Imaging Of HeLa Cells Research Articles

Bis-chalcone Fluorescent Probe for Hydrazine Ratio Sensing in Environment and Organism.

In this paper, four novel hydrazine fluorescent probes X1-X4 with bis-chalcone structure were designed and synthesized. Through the measurement of its optical properties, it is found that it can quickly identify hydrazine, high sensitivity, low detection limit, and good anti-interference ability. The recognition of hydrazine by probes X1-X4 is not affected in the pH range of 4-10, X2 has the highest sensitivity, and the detection limit is as low as 0.336 × 10-7 M. Through Gaussian quantization calculation of probe molecules and their reaction products with hydrazine, it is speculated that the recognition mechanism is the closure of intramolecular charge transfer effect. In addition, the cytotoxicity and imaging of HeLa cells were tested, which showed that probes X1-X4 could be used to detect hydrazine in cells.

Synthesis, characterization, fluorescent emission of sodium poly(ethylene glycol) and its uses in sensing, cell imaging and data encryptions

In contrast to conventional fluorescent polymers featuring large conjugated structures or classical chromophores, a class of fluorescent polymers, free of all conjugations and chromophores, has been gaining significance in immerging applications that conventional fluorescent materials cannot achieve. Poly(ethylene glycol) (PEG) is a popular biopolymer widely used in biomedical fields and reportedly fluorescent emissive under limited conditions. By simply reacting with concentrated NaOH, Na alkoxidation of low molecular weight PEG is achieved (Mn 200 to 600), and the highest alkoxidation is achieved with PEG of Mn 400. The formation of the sodium PEG (NaPEG) is confirmed through multiple perspectives: a distinct color change from colorless to dark brown for the reaction mixture, reaction materials balance, NMR and FTIR analyses, fluorescent emission and the detection of its cluster sizes, all in comparison with the starting material PEG. Comparing to PEG, the fluorescence emission of NaPEG is largely enhanced by more than 20 times, thanks to its enhanced clustering capacity based on cluster-triggered emission principle, confirmed by different testing. NaPEG is used for detections of Cr6+, Fe3+, H2O2 and antibiotic doxycycline (DOX), out-performing most of reported sensors. Practically nontoxic, NaPEG is also used for data encryption of different versions, HeLa cell imaging and monitoring of Fe3+ and DOX under physiological conditions. This work provides therefore a simple protocol for synthesis of a novel green polymer sensor of great potential for multipurpose applications, in particular as sensors in biomedical areas.

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.

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.

Synthesis of pH-Sensitive Nitrogen-Doped Carbon Dots with Biological Imaging Function and Their Application in Cu2+ and Fe2+ Determination by Ratiometric Fluorescent Probes.

pH-sensitive nitrogen-doped carbon dots (N-CDs) were synthesized using immature seeds of elm trees as a carbon source and ethylenediamine as a coreactant through a facile one-step hydrothermal method. The N-CDs were characterized using fluorescence spectroscopy, fluorescence lifetime, ultraviolet-visible absorption, X-ray photoelectron spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy, as well as transmission electron microscopy. The N-CDs displayed excellent fluorescence properties and responded to pH changes. The N-CDs exhibited low toxicity and good biocompatibility and had the potential to be used for the biological imaging of HeLa cells and mung bean sprouts. Utilizing the mechanism of fluorescence resonance energy transfer, ratiometric fluorescent probes were prepared by simple mixing of N-CDs and fluorexon in a Britton-Robinson buffer solution. The ratiometric fluorescent probe was used to detect Cu2+ and Fe2+. The linear equations were RCu = -0.0591[Q] + 3.505 (R2 = 0.992) and RFe = -0.0874[Q] + 3.61 (R2 = 0.999). The corresponding limits of detection were 0.5 and 0.31 μM, respectively. The good results had been obtained in the actual samples detection.

Enhanced Scanning Electron Microscopy Using Auto-Optimized Image Restoration With Constrained Least Squares Filter for Nanoscience.

The growing demands of nanoscience require the continuous improvement of visualization methods. The imaging performance of scanning electron microscopy (SEM) is fundamentally limited by the point spread function of the electron beam and degrades because of noise. This paper proposes an auto-optimization algorithm based on deconvolution for the restoration of SEM images. This algorithm uses a constrained least squares filter and does not dependent on the user's experience or the availability of nondegraded images. The proposed algorithm improved the quality of the SEM images of 10-nm Au nanoparticles, and achieved balance among the sharpness, contrast-to-noise ratio (CNR), and image artifacts. For the SEM image of 100-nm pitched line patterns, the analysis of the spatial frequencies allowed the 2.5-fold improvement of the intensity of 4-nm information, and the noise floor decreased approximately 32 times. Along with the results obtained by the application of the proposed algorithm to images of tungsten disulfide (WS2) flakes, carbon nanotubes (CNTs), and HeLa cells, the evaluation results confirm that the proposed algorithm can enhance the SEM imaging of nanoscale features that lie close to the microscope's resolution limit.

