconstructed by each method were used for cell recognition, and the cell images reconstructed by the proposed network had the highest cell recognition rate. The proposed network has brought insights into the atomic force microscope-based imaging of living cells and cell image reconstruction, which is of great significance in biological and medical research.
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The sensitive and reliable nanozyme-based sensor enables the detection of low concentrations of H2O2 in biological microenvironments, it has potential applications as an in-situ monitoring platform for cellular H2O2 release. The uniformly dispersed bimetallic sulfide (Zn2SnS4) nanoflowers were synthesized via a one-pot hydrothermal method and the two kinds of metal ions can serve as morphology and structure directing agents for each other in the synthetic process. The nanoparticles were utilized as nanozyme materials to fabricate a novel electrochemical sensor, and it exhibits a distinct electrochemical response towards H2O2 with excellent stability and detection capability (with a minimum detection limit of 1.79â nM (S/N=3)), the excellent characteristics facilitate the precise detection of low concentrations of H2O2 in biological microenvironments. Use the macrophages differentiated from leukemia THP-1 cells as a representative sensing model, the sensor was successfully utilized for real-time monitoring of the release of H2O2 induced by living cells, which has significant potential applications in clinical diagnosis and cancer treatment.
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Técnicas Electroquímicas , Peróxido de Hidrógeno , Límite de Detección , Sulfuros , Peróxido de Hidrógeno/química , Humanos , Técnicas Electroquímicas/métodos , Sulfuros/química , Zinc/química , Células THP-1 , Macrófagos/metabolismoRESUMEN
Nucleolus was an important cellular organelle. The abnormal morphology and number of the nucleolus have been considered as diagnostic biomarkers for some human diseases. However, the imaging agent based on nucleolus was limited. In this manuscript, a series of nucleolar fluorescent probes based on naphthalimide derivatives (NI-1 â¼ NI-5) had been designed and synthesized. NI-1 â¼ NI-5 could penetrate cell membranes and nuclear membranes, achieve clear nucleolar staining in living cells. These results suggested that the presence of amino groups on the side chains of naphthalimide backbone could enhance the targeting to the cell nucleolus. In addition, the molecular docking results showed that NI-1 â¼ NI-5 formed hydrogen bonds and hydrophobic interactions with RNA, and exhibited enhanced fluorescence upon binding with RNA. These results will provide favorable support for the diagnosis and treatment of nucleolus-related diseases in the future.
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Nucléolo Celular , Naftalimidas , Humanos , Nucléolo Celular/metabolismo , Simulación del Acoplamiento Molecular , ARN/metabolismoRESUMEN
Neurotransmitter exocytosis of living cells plays a vital role in neuroscience. However, the available amperometric technique with carbon fiber electrodes typically measures exocytotic events from one cell during one procedure, which requires professional operations and takes time to produce statistical results of multiple cells. Here, we develop a functionally collaborative nanostructure to directly measure the neurotransmitter dopamine (DA) exocytosis from living rat pheochromocytoma (PC12) cells. The functionally collaborative nanostructure is constructed of metal-organic framework (MOF)-on-nanowires-on-graphene oxide, which is highly sensitive to DA molecules and enables direct detection of neurotransmitter exocytosis. Using the microsensor, the exocytosis from PC12 cells pretreated with the desired drugs (e.g., anticoronavirus drug, antiflu drug, or anti-inflammatory drug) has been successfully measured. Our achievements demonstrate the feasibility of the functionally collaborative nanostructure in the real-time detection of exocytosis and the potential applicability in the highly efficient assessment of the modulation effects of medications on exocytosis.
