Cellular Imaging Applications Research Articles

Rapid conversion from common precursors to carbon dots in large scale: Spectral controls, optical sensing, cellular imaging and LEDs application

The commercial production of carbon dots will be concerned with the simplicity and energy consumption. Herein, maleic acid and m-phenylenediamine form elegantly simple sources for carbon dots. The two precursors are dissolved in formamid (abbreviated as FA) or N,N-dimethylformamide (abbreviated as DMF) and the dehydration-condensation processes have been performed at 30 min or 120 min under room temperature. No external energy/irradiations, reactants or high temperature will be required and the afforded carbon dots (abbreviated as CDs) are collected by extraction, centrifugation, dialysis and column chromatography. It has been found for the first time the choice of organic solvents has been correlated with emission color. The blue-emitting CDs (abbreviated as B-CDs) and green-emitting CDs (abbreviated as G-CDs) are yielded in FA and DMF respectively. Facts support that the increase of –CONH- units causes red-shift in emissions. The optical sensing of tetracycline has been explored and the detection limit of blue-emitting CDs is as low as 25 nM. Live cells exposed to B-CDs and G-CDs (0.5 mg/ml) show no apparent changes via both Cell Counting Kit-8 and Annexin V/7-AAD analysis. The blue and green fluorescent signals can be easily tracked in cells. It has been demonstrated that the two carbon dots can be fabricated as multiple-color light-emitting diodes (abbreviated as LEDs).

Glutathione functionalized copper nanoclusters as a fluorescence platform for specific biosensing of cysteine and application in cellular imaging

Herein, we have investigated a highly specific and very sensitive quantification of L-Cysteine (Cys) in human urine samples using highly fluorescent glutathione decorated copper nanoclusters (GSH-CuNCs) as a fluorescence probe. GSH-CuNCs exhibits emission peak at 613 nm with an excitation wavelength of 380 nm. Emission intensity of GSH-CuNCs was reduced after the addition of Cys. We expected that the quenching of emission intensity is due to the metal-thiol interaction between the GSH-CuNCs surface and thiol group contains the Cys analyte. Cys stimulated Cu-thiol interactions were verified by FT-IR and natural bond orbital (NBO) theory. The good linearity was achieved in the range from 3.00 × 10−8 to 42.00 × 10−8 mol dm−3 and the lowest limit of detection (LOD) was calculated as 8.63 × 10−9 mol dm−3 (S/N = 3). The effect of various potential coexistence substances such as other amino acids, metal ions and some biological relevant chemicals to GSH-CuNCs has also been investigated. Furthermore, it was found that even 10-fold higher concentrations of other thiol containing amino acids did not interfering the detection of Cys. In addition, the proposed strategies were successfully applied for the quantification of Cys in human urine samples and live cell imaging.

Upconversion nanoparticles as intracellular pH messengers

Upconversion nanoparticles (UCNPs) should be particularly well suited for measurement inside cells because they can be imaged down to submicrometer dimensions in near real time using fluorescence microscopy, and they overcome problems, such as photobleaching, autofluorescence, and deep tissue penetration, that are commonly encountered in cellular imaging applications. In this study, the performance of an UCNP modified with a pH-sensitive dye (pHAb) is studied. The dye (emission wavelength 580 nm) was attached in a polyethylene imine (PEI) coating on the UCNP and excited via the 540-nm UCNP emission under 980-nm excitation. The UC resonance energy transfer efficiencies at different pHs ranged from 25 to 30% and a Förster distance of 2.56 nm was predicted from these results. Human neuroblastoma SH-SY5Y cells, equilibrated with nigericin H+/K+ ionophore to equalize the intra- and extracellular pH‚ showed uptake of the UCNP-pHAb conjugate particles and, taking the ratio of the intensity collected from the pHAb emission channel (565–630 nm) to that from the UCNP red emission channel (640–680 nm), produced a sigmoidal pH response curve with an apparent pKa for the UCNP-pHAb of ~ 5.1. The UCNP-pHAb were shown to colocalize with LysoBrite dye, a lysosome marker. Drug inhibitors such as chlorpromazine (CPZ) and nystatin (NYS) that interfere with clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively, were investigated to elucidate the mechanism of nanoparticle uptake into the cell. This preliminary study suggests that pH indicator–modified UCNPs such as UCNP-pHAb can report pH in SH-SY5Y cells and that the incorporation of the nanoparticles into the cell occurs via clathrin-mediated endocytosis.Graphical abstract

