Fluorescence Imaging Of Cells Research Articles

Infectious Virus Tracking by Fluorescent Live Cell Imaging in Primary Cells.

To successfully infect a cell, HIV-1 has to overcome several host barriers while exploiting cellular cofactors. HIV-1 infection is highly inefficient with the great majority of viral particles not being able to successfully integrate into the target cell genome. Nonproductive HIV-1 particles are degraded or accumulated in cellular compartments. Thus, it becomes hard to distinguish between viral behaviors that lead to effectively infecting the cell from the ones that do not by using traditional methods. Here, we describe the infectious virus tracking method that detects and quantifies individual fluorescent viral particles over time and links viral particle behavior to its infectivity. This method employs live-cell imaging at ultra-low MOIs to detect the outcome of infection for every HIV-1 particle.

Designing functionalized nanodiamonds with hyaluronic acid-phospholipid conjugates for enhanced cancer cell targeting and fluorescence imaging capabilities.

Nanomedicine aims to develop smart approaches for treating cancer and other diseases to improve patient survival and quality of life. Novel nanoparticles as nanodiamonds (NDs) represent promising candidates to overcome current limitations. In this study, NDs were functionalized with a 200 kDa hyaluronic acid-phospholipid conjugate (HA/DMPE), enhancing the stability of the nanoparticles in water-based solutions and selectivity for cancer cells overexpressing specific HA cluster determinant 44 (CD44) receptors. These nanoparticles were characterized by diffuse reflectance Fourier-transform infrared spectroscopy, Raman spectroscopy, and photoluminescence spectroscopy, confirming the efficacy of the functionalization process. Scanning electron microscopy was employed to evaluate the size distribution of the dry particles, while dynamic light scattering and zeta potential measurements were utilized to evaluate ND behavior in a water-based medium. Furthermore, the ND biocompatibility and uptake mediated by CD44 receptors in three different models of human adenocarcinoma cells were assessed by performing cytofluorimetric assay and confocal microscopy. HA-functionalized nanodiamonds demonstrated the advantage of active targeting in the presence of cancer cells expressing CD44 on the surface, suggesting higher drug delivery to tumors over non-tumor tissues. Even CD44-poorly expressing cancers could be targeted by the NDs, thanks to their good passive diffusion within cancer cells.

A lipid droplet-targeted fluorescent probe for fluorescence imaging of cell and zebrafish viscosity

Lipid droplets (LDs) represent highly dynamic organelles found in almost all organisms and play a pivotal role in regulating the balance of intracellular lipid homeostasis.

Monoamine oxidase A probe synthesis and fluorescence imaging in cells and zebrafish based on AIE mechanism

Monoamine oxidase A (MAO-A) is a crucial mammalian flavoenzyme that plays a significant role in various neurodegenerative diseases such as depression, Parkinson’s disease, schizophrenia, and epilepsy, as well as cancer, cardiac disorders, and liver disease. Thus, a real-time and targeted MAO-A detection approach is desperately needed to study the onset and management of disorders linked to MAO-A. In this study, we have successfully synthesized a red-emitting fluorescent probe AIE-M, which is based on the specificity of MAOs for the structural catalysis of propylamine group substrates. This probe utilizes quinoline-malononitrile (QM) with AIE properties as the fluorescent master. The probe containing QM exhibits excellent aggregation-induced luminescence (AIE), biocompatibility, and high photostability, enabling specific recognition of MAO-A. Moreover, it can be employed for the detection and imaging of endogenous MAO-A in neuroblastoma cells and zebrafish, thereby offering a novel research instrument for diagnosing associated ailments.

Real-time imaging of individual electropores proves their longevity in cells

With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to −400 mV pulse between the patch pipette and ITO evoked focal Ca2+ transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients (“Ca2+ plumes”) faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to −50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.