Red emission upconversion of NaYF4: Yb3+/Er3+ nanoparticles: Rapid preparation via single-mode focused microwave method and their application in cell imaging

The red emission of upconversion nanoparticles (UCNPs) is located in an “optical window” range of the biological tissue, which has a promising prospect in biology application. In this work, novel red-emitting NaYF4:Yb3+/Er3+ UCNPs with CH3COO− modifications were synthesized by single-mode focused microwave method. The morphology, structure, and luminescence properties of as-prepared UCNPs were characterized. The results showed that single-mode focused microwave method was an effective approach to prepare hydrophilic and strongly red emission UCNPs within a drastically reduced reaction time (only 10 min) compared to the traditional methods. Importantly, we also demonstrated that the CH3COO− modified NaYF4:Yb3+/Er3+ UCNPs could be successfully applied for in vitro imaging of HeLa cells. This work not only provides a rapidly synthetic methodology for the preparation of red-emitting UCNPs but also offers an effective material for cancer cell imaging.

Strategy of eudragit coated curcumin nanoparticles delivery system: Release and cell imaging studies in simulated gastrointestinal microenvironments

Curcumin has a broad-spectrum anti-tumor effect and has no toxic side effects. However, the unique diketone structure of curcumin will undergo diketo-enol tautomerism under different acid-base conditions, resulting in its instability under physiological conditions. In addition, the low biocompatibility and absorption rate of curcumin also limit the use of curcumin drugs. In this paper, curcumin was modified by substitution of acryloyl and acrylsulfonyl groups, and four kinds of nanoparticles with regular morphology were prepared using non-toxic and non-irritating acrylic resin as coating material to improve the stability and bioavailability of the compounds. Zeta potential testing shows that the composites surface carries positive charges and have good stability. In the release experiment, four complexes have the potential for slow and controlled release. Imaging of Hela cells with different channels was performed, and the imaging results showed that the complexes could enter the cells and be absorbed by them, demonstrating good imaging performance. MTT experiments have shown that the complexes have certain anti-tumor activity and low cytotoxicity. In general, the complexes synthesized in this paper have potential in the field of drug fluorescence imaging detection. At the same time, this experiment provides a new idea for the design of slow and controlled release of drugs.

A novel colorimetric/fluorescent dual-signal probe based on silver nanoparticles functionalized with L-cysteine and rhodamine 6G derivatives for copper ion detection and cell imaging

The dual-signal probe utilizing functionalized silver nanoparticles (AgNPs) is a promising sensing tool. Herein, a novel colorimetric/fluorescent dual-signal probe (AgNPs-L-Cys-Rh6G2) was fabricated for copper ion (Cu2+) detection and cell imaging by using L-cysteine as a “bridge” to connect AgNPs and rhodamine 6G derivatives. The AgNPs-L-Cys-Rh6G2 probe exhibits a dual-signal response to Cu2+ due to Rh6G2 hydrolysis, resulting in a high fluorescence response and a significant change in color from light yellow to pink under sunlight. The linear detection ranges of the AgNPs-L-Cys-Rh6G2 probe for Cu2+ were 100–450 μM and 150–650 μM using fluorescent and colorimetry methods, respectively. The detection limits were as low as 0.169 μM and 1.36 μM, respectively. Meanwhile, the proposed probe was applied to detect Cu2+ in the actual sediment with satisfactory recovery and low relative standard deviation. Furthermore, the probe was further employed for fluorescence imaging in HeLa cells. In brief, the developed AgNPs-L-Cys-Rh6G2 sensing platform can be used for simultaneous Cu2+ determination and cell imaging.