Luminescent anticancer Ru(II)-arenebipyridine and phenanthroline complexes: Synthesis, characterization, DFT studies, biological interactions and cellular imaging application

A series of ruthenium(II)-arene complexes of several bipyridine and phenanthroline derivatives have been synthesized by employing a green and efficient protocol involving water as a solvent under sonication. The structures of all the complexes were elucidated by the spectroscopic analysis. The geometry of the chlorido and PTA (1,3,5-Triaza-7-phosphaadamantane) complexes were further confirmed by DFT and single crystal XRD. The stability study in various solvents, specifically in the intracellular one was conducted. Most of the compounds exhibited significant potency and selectivity against MCF7 and HeLa cell lines with respect to normal HEK-293 cells compared to cisplatin and RAPTA-C (Ruthenium(II)-arene PTA complex). Complex [(η6-hexamethylbenzene)RuCl(κ2-N,N-4,4′-di-n-nonyl-2,2′-bpy)]Cl (3e) presented best anticancer profiles against all the human cancer cells. Interestingly, few complexes turned up to be highly fluorescent depicted by the quantum yield values. Remarkably, [(η6-p-cymene)RuCl(κ2-N,N-bpy)]Cl (3i) was identified as most significant anticancer theranostic agent interms of potency, selectivity and fluorescence quantum yield. This complex also represented itself as significant cellular imaging agent in live U-87 MG cells which was monitored by confocal microscope. Absorption and emission spectral studies of bypyridine and phenanthroline complex series revealed that the complexes interacted with calf thymus DNA through groove binding as well as intercalative mode. In addition to this, strong binding efficacy of these scaffolds wih BSA (Bovin Serum Albumin) also enhanced their transportation property inside the cells.

Excitation spectrum, nanoparticles, and their applications in cellular optical imaging

ABSTRACT The attractive photophysical properties of luminescent nanoparticles have prompted numerous studies on their synthesis and biological applications. Due to the complexity of components, sizes, morphologies, and strong light scattering of the nanoparticles the absorption spectrum alone is not enough for characterising and analysing the real absorption band of a nanoparticle product. Instead, the excitation spectrum is necessary for this purpose. To address the importance of the excitation spectrum in the utilisation of the photophysical properties of nanoparticles for cellular imaging, we compared the absorption and excitation spectra of a small organic molecule (rhodamine 6G) and commercially available nanoparticles (semiconductor quantum dots). Rhodamine 6G shows a good superimposition between its absorption and excitation spectra. However, the quantum dots do not, due to the presence of impurities and the polydispersity in nanoparticle size and shape. We demonstrated, by either spectrometer or cellular imaging, that excitation of the quantum dots at the maximum absorption peak will not result in the highest emission intensity. Instead, the best excitation wavelength for the nanoparticles can be revealed by the excitation spectrum. This has usually been overlooked by chemists and biologists.

Recent Advances in Luminescence Imaging of Biological Systems Using Lanthanide(III) Luminescent Complexes.

The use of luminescence in biological systems allows one to diagnose diseases and understand cellular processes. Molecular systems, particularly lanthanide(III) complexes, have emerged as an attractive system for application in cellular luminescence imaging due to their long emission lifetimes, high brightness, possibility of controlling the spectroscopic properties at the molecular level, and tailoring of the ligand structure that adds sensing and therapeutic capabilities. This review aims to provide a background in luminescence imaging and lanthanide spectroscopy and discuss selected examples from the recent literature on lanthanide(III) luminescent complexes in cellular luminescence imaging, published in the period 2016–2020. Finally, the challenges and future directions that are pointing for the development of compounds that are capable of executing multiple functions and the use of light in regions where tissues and cells have low absorption will be discussed.