A novel “turn-on” fluorescent probe based on thiocarbamoyl-DHP for Hg2+ detection in water samples and living cells

In this study, two fluorescent sensing probes, dihydropyridine (DHP) derivatives (DHP-CT1 and DHP-CT2) bearing phenoxy thiocarbonyl group, have been developed for Hg2+ detection. The tandem trimerization-cyclization of methylpropiolate with ammonium acetate gave 1.4-DHP and 1,2-DHP derivatives, which were reacted with O-phenylcarbonochloridothioate to produce DHP-CT1 and DHP-CT2, respectively. DHP-CT1 exhibits superior sensitivity and selectivity of fluorescence enhancement towards Hg2+ in aqueous media. The fluorescence intensity shows a good linear relationship with the concentration of Hg2+ in the range of 0–10 µM providing the extremely low LOD of 346 nM (69.4 ppb). The fluorescence enhancement is caused by the Hg2+ promoted hydrolysis of the thioamide bond releasing the fluorescent 1,4-DHP that was confirmed by NMR and HRMS. The quantitative analysis of Hg2+ in water samples using DHP-CT1 probe was demonstrated in aqueous solution and paper-based sensing strips. Furthermore, DHP-CT1 was also applied for monitoring intracellular Hg2+ in living RAW264.7 macrophages through fluorescence cell imaging.

Dynamic altruistic cooperation within breast tumors

BackgroundSocial behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism’s survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance.MethodsMicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells.ResultsOpposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKβ. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit.ConclusionsOur data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

A structural optimized fluorescent probe for monitoring hydrogen sulfide in cells and zebrafish

In this work, we reported a fluorescent probe Fur-SH, a derivative of benzofuranone, which was used to detect H2S in living cells and zebrafish. Based on the three structural characteristics of the probe, the effects of different structural modifications on the optical properties of the fluorophore were compared. Then, the fluorophore Fur-OH was synthesized by modifying diethylamino group with benzofuranone as the main skeleton. With 2,4-dinitrofluorobenzene as the recognition group and diethylamino as the electron donor, the push–pull electron effect occurred with nitro group, which led to fluorescence quenching, and an openable fluorescent probe Fur-SH was formed. The probe Fur-SH (λex = 510 nm; λem = 570 nm) had the advantages of smaller full width at half maxima, rapid response (5 min) and wide pH window. The quantitative properties of the probe were excellent, reaching saturation at 50 equivalents of substrate. The probe Fur-SH showed high sensitivity to H2S, with LOD of 48.9 nM and LOQ of 50 nM. At present, the probe Fur-SH had been applied to fluorescence imaging of MCF-7 cells and zebrafish. By comparing the effects of different structures on the optical properties of fluorophores, this work was expected to be helpful to the development of fluorescent probes in the future.

The Na/K-ATPase α1/Src Signaling Axis Regulates Mitochondrial Metabolic Function and Redox Signaling in Human iPSC-Derived Cardiomyocytes.

Na/K-ATPase (NKA)-mediated regulation of Src kinase, which involves defined amino acid sequences of the NKA α1 polypeptide, has emerged as a novel regulatory mechanism of mitochondrial function in metazoans. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To assess the impact of the proposed NKA/Src regulatory axis on cardiac mitochondrial metabolic function, we used a gene targeting approach in human cardiac myocytes. Human induced pluripotent stem cells (hiPSC) expressing an Src-signaling null mutant (A420P) form of the NKA α1 polypeptide were generated using CRISPR/Cas9-mediated genome editing. Total cellular Na/K-ATPase activity remained unchanged in A420P compared to the wild type (WT) hiPSC, but baseline phosphorylation levels of Src and ERK1/2 were drastically reduced. Both WT and A420P mutant hiPSC readily differentiated into cardiac myocytes (iCM), as evidenced by marker gene expression, spontaneous cell contraction, and subcellular striations. Total NKA α1-3 protein expression was comparable in WT and A420P iCM. However, live cell metabolism assessed functionally by Seahorse extracellular flux analysis revealed significant reductions in both basal and maximal rates of mitochondrial respiration, spare respiratory capacity, ATP production, and coupling efficiency. A significant reduction in ROS production was detected by fluorescence imaging in live cells, and confirmed by decreased cellular protein carbonylation levels in A420P iCM. Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This signaling axis in cardiac myocytes may provide a new approach to counteract mitochondrial dysfunction in cardiometabolic diseases.

Multifunctional phthalocyanine NIR sensor for fluorescent and colorimetric dual-imaging and removal of intracellular and environmental Cu2+

Cu2+ sensing and removal are of great significance for environment protection and human health. Herein, multifunctional Cu2+ sensor system for both detection and removal of intracellular and environmental Cu2+ based on trans/cis interconversion of silicon phthalocyanine (SiPc) was developed. This phthalocyanine based Cu2+ sensor shows remarkably dual-mode NIR fluorescence and colorimetric responses for recognition of Cu2+, resulting in insoluble chelate for Cu2+ removal as well as the extremely high anti-interference in the presence of various anions and cations. The binding mechanism and stoichiometry ratio were systematically revealed. Intracellular fluorescence imaging in MCF-7 cells demonstrated that Cu2+ could be efficiently detected in living cells and form insoluble chelate for clearance. As the result, this novel phthalocyanine based multifunctional sensor can be used for dual-mode NIR fluorescence/colorimetric detection and removal of Cu2+ in either complicated environment or biological systems.