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Dopamina , Nanoestructuras , Animales , Ratas , Electrodos , Exocitosis/fisiología , NeurotransmisoresRESUMEN
To enhance our understanding of electroporation and optimize the pulses used within the frequency range of 1 kHz to 100 MHz, with the aim of minimizing side effects such as muscle contraction, we introduce a novel electrical model, structured as a 2D representation employing exclusively lumped elements. This model adeptly encapsulates the intricate dynamics of living cells' impedance variation. A distinguishing attribute of the proposed model lies in its capacity to decipher the distribution of transmembrane potential across various orientations within living cells. This aspect bears critical importance, particularly in contexts such as electroporation and cellular stimulation, where precise knowledge of potential gradients is pivotal. Furthermore, the augmentation of the proposed electrical model with the Hodgkin-Huxley (HH) model introduces an additional dimension. This integration augments the model's capabilities, specifically enabling the exploration of muscle cell stimulation and the generation of action potentials. This broader scope enhances the model's utility, facilitating comprehensive investigations into intricate cellular behaviors under the influence of external electric fields.
In our research, we've introduced an enhanced electrical model for living cells. This model simplifies cell behavior using only basic electrical components like resistors and capacitors. It's designed to mimic the real electrical properties of cells, particularly the cell membrane, which can change in response to electricity at different frequencies, ranging from 1 kHz to 100 MHz. This frequency range is essential for studying processes like electroporation, a technique used in various medical applications.Our model is represented in a two-dimensional structure, making it a handy tool for identifying transmembrane potential distributions, a critical factor in electroporation procedures. This means we can better understand how cells react to electrical impulses, which is crucial for improving electroporation techniques.Additionally, we've extended our model to include muscle cells by incorporating the Hodgkin-Huxley model, a well-established model for understanding electrical behavior in muscle cells. This allows us to study how muscles contract when exposed to different electrical pulses, a common side effect of electroporation procedures. By examining various pulse characteristics, we can determine which ones are best for minimizing muscle contractions during electroporation.In summary, our research has led to the development of a versatile electrical model for living cells. It not only helps us understand how cells respond to electricity in the context of electroporation but also provides insights into muscle contractions and how to optimize electrical pulses for medical treatments.
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Potenciales de Acción , Modelos Biológicos , Electroporación , Supervivencia Celular/efectos de la radiación , Electricidad , HumanosRESUMEN
The analysis and regulation of proteins are of great significance for the development of disease diagnosis and treatment. However, complicated analytical environment and complex protein structure severely limit the accuracy of their analysis results. Nowadays, ascribing to the editability and bioactivity of peptides, peptide-based probes could meet the requirements of good selectivity and high affinity to overcome the challenges. In this review, we summarize the advances in the use of modular peptide probes for proteins analysis. It focuses on how to design and optimize the structure of probes, as well as their performance. Then, the strategies and application to improve the analysis result of modular peptide probes are introduced. Finally, we also discuss current challenge and provide some ideas for the future direction for modular peptide probes, hoping to accelerate their clinical transformation.
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Colorantes Fluorescentes , Péptidos , Colorantes Fluorescentes/química , Péptidos/química , ProteínasRESUMEN
Many diseases are related to changes in the biomechanical properties of cells; their study can provide a theoretical basis for drug screening and can explain the internal working of living cells. In this study, the biomechanical properties of nephrocytes (VERO cells), hepatocytes (HL-7702 cells), and hepatoma cells (SMCC-7721 cells) in culture were detected by atomic force microscopy (AFM) to analyse the side effects of colchicine at different concentrations (0.1 µg/mL (A) and 0.2 µg/mL (B)) at the nanoscale for 2, 4 and 6 h. Compared with the corresponding control cells, the damage to the treated cells increased in a dose-dependent manner. Among normal cells, the injury of nephrocytes (VERO cells) was markedly worse than that of hepatocytes (HL-7702 cells) in both colchicine solutions A and B. Based on the analyses of biomechanical properties, the colchicine solution reduced the rate of division and inhibited metastasis of SMCC-7721 cells. By comparing these two concentrations, we found that the anticancer effect of colchicine solution A was greater than that of solution B. Studying the mechanical properties of biological cells can help understand the mechanism of drug action at the molecular level and provide a theoretical basis for preventing the emergence and diagnosis of diseases at the nanoscale.