Uniform Spheres of α-NaYF4:RE3+ (RE=Eu, Tb, Ce, Er, and Tm): Template-Free Synthesis, Multi-Color Photoluminescence, and Their Application in Cellular Imaging

Uniformly dispersed luminescent probes with a high brightness and high resolution are desired in bio imaging fields. Here, ~100 nm sized and well-dispersed spheres of RE3+ doped α-NaYF4 (rare earth (RE) = Eu, Tb, Ce, Er, and Tm) have been facile synthesized through hydrothermal processing in the absence of a template, followed by a proper annealing. The processing window of the cubic structured spheres is wide, because the hydrothermal products are independent of the processing conditions, including reaction time and temperature. The original morphology and crystal structure can be well retained with a calcination temperature up to 600 °C. However, calcination gives rise to a reduction of particle sizes, as a result of the crystallite growth and densification. Under ultraviolet radiation, α-NaYF4:RE3+ spheres show characteristic f-f emissions of RE3+ (RE = Eu, Tb, Ce, Er, and Tm), and exhibit orange red, green, ultraviolet (UV), blue green, and blue emissions, respectively. Mainly because of the near-infrared emission at ~697 nm (5D0→7F4 transitions of Eu3+), the successful imaging of macrophages was achieved by NH2-NaYF4:Eu3+ probes, indicating their excellent imaging capacity for cells in vitro.

Dual-Self-Restricted GFP Chromophore Analogues with Significantly Enhanced Emission.

The tremendous gap of fluorescence emission of synthetic green fluorescent protein (GFP) chromophore to the protein itself makes it impossible to use for applications in molecular and cellular imaging. Here, we developed an efficient methodology to enhance the photoluminescence response of synthetic GFP chromophore analogues by constructing dual-self-restricted chromophores. Single self-restricted chromophores were first generated with 2,5-dimethoxy substitution on the aromatic ring, which were further modified with phenyl or 2,5-dimethoxy phenyl to form dual-self-restricted chromophores. These two chromophores showed an obvious solvatofluorochromic color palette across blue to yellow with a maximum emission Stokes shift of 95 nm and dramatically enhanced fluorescence emission in various aprotic solvents, especially in hexane, where the QY reached around 0.6. Importantly, in acetonitrile and dimethyl sulfoxide, the fluorescence QYs of both chromophores were over 0.22, which were the highest reported so far in high polar organic solvents. Meanwhile, the fluorescence lifetimes also improved obviously with the maximum of around 4.5 ns. Theoretical calculations revealed a more favorable Mülliken atomic charge translocation over the double-bond bridge and illustrated much higher energy barriers for the Z/E photoisomerization together with larger bond orders compared with the GFP core chromophore, p-HBDI. Our work significantly improved the fluorescence emission of synthetic GFP chromophore analogues in polar solvents while reserved the multicolor emitting function, providing a solid molecular motif for engineering high-performance fluorescent probes.

Functionalisation of gold nanoparticles with ruthenium(II) polypyridyl complexes for their application in cellular imaging.

Two new dinuclear Ru(ii) polypyridyl complexes containing an alkyl disulphide functionalised bipyridine-based ligand and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesised and characterised. Their attachment onto the surface of gold nanoparticles (AuNPs, average diameter of ca. 2.5 nm) resulted in the formation of two new water-soluble Ru(ii)-AuNP conjugates that combine the advantageous properties of both moieties. Both free complexes show the attractive photophysical properties of Ru(ii) polypyridyl complexes and a rapid cellular uptake in HeLa cervical cancer cells. However, their corresponding gold conjugates displayed lower quantum yields than those determined for the free complexes presumed to be due to an energy transfer quenching of the Ru(ii) luminescence by interaction with the gold surface. Despite their diminished luminescence, confocal fluorescence microscopy studies revealed that the Ru(ii)-AuNP conjugates are successfully internalised into HeLa cells and better tolerated than their free complex counterparts after 24 h incubation, which makes them potential luminescent nanomaterials for bioimaging applications.

One-step synthesis of green emission carbon dots for selective and sensitive detection of nitrite ions and cellular imaging application

In recent years, carbon dot (CD)-based fluorescent sensors for selective ions or small biomolecules have drawn great attention. In this work, highly fluorescent CDs (QY = 21%) were prepared from 2,3-diamino pyridine as the precursor through a facile solvothermal process. The CDs showed high stability and a green emission in aqueous, and the optimal emission wavelength of CDs is 508 nm under the excitation wavelength of 438 nm. Interestingly, a CDs-based nanoprobe was developed for a selective and sensitive fluorescence quenching response to NO2− in water, and the quenching mechanism was investigated in the work. Besides, the recovery rates of NO2− in the range of 98–103.5% were found to be acceptable, indicating that the proposed CDs could be act as potential candidates for determination of nitrite ions in real samples. Meanwhile, the nanoprobe was also successfully employed in a visualization biosensing platform for determination of NO2− in living cells due to its eminent biocompatibility.