Synthesis of a super-low detection limit fluorescent probe for Al3+ and its application in fluorescence imaging of zebrafish and cells

A novel fluorescent probe N'-(2-hydroxybenzylidene)-indole-3-formylhydrazine (JHK) was designed and synthesized based on the condensation reaction of indole-3-formylhydrazine and salicylaldehyde. The probe JHK solution could highly selectively recognize Al3+ by the obvious fluorescence enhancement (288-fold) after adding Al3+. And the probe solution with Al3+ had a very high fluorescence quantum yield (89.29 %). The detection limit was calculated to be 1.135 nM, which was significantly lower than many reported detection limits, indicating that the probe JHK had pretty good sensitivity. The ratio of JHK to Al3+ (1:1) and the sensing mechanism were determined by Job's plot, 1H NMR spectra, FTIR spectra, ESI-MS and Gaussian calculation. The probe solution and medium-speed filter paper were successfully used to make test papers for more convenient detection of Al3+. Furthermore, the probe JHK had been successfully applied to the detection of Al3+ in real water, zebrafish and living cells.

Relay recognition of Cu2+ and S2− using naphthalimide-based fluorescent probe and its applications in molecular logic gate and bioimaging

A fluorescent chemosensor based on naphthalimide derivative (HL) was prepared and characterized. HL exhibited strong fluorescence emission at 528 nm, which was attributed to naphthalimide moiety, while the addition of Cu2+ almost completely quenched the fluorescence emission. However, with the addition of S2−, the fluorescence at 528 nm reoccurred remarkably. The detection limits for Cu2+ and S2− were measured to be 1.72 × 10−7 M and 2.66 × 10−7 M, respectively. As determined, the fluorescence behavior of HL in the presence of Cu2+ and S2− could be manufactured to IMPLICATION type logic gate. Moreover, confocal fluorescence imaging in HepG2 cells indicated that the HL could act as a useful fluorescent probe for intracellular Cu2+ and S2− detection with negligible cytotoxicity.

A triple-targeting fluorescent probe reveals the glutathione and viscosity crosstalk in mitochondria, endoplasmic reticulum, and nucleoli in cells during ferroptosis

Ferroptosis, a recently termed cell death form, is featured by iron accumulation and lipid peroxidation, which plays various roles in physiological and pathological processes such as cancer evolution and therapy. Redox homeostasis profoundly varies in cells during ferroptosis, but many events remain to be explored, especially at the sub-cellular level. Glutathione (GSH) is the most abundant bio-reductant and mainly distributes in mitochondria (Mito), endoplasmic reticulum (ER), and nucleoli (Nuc) except the cytoplasm. The GSH level is intrinsically related to the redox equilibriums in the three organelles. Viscosity is an index of the intracellular micro-environments related to redox homeostasis. The dynamics of the GSH and viscosity levels in organelles are vital to understanding cell ferroptosis but are in a veil due to the lack of the appropriate tools. Here we report a fluorescent probe (FPY) with dual-response to GSH and viscosity, characteristic of the triple-targeting to Mito, ER, and Nuc. We apply FPY in fluorescent imaging of cells during ferroptosis and disclose the GSH-viscosity crosstalk at the sub-cellular level. We discover an abnormal GSH recruitment in Nuc from the cytoplasm in the late stage of ferroptosis.

Lysosome-targeted dual-emissive carbon dots for ratiometric optical dual-readout and smartphone-assisted visual determination of Hg2+ and SO32−