ESIPT-based fluorescent enhanced probes prompted by methylated β-cyclodextrin for the detection of thiophenols

Thiophenol and its derivatives are compounds with high toxicity to organisms and environmental pollution, so it is necessary to detect the level of thiophenols in the environment and biological samples. The probes 1a-b were obtained by introducing the 2,4-dinitrophenyl ether group into diethylcoumarin–salicylaldehyde based compounds. And they can form host-guest compounds with methylated β-cyclodextrin (M-β-CD), the association constants of inclusion complexes are 49.2 M−1, 125 M−1 respectively. The fluorescence intensities of probes 1a-b at 600 nm (1a) and 670 nm (1b) increased significantly in thiophenols detection. Meanwhile, with the addition of M-β-CD, the hydrophobic cavity of M-β-CD significantly increased the fluorescence intensity of probes 1a-b, thus the detection limits of probes 1a-b to thiophenols were reduced from 410 nM, 365 nM to 62 nM, 33 nM respectively. Whereas, the good selectivity and short response time of probes 1a-b towards thiophenols was not affected in the presence of M-β-CD. Moreover, probes 1a-b were used for further water sample detection and HeLa cell imaging experiments due to their good response to thiophenols and the results suggested that probes 1a-b had the potential to detect the content of thiophenols in water samples and living cells.

The Rho1 GTPase controls anillo-septin assembly to facilitate contractile ring closure during cytokinesis

Animal cell cytokinesis requires activation of the GTPase RhoA (Rho1 in Drosophila), which assembles an F-actin- and myosin II-dependent contractile ring (CR) at the equatorial plasma membrane. CR closure is poorly understood, but involves the multidomain scaffold protein, Anillin. Anillin binds many CR components including F-actin and myosin II (collectively actomyosin), RhoA and the septins. Anillin recruits septins to the CR but the mechanism is unclear. Live imaging of Drosophila S2 cells and HeLa cells revealed that the Anillin N-terminus, which scaffolds actomyosin, cannot recruit septins to the CR. Rather, septin recruitment required the ability of the Anillin C-terminus to bind Rho1-GTP and the presence of the Anillin PH domain, in a sequential mechanism occurring at the plasma membrane, independently of F-actin. Anillin mutations that blocked septin recruitment, but not actomyosin scaffolding, slowed CR closure and disrupted cytokinesis. Thus, CR closure requires coordination of two Rho1-dependent networks: actomyosin and anillo-septin.

Following nanoparticle uptake by cells using high-throughput microscopy and the deep-learning based cell identification algorithm Cellpose

How many nanoparticles are taken up by human cells is a key question for many applications, both within medicine and safety. While many methods have been developed and applied to this question, microscopy-based methods present some unique advantages. However, the laborious nature of microscopy, in particular the consequent image analysis, remains a bottleneck. Automated image analysis has been pursued to remedy this situation, but offers its own challenges. Here we tested the recently developed deep-learning based cell identification algorithm Cellpose on fluorescence microscopy images of HeLa cells. We found that the algorithm performed very well, and hence developed a workflow that allowed us to acquire, and analyse, thousands of cells in a relatively modest amount of time, without sacrificing cell identification accuracy. We subsequently tested the workflow on images of cells exposed to fluorescently-labelled polystyrene nanoparticles. This dataset was then used to study the relationship between cell size and nanoparticle uptake, a subject where high-throughput microscopy is of particular utility.

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels using Live Imaging in HeLa Cells.

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Anthracenemethyl Glycosides as Supramolecular Synthons for Chiral Self-Assembly and as Probes in Cell Imaging.

Chiral self-assembly of molecules warrants optimal structural features of synthons that promote formation of such self-assembled structures. A polyaromatic moiety coupled with hydrophilic, chiral-rich carbohydrates leads to segmentation of the regions and the self-assembly to supramolecular structures. Thermodynamic stability is augmented further through chiral self-assembly of the molecules, and formation of the desired chiral supramolecular structures is achieved. In the present study, we develop anthracene glycosides as efficient synthons that, in aqueous solutions, undergo facile self-assembly and lead to chiral supramolecular structures. Anthracenemethyl O-glycosides, installed with mono- and disaccharides, are studied for their self-assembly properties. Emerging chiral structures follow the configuration of the attached sugar moiety. Monosaccharide d- and l-glycopyranoside-containing derivatives alternate between left- and right-handed chiral structures, respectively. Disaccharide-containing derivatives do not exhibit chirality, even when self-assembly occurred. Photochemical [4π + 4π] cycloaddition occurs in the self-assembled structure in aqueous solution. Cell viability assay using HeLa cells shows above 80% viable cells at a concentration of 50 μM. Bioimaging assays reveal a significant imaging of HeLa cells for anthracenemethyl d-glucopyranoside; bright imaging was observed at the perinuclear region of the cells, suggestive of an active transport of the molecules through the cell membrane. d-Galactopyranoside and l-glucopyranoside-containing derivatives show weak imaging potencies.