A novel "on-off-on" fluorescence assay for the discriminative detection of Cu(ii) and l-cysteine based on red-emissive Si-CDs and cellular imaging applications.

Copper ions (Cu2+) and l-cysteine (l-Cys) in the human body always play critical roles in various physiological processes, while abnormal Cu2+ and l-Cys concentrations in the biological system lead to many diseases. In this manuscript, Si-doped carbon dots (Si-CDs) with near-infrared fluorescence were designed for the detection of Cu2+ and l-Cys through the fluorescence "on-off-on" mode. The carbon dots exhibited not only excellent optical merits including good stability against photobleaching and high chemical stability, but also superior biological compatibility. Interestingly, due to the abundant amino groups distributed on the surface of Si-CDs, they could bind to copper ions to form cupric amine complexes and then quench the fluorescence of Si-CDs due to an electron transfer process. In addition, upon the addition of l-Cys, the FL intensity of Si-CDs could be effectively recovered accompanied with complexation between Cu2+ and the functional groups in l-Cys, due to which Cu2+ was removed from the surface of Si-CDs. Notably, as far as we know, these are the first red-emitting carbon dots for copper ion and l-Cys assays in water samples and human plasma samples. Furthermore, this strategy was successfully applied to the determination of Cu2+ and l-Cys in living systems, demonstrating great practicability in biomedical applications.

PH-Controlled fluorescence switching in water-dispersed polymer brushes grafted to modified boron nitride nanotubes for cellular imaging.

pH-Switchable, fluorescent, hybrid, water-dispersible nanomaterials based on boron nitride nanotubes (BNNTs) and grafted copolymer brushes (poly(acrylic acid-co-fluorescein acrylate) – P(AA-co-FA)) were successfully fabricated in a two-step process. The functionalization of BNNTs was confirmed by spectroscopic, gravimetric and imaging techniques. In contrast to “pure” BNNTs, P(AA-co-FA)-functionalized BNNTs demonstrate intense green fluorescence emission at 520 nm. Under neutral or alkaline pH values, P(AA-co-FA)-functionalized BNNTs are highly emissive in contrast to acidic pH conditions where the fluorescent intensity is absent or low. No increase in the absorption was observed when the suspension pH was increased from 7 to 10. The functionalized BNNTs are easily taken up by human normal prostate epithelium (PNT1A) and human prostate cancer cell lines (DU145) and are suitable for further evaluation in cellular imaging applications.

Preparation of Carbon Dots with High-Fluorescence Quantum Yield and Their Application in Dopamine Fluorescence Probe and Cellular Imaging

Carbon dots represent a kind of fluorescent nanomaterial and have broad application prospects in the field of biosensing and optoelectronics. Here, we explored carbon dots with a high-fluorescence quantum yield rate synthesized from L-cysteine and citric acid by the microwave-assisted method. The characteristics of the carbon dots were studied using a transmission electron microscope, an X-ray diffractometer, X-ray photoelectron spectra, a UV-Vis absorption spectrometer, a FTIR spectrometer, and a fluorescence spectrophotometer. The obtained carbon dots exhibited a high-fluorescence quantum yield (up to 85%), which is due to the combination of amidogens and sulfydryl with carbon dots, and hence bring the improved fluorescence property. We used carbon dots for in vitro imaging of CRL-5822 cells and human umbilical vein endothelial cells, which showed the low inhibitory rate (0.8%) of cells for 48 h with good biocompatibility demonstrated by the cell viability assay. The image of cells can be observed clearly under UV light. The Stern-Volmer equation was introduced to describe the quenching effect between the fluorescence intensity of carbon dots and the concentration of aqueous dopamine (DA).

Fluorescent Nanoparticles Synthesized by Carbon-Nitride-Stabilized Pickering Emulsion Polymerization for Targeted Cancer Cell Imaging.