Smartphone-assisted visual assay not only expands quantitative analysis but also enhance real-time on-site sensing capabilities. Herein, lysosome-targeted dual-emissive carbon dots (L-CDs) can not only recognize Hg2+ and SO32− by ratiometric fluorescence and ratiometric absorption, but also visually quantify Hg2+ and SO32− by smartphone-assisted method. With monitoring of intrinsic ratiometric fluorescent variation (I580/I468), L-CDs are developed as an effective sensing platform for ratiometric fluorescent successive identification of Hg2+ and SO32− accompanying with continuous fluorescence variation of blue, purplish pink, pink, and light yellow. With detecting of inherent ratiometric absorption change (A538/A206), L-CDs are also constructed as an efficacious sensing terrace for ratiometric absorption consecutive discrimination of Hg2+ and SO32−. More crucially, integrating continuous fluorescence color change and color recognizer APP in the smartphone, visual quantification of Hg2+ and SO32− can be accomplished on the basis of the ratio of red value and blue value (R/B) with linear ranges of 0–130 and 0–850 µM, respectively, as well as LOD of 8.4 and 12.9 nM, respectively. More interestingly, confocal fluorescent imaging of HeLa cells further verifies that L-CDs can be regarded as favorable biosensors for on-site, real-time differentiation of Hg2+ and SO32− in vitro and in vivo with ratiometric manners.

Ratiometric fluorescence detection of the angiotensin-converting enzyme via single-excitation and double-emission biomass-derived carbon quantum dots

Efficient and rapid detection of angiotensin-converting enzyme (ACE) activity is important for preventing hypertension and the discovery of new angiotensin-converting enzyme inhibitors (ACEI). In this work, a single-excitation and double-emission biomass-derived carbon quantum dots (CQDs) was prepared and applied for ratiometric fluorescence detection of ACE. Fresh banyan leaves were extracted with ethanol and acetone, and the extracted solution was used as the precursor to produce the carbon quantum dots (BL-CQDs) with single-excitation and double-emission properties. The synthesized BL-CQDs is about 1.7 nm, has a graphene-like structure, contains a variety of hydrophilic functional groups on the surface, and has good fluorescence properties. Its fluorescence intensity ratio (I677/I460) is linear with ACE activity in the range of 0.02–0.8 U l−1. The regression equation is △F=2.5371 C ACE -0.0311. The method was successfully applied to the determination of ACE activity in pig lung and human serum, and the inhibitory efficiency of the flavonoid extract and captopril tablets on ACE activity was also investigated, which can be applied to the screening of ACEI. The survival rate and fluorescence imaging of Bel-7404 cells under the condition of high concentration BL-CQDs showed BL-CQDs had low cytotoxicity and good biocompatibility. These results indicate that the BL-CQDs can be used as an excellent fluorescent probe, providing a new method for screening ACE activity and plant-derived ACEI.

Tocotrienol-Rich Fraction Attenuates Blue Light-Induced Oxidative Stress and Melanogenesis in B16-F1 Melanocytes via Anti-Oxidative and Anti-Tyrosinase Properties.

Our skin is constantly exposed to blue light (BL), which is abundant in sunlight and emitted by digital devices. Prolonged exposure to BL can lead to oxidative stress-induced damages and skin hyperpigmentation. For this study, we used a cell line-based model to examine the protective effects of tocotrienol-rich fraction (TRF) on BL-induced oxidative stress and hyperpigmentation in B16-F1 melanocytes. Alpha-tocopherol (αTP) was used as a comparator. Molecular assays such as cell viability assay, flow cytometry, western blotting, fluorescence imaging, melanin and tyrosinase analysis were performed. Our results showed that TRF effectively suppressed the formation of reactive oxygen species and preserved the mitochondrial membrane potential. Additionally, TRF exhibited anti-apoptotic properties by reducing the activation of the p38 mitogen-activated protein kinase molecule and downregulating the expression of cleaved caspase-3. Moreover, TRF modulated tyrosinase activity, resulting in a lowered rate of melanogenesis and reduced melanin production. In contrast, αTP did not exhibit significant protective effects against skin damages and pigmentation in BL-induced B16-F1 cells. Therefore, this study indicates that TRF may offer superior protective effects over αTP against the effects of BL on melanocytes. These findings demonstrate the potential of TRF as a protective natural ingredient that acts against BL-induced skin damages and hyperpigmentation via its anti-oxidative and anti-melanogenic properties.

2-λ5-Phosphaquinolin-2-ones as Non-cytotoxic, Targetable, and pH-Stable Fluorophores.

Several phosphaquinolinone derivatives have been synthesized and characterized to explore their usefulness in the realm of cell imaging. Solution-state photophysical properties in both aqueous and organic solutions were collected for these derivatives. Additionally, CCK-8 cell viability assays and fluorescent imaging in HeLa cells incubated with the new heterocyclic derivatives show evidence of favorable cell permeability, cell viability, and moderate intracellular localization when appended with the well-known morpholine targeting motif.