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Colchicina , Hepatocitos , Animales , Chlorocebus aethiops , Colchicina/toxicidad , Fenómenos Biomecánicos , Células Vero , Microscopía de Fuerza AtómicaRESUMEN
A reactive fluorescent "turn-on" probe (di-PIP) with imine-linked dual phenanthro[9,10-d]imidazole luminophore have been conveniently prepared as an Al3+ and H+ dual functional receptor. di-PIP displayed high selectivity and sensitivity towards Al3+ ion in DMF/HEPES accompanied by fluorescence blue-shift and a good linear relationship as well as a low detection limit of 30.5 nmol·L-1, which can root from the synergetic functions of the decomposition reaction of di-PIP promoted by acidic Al3+ and the coordination effect between decomposition product and Al3+. Intriguingly, it was found that hydrogen ion H+ can be sufficient for simulating the fluorescence enhancing of di-PIP. 1H NMR titration and MS analyses for elucidation of the intermediate structure further revealed that the acid-triggered decomposition reaction resulted in the rapid, and sensitive sensing to Al3+ and H+. In addition, the probe di-PIP could be successfully applied to the detection of Al3+ in real water samples, and also utilized to visualize Al3+ and H+ in the living cells.
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Colorantes Fluorescentes , Protones , Colorantes Fluorescentes/química , Aluminio/análisis , Espectrometría de Fluorescencia , AguaRESUMEN
Heavy metal ion pollution harms human health and the environment and continues to worsen. Here, we report the synthesis of boron (B), phosphorous (P), nitrogen (N), and sulfur (S) co-doped carbon dots (BP/NS-CDs) by a one-step facile hydrothermal process. The optimum synthetic parameters are of 180 °C temperature, 12 h reaction time and 15% of PBA mass. The as-synthesized BP/NS-CDs exhibits excellent water solubility, strong green photoluminescence (PL) at 510 nm, and a high quantum yield of 22.4%. Moreover, BP/NS-CDs presented high monodispersity (7.2 ± 0.45 nm), excitation-dependent emission, PL stability over large pH, and high ionic strength. FTIR, XRD, and XPS are used to confirm the successful B and P doping of BP/NS-CDs. BP/NS-CD photoluminescent probes are selectively quenched by Cu2+ and Fe3+ ions but showed no response to the presence of other metal cations. The PL emission of BP/NS-CDs exhibited a good linear correlation with Cu2+ and Fe3+ concentrations with detection limits of 0.18 µM and 0.27 µM for Cu2+ and Fe3+, respectively. Furthermore, the HCT116 survival cells kept at 99.4 ± 1.3% and cell imaging capability, when the BP/NS-CDs concentration is up to 300 µg/mL by MTT assay. The proposed sensor is potential applications for the detection of Cu2+ and Fe3+ ions in environmental water samples.
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Carbono , Azufre , Humanos , Temperatura , Iones , Agua , NitrógenoRESUMEN
Nitric oxide (NO), as a vital signaling molecule related to different physiological and pathological processes in living systems, is closely associated with cancer and cardiovascular disease. However, the detection of NO in real-time remains a difficulty. Here, PtBi alloy nanoparticles (NPs) were synthesized, dealloyed, and then fabricated to NP-based electrodes for the electrochemical detection of NO. Transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and nitrogen physical adsorption/desorption show that dealloyed PtBi alloy nanoparticles (dPtBi NPs) have a porous nanostructure. Electrochemical impedance spectroscopy and cyclic voltammetry results exhibit that the dPtBi NP electrode possesses unique electrocatalytic features such as low charge transfer resistance and large electrochemically active surface area, which lead to its excellent NO electrochemical sensing performance. Owing to the higher density of catalytical active sites formed PtBi bimetallic interface, the dPtBi NP electrode displays superior electrocatalytic activity toward the oxidation of NO with a peak potential at 0.74 V vs. SCE. The dPtBi NP electrode shows a wide dynamic range (0.09-31.5 µM) and a low detection limit of 1 nM (3σ/k) as well as high sensitivity (130 and 36.5 µA µM-1 cm-2). Moreover, the developed dPtBi NP-based electrochemical sensor also exhibited good reproducibility (RSD 5.7%) and repeatability (RSD 3.4%). The electrochemical sensor was successfully used for the sensitive detection of NO produced by live cells. This study indicates a highly effective approach for regulating the composition and nanostructures of metal alloy nanomaterials, which might provide new technical insights for developing high-performance NO-sensitive systems, and have important implications in enabling real-time detection of NO produced by live cells.