Graphitic carbon nitride (CN) has been found to possess amphiphilic properties, enabling it to act as a solid emulsifier to stabilize the Pickering emulsion and subsequent polymerization. Herein, we used CN-stabilized Pickering emulsion polymerization to prepare molecularly imprinted polymer nanoparticles, which showed selective binding toward cancer cells. Taking advantage of its intrinsic amphiphilicity and emission properties, CN served as both the emulsifier in the polymerization process and emissive probe for cellular imaging application. Sialic acid (SA), as an important indicator of some certain cancers, was introduced into the polymer nanoparticles by forming reversible bonds with 4-vinylphenylboronic acid comonomer and removed by adjusting the pH value. The obtained SA-imprinted polymer nanoparticles exhibited good biocompatibility and excellent targeted imaging of DU 145 cells, which are prostate cancer cells having an SA-overexpressed surface. This work provides an easy, low-cost, low-toxicity, and large-scale strategy to prepare various functional polymer nanoparticles.

Translational AI and Deep Learning in Diagnostic Pathology.

There has been an exponential growth in the application of AI in health and in pathology. This is resulting in the innovation of deep learning technologies that are specifically aimed at cellular imaging and practical applications that could transform diagnostic pathology. This paper reviews the different approaches to deep learning in pathology, the public grand challenges that have driven this innovation and a range of emerging applications in pathology. The translation of AI into clinical practice will require applications to be embedded seamlessly within digital pathology workflows, driving an integrated approach to diagnostics and providing pathologists with new tools that accelerate workflow and improve diagnostic consistency and reduce errors. The clearance of digital pathology for primary diagnosis in the US by some manufacturers provides the platform on which to deliver practical AI. AI and computational pathology will continue to mature as researchers, clinicians, industry, regulatory organizations and patient advocacy groups work together to innovate and deliver new technologies to health care providers: technologies which are better, faster, cheaper, more precise, and safe.

Synthesis and characterization of Mono-disperse Carbon Quantum Dots from Fennel Seeds: Photoluminescence analysis using Machine Learning

Herein, we present the synthesis of mono-dispersed C-QDs via single-step thermal decomposition process using the fennel seeds (Foeniculum vulgare). As synthesized C-QDs have excellent colloidal, photo-stability, environmental stability (pH) and do not require any additional surface passivation step to improve the fluorescence. The C-QDs show excellent PL activity and excitation-independent emission. Synthesis of excitation-independent C-QDs, to the best of our knowledge, using natural carbon source via pyrolysis process has never been achieved before. The effect of reaction time and temperature on pyrolysis provides insight into the synthesis of C-QDs. We used Machine-learning techniques (ML) such as PCA, MCR-ALS, and NMF-ARD-SO in order to provide a plausible explanation for the origin of the PL mechanism of as-synthesized C-QDs. ML techniques are capable of handling and analyzing the large PL data-set, and institutively recommend the best excitation wavelength for PL analysis. Mono-disperse C-QDs are highly desirable and have a range of potential applications in bio-sensing, cellular imaging, LED, solar cell, supercapacitor, printing, and sensors.

Synthesis of PEGylated Salicylaldehyde Azine via Metal-free Click Chemistry for Cellular Imaging Applications

In this work, two kinds of PEGylated salicylaldehyde azine(SA) polymers were prepared and investigated for cellular imaging applications. First, a diazido derivative of SA was synthesized and subsequently PEGylated with polyethylene glycol monomethyl ether(mPEG) by metal-free azide-alkyne 1,3-dipolar cycloaddition reaction. The formed triazole group in mPEG-SA was then converted into cationic triazolium group by N-alkylation reaction. Both the synthesized polymers, mPEG-SA and N-alkylated mPEG-SA, showed good dispersibility in water, but differences in self-assembly of nanostructures. The mPEG-SA with triazole groups self-assembled into micelles, while the N-alkylated mPEG-SA with triazolium groups self-assembled into vesicles. Furthermore, mPEG-SA and N-alkylated mPEG-SA nanoparticles showed bright fluorescence due to the aggregation of AIE-active SA molecules in the nano-particles and could be successfully used as fluorescent nanoprobes for bioimaging applications in HeLa cancer cells. Finally, both the synthesized polymers showed minimal cytotoxicity and low hemolytic activity. Therefore, these PEGylated SA polymers proved to be promising bioimaging nanoprobes or traceable drug delivery vehicles.