Dual-Stimuli-Activatable Hybrid Prodrug for the Self-Immolative Delivery of an Anticancer Agent and Hydrogen Sulfide with Turn-On Fluorescence.

Stimuli-responsive fluorogenic prodrugs are advantageous for the targeted drug delivery enabling real-time non-invasive monitoring with turn-on fluorescence. We report herein the dual-stimuli (ROS and CA)-responsive thiocarbamate-based prodrug (AM-TCB) for the turn-on fluorogenic delivery of the naphthalimide-based anticancer agent amonafide along with the gasotransmitter hydrogen sulfide (H2 S). A carbamate-based prodrug AM-CB was also designed, capable of releasing the anticancer agent amonafide without any H2 S. The prodrugs were synthesized using multi-step organic synthesis. UV-Vis and fluorescence spectroscopic studies revealed selective reactivity of the boronate ester group of prodrugs towards ROS (primarily H2 O2 ) with the release of amonafide and COS/CO2 via self-immolative processes. Hydrolysis of the generated COS by carbonic anhydrase (CA) produces H2 S. While the prodrug AM-TCB retained the anticancer activity of free amonafide in cancer cells (MDA-MB-231 and HeLa), unlike amonafide, it enhanced the cellular viability of the non-malignant cells (HEK-293). Fluorescence imaging in HeLa cells revealed the simultaneous delivery of the anticancer agent and H2 S from AM-TCB with turn-on fluorescence. Western blot studies further revealed the cytoprotective effects of the released H2 S from AM-TCB. The present adjuvant strategy therefore would be helpful in future for ameliorating the anticancer drug-induced side-effects.

Gram-scale synthesis of graphene quantum dots via energetic material deflagration for bioimaging application

Graphene quantum dots, which possesses unique photoluminescence property, have received enormous attention for bio-sensor, photovoltaic devices and bioimaging application. However, synthesizing high-quality of GQDs remains a formidable obstacle in terms of the two conventional methods since the “top-down” method usually requires expensive raw materials while the “bottom-up” method suffers from incompatibility between quality and production. Thus, developing new strategies to trade-off these issues is promising for GQDs production. Here, we reported an ultrafast deflagration strategy by igniting Al and pentafluoropyridine mixture to prepare high-quality of carbon nanomaterials (CNs) with high graphitization, reaching a high yield of ∼3 g. Combining with a subsequent Hummer's exfoliation and dialyze method, the CNs materials were successfully exfoliated into the uniform-dispersed graphene quantum dots (D-GQDs) with size of 1∼2 nm. The as-prepared D-GQDs derivatives shows remarkable optical properties and it is successfully applied in fluorescence imaging of HeLa cells. Our results in this study highlighted the high efficiency of deflagration technique to prepare functional carbon-based nanomaterials, and proposed a facile and fast strategy for gram-scale synthesis.

6-azido and 6-azidomethyl uracil nucleosides

Azido nucleosides have been utilized for click reactions, metabolic incorporation into cellular DNA, and fluorescent imaging of live cells. Two classes of 6-azido modified uracil nucleosides; one with azido group directly attached to uracil ring and second with azido group attached via methylene linker are described. The 6-azido-2’-deoxyuridine (6-AdU) was prepared in 55% overall yield by lithiation-based regioselective C6-iodination of silyl protected 2’-deoxyuridine followed by treatment with sodium azide and deprotection with TBAF. Lithiation-based C6-alkylation of the protected uridine with methyl iodide followed by the oxidation of the 6-methyl product with selenium dioxide and the subsequent mesylation and azidation of the resulting 6-hydroxymethyl group gave after deprotection 6-azidomethyluridine (6-AmU) in 61% overall yield. Direct lithiation-based C6-hydroxymethylation followed by mesylation/azidation sequence and deprotection provided 6-AmU or 6-azidomethyl-2’-deoxyuridine (6-AmdU). Yields for the lithiation-based regioselective C6-iodination and alkylation were higher for uridine than 2’-deoxyuridine derivatives and they appear to be less dependent on the sugar protection group used. Strain promoted click reactions of 6-AdU and 6-AmdU with symmetrically fused cyclopropyl cyclooctyne (OCT) provided fluorescent triazoles. DFT-calculated dihedral angles and energy differences for the favored anti and syn conformation of 6-AdU and 6-AmdU versus their C5 azido counterparts are discussed.