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Nanopartículas , Óxido Nítrico , Reproducibilidad de los Resultados , Dispersión del Ángulo Pequeño , Difracción de Rayos X , AleacionesRESUMEN
A novel aptasensor has been designed for quantitative monitoring of epinephrine (EP) based on cerium metal-organic framework (CeMOF) loaded gold nanoparticles (AuNPs). The aptamer, specific to EP, is immobilized on a flexible screen-printed electrode modified with AuNPs@CeMOF, enabling highly selective binding to the target biomolecule. Under optimized operational conditions, the peak currents using voltammetric detection measured at voltage of 83 mV (vs. Ag/AgCl) for epinephrine exhibit a linear increase within concentration in the range 1 pM-10 nM. Following this detection strategy, a boasted limit of detection of 0.3 pM was achieved, surpassing the sensitivity of most reported methods. The developed biosensor showcased exceptional performance in detection of EP in spiked serum sample, with remarkable recovery range of 95.8-113% and precision reflected by low relative standard deviation (RSD) ranging from 2.23 to 6.19%. These results indicate the potential utility of this biosensor as a valuable clinical diagnostic tool. Furthermore, in vitro experiments were carried out using the presented biosensor to monitor the release of epinephrine from PC12 cells upon extracellular stimulation with K+ ions.
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Aptámeros de Nucleótidos , Nanopartículas del Metal , Estructuras Metalorgánicas , Oro/química , Nanopartículas del Metal/química , Aptámeros de Nucleótidos/química , Estructuras Metalorgánicas/química , EpinefrinaRESUMEN
As the third gasotransmitter, hydrogen sulfide (H2S) is involved in a variety of physiological and pathological processes wherein abnormal levels of H2S indicate various diseases. Therefore, an efficient and reliable monitoring of H2S concentration in organisms and living cells is of great significance. Of diverse detection technologies, electrochemical sensors possess the unique advantages of miniaturization, fast detection, and high sensitivity, while the fluorescent and colorimetric ones exhibit exclusive visualization. All these chemical sensors are expected to be leveraged for H2S detection in organisms and living cells, thus offering promising options for wearable devices. In this paper, the chemical sensors used to detect H2S in the last 10 years are reviewed based on the different properties (metal affinity, reducibility, and nucleophilicity) of H2S, simultaneously summarizing the detection materials, methods, linear range, detection limits, selectivity, etc. Meanwhile, the existing problems of such sensors and possible solutions are put forward. This review indicates that these types of chemical sensors competently serve as specific, accurate, highly selective, and sensitive sensor platforms for H2S detection in organisms and living cells.
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Sulfuro de Hidrógeno , Metales , Colorantes Fluorescentes/química , ColorimetríaRESUMEN
A random laser carrying the scattering information on a biological host is a promising tool for the characterization of biophysical properties. In this work, random lasing from label-free living cells is proposed to achieve rapid cytometry of apoptosis. Random lasing is achieved by adding biocompatible gain medium to a confocal dish containing cells under optically pumped conditions. The random lasing characteristics are distinct at different stages of cell apoptosis after drug treatment. By analyzing the power Fourier transform results of the random lasing spectra, the percentage of apoptotic cells could be distinguished within two seconds, which is more than an order of magnitude faster than traditional flow cytometry. These results provide a label-free approach for rapid cytometry of apoptosis, which is advantageous for further research of random lasers in the biological field.