Facile Incorporation of "Aggregation-Induced Emission"-Active Conjugated Polymer into Mesoporous Silica Hollow Nanospheres: Synthesis, Characterization, Photophysical Studies, and Application in Bioimaging.

Typical aggregation-induced emission (AIE) luminogens tetraphenylethylene (TPE) and triphenylamine have been used to construct an AIE-active conjugated polymer, namely, poly(N,N-diphenyl-4-(4-(1,2,2-triphenylvinyl)styryl)aniline) (PTPA), which consist of D-π-A architecture by Wittig polymerization. We fabricated mesoporous silica hollow nanospheres (MSHNs) which were encapsulated with the AIE-active polymer for applications in cellular imaging. It exhibits a positive solvatochromism effect by increasing solvent polarity, supported by theoretical calculation using density functional theory. The structure of the monomers and polymer was confirmed by Fourier transform infrared, nuclear magnetic resonance, and high-resolution mass spectrometry techniques. Considering the advantage of high brightness in the fluorescence of PTPA, it was encapsulated into MSHNs by a noncovalent approach, and the surface was functionalized with an anti-EpCAM (antiepithelial cell adhesion molecule) aptamer through conjugation with γ-glycidoxypropyltrimethoxysilane for targeting cancer cells specifically. The aptamer-functionalized Apt-MSHNs exhibited excellent biocompatibility with the liver cancer-Huh-7 cells used for this study and was efficiently internalized by these cells. Because EpCAM are overexpressed in multiple carcinomas, including liver cancer, these aptamer-conjugated AIE MSHNs are therefore good candidates for targeted cellular imaging applications.

Electrochemical Synthesis and Characterization of ZnO Nanocomposite Copolymer Containing Fluorescent Feature Dye

BODIPY (boron dipyrrolmethene) has attracted the attention of scientists although the synthesis of BODIPY from the fluorescent dye class is challenging, there are many applications such as fluorescence, ion / molecule and pH probes, sensors, redox active molecules, metal chelators, cellular imaging, photodynamic therapy, drug delivery materials and photovoltaic applications and energy storage. Firstly, it is synthesized with difficulty in synthesis but attractive fluorescence active monomer. Then, by electrochemical method, a conductive copolymer, which is an innovative material, was synthesized. The nanocomposite was synthesized with the help of a potentiostat using Zno nanoparticles to increase the strength and conductivity properties of the obtained semiconducting material and its existence was determined by advanced technological devices such as SEM-EDX, AFM-Raman. Characteristic peaks of inorganic and nano-scale compounds such as ZnO are observed in the fingerprint region and weak / medium in the conventional FT-IR device. This makes it difficult to qualitatively diagnose the compounds by infrared spectrometry. Raman spectroscopy, however, has been preferred for the determination of nanoparticles because it is relatively laborious, such as ICP-MS, and is not a complex spectral region such as FT-IR.

Synthesis of DNA-templated copper nanoparticles with enhanced fluorescence stability for cellular imaging.

Fluorescence of DNA-templated copper nanoparticles (DNA-CuNPs) is not stable over time which limits applications in cellular imaging. This is due to the presence of oxygen during synthesis which oxidizes Cu(0) to Cu(II) and also produces the free hydroxyl radical. The authors have prepared DNA-CuNPs with enhanced temporal stability of fluorescence by optimizing the reaction conditions so as to minimize the deleterious effects of oxygen. The operational lifetime of DNA-CuNPs was increased from 25min to 200min. Fluorescence spectra of DNA-CuNPs in optimized condition show an emission peak at 650nm when excited at 340nm. DNA-CuNPs synthesized in this manner were used for cell imaging. As a proof of concept, the nucleus of a human colon cell line (HCT116) was stained. The method does not involve any chemicals other that copper sulfate and ascorbate. This new approach for generating DNA-CuNPs improves imaging of biological processes and provides a basis for developing other types of DNA-templated nanomaterials. Graphical abstract Schematic presentation of the formation of fluorescent DNA-templated copper nanoparticles (DNA-CuNPs). A large amount of ascorbate provides long operational lifetime for cellular imaging under the condition exposed to oxygen. *Asc- and **DHA stand for ascorbate and dehydroascorbic acid.