Rational N,P-Codoped pH-Activatable Red Carbon Dot for In Vitro and In Vivo Tumor Imaging.

Tumor detection and imaging via tumor microenvironmental indicators can have practical value. Here, a low-pH-responsive red carbon dot (CD) was prepared via a hydrothermal reaction for specific tumor imaging in vitro and in vivo. The probe responded to the acidic tumor microenvironment. The CDs are codoped by nitrogen and phosphorene and contain anilines on the surface. These anilines are efficient electron donors and modulate the pH response: Fluorescence is undetectable at common physical pH (>7.0), but red fluorescence (600-720 nm) increases with decreasing pH. The inactivation of fluorescence is due to three aspects: photoinduced electron transfer from anilines, deprotonation-induced energy states changing, and particle aggregation-induced quenching. It is believed that this pH-responsive character of CD is better than other reported CDs. Thus, in vitro images of HeLa cells show strong fluorescence that is 4-fold higher than normal cells. Subsequently, the CDs are used for in vivo imaging of tumors in mice. Tumors can be clearly observed within 1 h, and clearance of CDs will be finished within 24 h due to the small size of the CDs. The CDs offer excellent tumor-to-normal tissue (T/N) ratios and have great potential for biomedical research and disease diagnosis.

Programmable Aperture Light‐Field Microscopy

AbstractA computational three‐dimensional (3D) microscopy technique, termed programmable aperture light‐field microscopy (PALFM), for motion‐free, high‐resolution volumetric imaging of fluorescent or self‐luminous samples is proposed. The well‐known Fourier slice theorem is extended to incoherent tomographic imaging, which states that a detected image under an ideal aperture corresponds to a central slice in the 3D object spectrum, so the spectrum coverage can be accomplished based on the motion‐free aperture modulation. When further considering frequency extension and coverage, a hybrid aperture modulation scheme is designed consisting of non‐centrosymmetric circular and annular apertures for high‐efficiency, non‐ambiguous depth discrimination. A PALFM system with an easy‐to‐build programmable aperture module attached to an off‐the‐shelf inverted fluorescence microscope is constructed, where annular apertures can manipulate Bessel‐like beams for spectrum modulation. Experimental results on near‐diffraction‐limited imaging of a resolution target across a large depth range and high‐resolution, multi‐color 3D imaging of a mouse kidney section verify the validity and effectiveness of PALFM. High‐speed, long‐term time‐lapse volumetric imaging of HeLa cells in vitro further demonstrates that PALFM is a promising tomographic imaging tool for studying dynamic cellular processes and events without requiring complicated sample rotation or beam scanning.

Multifunctional carbon dots for glutathione detection and Golgi imaging

Glutathione (GSH) is present in almost every cell in the body and plays various integral roles in many biological processes. The Golgi apparatus is a eukaryotic organelle for the biosynthesis, intracellular distribution, and secretion of various macromolecules; however, the mechanism of GSH in the Golgi apparatus has not been fully elucidated. Here, specific and sensitive sulfur-nitrogen co-doped carbon dots (SNCDs) with orange-red fluorescence was synthesized for the detection of GSH in the Golgi apparatus. The SNCDs have a Stokes shift of 147 nm and excellent fluorescence stability, and they exhibited excellent selectivity and high sensitivity to GSH. The linear response of the SNCDs to GSH was in the range of 10–460 μM (LOD = 0.25 μΜ). More importantly, we used SNCDs with excellent optical properties and low cytotoxicity as probes, and successfully realized golgi imaging in HeLa cells and GSH detection at the same time.

Fluorescent Carbon Dots Derived From Soy Sauce for Picric Acid Detection and Cell Imaging.

Picric acid (PA) is a powerful nitro-aromatic explosive that harms the environment and human health. Developing non-toxic and low-cost sensors for the rapid detection of PA is essential. An environment-friendly fluorescent probe for PA detection is designed based on carbon dots (CDs) directly separated from edible soy sauce by silica gel column chromatography. Neither organic reagents nor heating process was needed to prepare CDs. The obtained CDs exhibit bright blue fluorescence, good water solubility, and photostability. The fluorescent probe for PA was developed according to the CD's fluorescence can be significantly quenched via the inner filter effect between CDs and PA. The linear range was 0.2-24µM with a limit of detection of 70nM. This proposed method was successfully employed to detect PA in the real water samples with satisfactory recoveries between 98.0-104.0%. Moreover, the CDs were suitable for fluorescence imaging of HeLa cells owing to their low toxicity and good biocompatibility.