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Rayos Láser , Luz , ApoptosisRESUMEN
A conditional entropic approach is discussed for nonequilibrium complex systems with a weak correlation between spatiotemporally fluctuating quantities on a large time scale. The weak correlation is found to constitute the fluctuation distribution that maximizes the entropy associated with the conditional fluctuations. The approach is illustrated in diffusion phenomenon of proteins inside bacteria. A further possible illustration is also presented for membraneless organelles in embryos and beads in cell extracts, which share common natures of fluctuations in their diffusion.
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Förster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS-SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3-cube FRET imaging in OS-SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS-SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS-SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS-SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were EC32VOSF=0.32±0.02,EC17VOSF=0.38±0.02 , and EC5VOSF=0.45±0.03 , and the measured acceptor-to-donor concentration ratios ( Rc ) were RC32VOSF=1.07±0.03 , RC17VOSF=1.09±0.03 , and RC5VOSF=1.02±0.04 , consistent with the reported values. Collectively, our data demonstrates that OS-SIM can be integrated into FRET microscopy to build an OS-SIM-FRET with confocal microscopy-like resolution.
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Transferencia Resonante de Energía de Fluorescencia , Iluminación , Transferencia Resonante de Energía de Fluorescencia/métodos , Células HeLa , Humanos , Microscopía Confocal/métodosRESUMEN
As an important cellular microenvironmental parameter, viscosity could reflect the status of living cells. Small molecular fluorescent probes are a vital tool to measure the change of viscosity in living cells. A novel fluorescence probe ZL-1 with a large Stokes shift (in methanol it reached to 153 nm and in glycerol it reached to 125 nm) and excellent sensitivity toward viscosity was developed. The sharp enhancement of the emission intensity for the probe ZL-1 from low viscous methanol to high viscous glycerol indicated that the probe ZL-1 could respond to the viscosity variations. Moreover, the probe ZL-1 has been successfully utilized to detect of the viscosity variations in living cells.
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Colorantes Fluorescentes , Metanol , Glicerol , Células HeLa , Humanos , Imagen Óptica/métodos , ViscosidadRESUMEN
Cell, enzyme, and tissue activity in living organisms are closely related to intracellular pH. Detecting the changes of intracellular pH is important to understanding the physiological and pathological changes in the process of crucial cell metabolism. A pH probe (HTBI) based on hemicyanine was synthesized. The probe solution displayed a marked colour change from yellow to amaranth with the pH increase from neutral to basic; simultaneously, the emission spectra showed a significant red shift. The probe exhibited a ratiometric fluorescence emission (F586nm /F542nm ) characteristic of pKa 8.82. As expected, HTBI exhibited high sensitivity and selectivity for pH, fine photostability, reversibility, and low cytotoxicity. Therefore, it would be a very useful tool for measuring the intracellular pH changes.
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Colorimetría , Colorantes Fluorescentes , Colorantes Fluorescentes/química , Concentración de Iones de Hidrógeno , Imagen Óptica , Espectrometría de FluorescenciaRESUMEN
Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment. In more detail, Raster Image Correlation Spectroscopy (RICS) performed at fast timescales (i.e., microseconds) reveals the fast motion of fluorescently labeled molecules within two exemplary dynamic subcellular nanostructures of biomedical interest, the lysosome and the insulin secretory granule (ISG). The measurement of molecular diffusion is then used to extract information on the average properties of subcellular nanostructures, such as macromolecular crowding or molecular aggregation. Concerning the lysosome, fast RICS on a fluorescent tracer allowed us to quantitatively assess the increase in organelle viscosity in the pathological condition of Krabbe disease. In the case of ISGs, fast RICS on two ISG-specific secreting peptides unveiled their differential aggregation propensity depending on intragranular concentration. Finally, a combination of fast RICS and feedback-based 3D orbital tracking was used to subtract the slow movement of subcellular nanostructures from the fast diffusion of molecules contained within them and independently validate the results. Results presented here not only demonstrate the acquired ability to address the dynamic behavior of molecules in moving, nanoscopic reference systems, but prove the relevance of this approach to advance our knowledge on cell function at the subcellular scale.