New Tools for Imaging Neutrophils: Work Function Mapping and Element-Specific, Label-Free Imaging of Cellular Structures.

Photoemission electron microscopy and imaging X-ray photoelectron spectroscopy are today frequently used to obtain chemical and electronic states, chemical shifts, work function profiles within the fields of surface- and material sciences. Lately, because of recent technological advances, these tools have also been valuable within life sciences. In this study, we have investigated the power of photoemission electron microscopy and imaging X-ray photoelectron spectroscopy for visualization of human neutrophil granulocytes. These cells, commonly called neutrophils, are essential for our innate immune system. We hereby investigate the structure and morphology of neutrophils when adhered to gold and silicon surfaces. Energy-filtered imaging of single cells are acquired. The characteristic polymorphonuclear cellular nuclei divided into 2-5 lobes is visualized. Element-specific imaging is achieved based on O 1s, P 2p, C 1s, Si 2p, and N 1s core level spectra, delivering elemental distribution with submicrometer resolution, illustrating the strength of this type of cellular morphological studies.

Tailorable Membrane-Penetrating Nanoplatform for Highly Efficient Organelle-Specific Localization.

Given the breadth of currently arising opportunities and concerns associated with nanoparticles for biomedical imaging, various types of nanoparticles have been widely exploited, especially for cellular/subcellular level probing. However, most currently reported nanoparticles either have inefficient delivery into cells or lack specificity for intracellular destinations. The absence of well-defined nanoplatforms remains a critical challenge hindering practical nano-based bio-imaging. Herein, the authors elaborate on a tailorable membrane-penetrating nanoplatform as a carrier with encapsulated actives and decorated surfaces to tackle the above-mentioned issues. The tunable contents in such a versatile nanoplatform offer huge flexibility to reach the expected properties and functions. Aggregation-induced emission luminogen (AIEgen) is applied to achieve sought-after photophysical properties, specific targeting moieties are installed to give high affinity towards different desired organelles, and critical grafting of cell-penetrating cyclic disulfides (CPCDs) to promote cellular uptake efficiency without sacrificing the specificity. Hereafter, to validate its practicability, the tailored nano products are successfully applied to track the dynamic correlation between mitochondria and lysosomes during autophagy. The authors believe that the strategy and described materials can facilitate the development of functional nanomaterials for various life science applications.

Activatable MRI probes for the specific detection of bacteria.

Activatable fluorescent probes have been successfully used as molecular tools for biomedical research in the last decades. Fluorescent probes allow the detection of molecular events, providing an extraordinary platform for protein and cellular research. Nevertheless, most of the fluorescent probes reported are susceptible to interferences from endogenous fluorescence (background signal) and limited tissue penetration is expected. These drawbacks prevent the use of fluorescent tracers in the clinical setting. To overcome the limitation of fluorescent probes, we and others have developed activatable magnetic resonance probes. Herein, we report for the first time, an oligonucleotide-based probe with the capability to detect bacteria using magnetic resonance imaging (MRI). The activatable MRI probe consists of a specific oligonucleotide that targets micrococcal nuclease (MN), a nuclease derived from Staphylococcus aureus. The oligonucleotide is flanked by a superparamagnetic iron oxide nanoparticle (SPION) at one end, and by a dendron functionalized with several gadolinium complexes as enhancers, at the other end. Therefore, only upon recognition of the MRI probe by the specific bacteria is the probe activated and the MRI signal can be detected. This approach may be widely applied to detect bacterial infections or other human conditions with the potential to be translated into the clinic as an activatable contrast agent.

Light-Up Lipid Droplets Dynamic Behaviors Using a Red-Emitting Fluorogenic Probe.

Intracellular lipid metabolism occurs in lipid droplets (LDs), which is critical to the survival of cells. Imaging LDs is an intuitive way to understand their physiology in live cells. However, this is limited by the availability of specific probes that can properly visualize LDs in vivo. Here, an LDs-specific red-emitting probe is proposed to address this need, which is not merely with an ultrahigh signal-to-noise (S/N) ratio and a large Stokes shift (up to 214 nm) but also with superior resistance to photobleaching. The probe has been successfully applied to real-time tracking of intracellular LDs behaviors, including fusion, migration, and lipophagy processes. We deem that the proposed probe here offers a new possibility for deeper understanding of LDs-associated behaviors, elucidation of their roles and mechanisms in cellular metabolism, and determination of the transition between adaptive lipid storage and lipotoxicity as well.

Graphene Decorated with Iron Oxide Nanoparticles for Highly Sensitive Interaction with Volatile Organic Compounds.

Gases, such as nitrogen dioxide, formaldehyde and benzene, are toxic even at very low concentrations. However, so far there are no low-cost sensors available with sufficiently low detection limits and desired response times, which are able to detect them in the ranges relevant for air quality control. In this work, we address both, detection of small gas amounts and fast response times, using epitaxially grown graphene decorated with iron oxide nanoparticles. This hybrid surface is used as a sensing layer to detect formaldehyde and benzene at concentrations of relevance (low parts per billion). The performance enhancement was additionally validated using density functional theory calculations to see the effect of decoration on binding energies between the gas molecules and the sensor surface. Moreover, the time constants can be drastically reduced using a derivative sensor signal readout, allowing the sensor to work at detection limits and sampling rates desired for air quality monitoring applications.

Hybrid Rhodamine Fluorophores in the Visible/NIR Region for Biological Imaging.

Fluorophores and probes are invaluable for the visualization of the location and dynamics of gene expression, protein expression, and molecular interactions in complex living systems. Rhodamine dyes are often used as scaffolds in biological labeling and turn-on fluorescence imaging. To date, their absorption and emission spectra have been expanded to cover the entire near-infrared region (650-950 nm), which provides a more suitable optical window for monitoring biomolecular production, trafficking, and localization in real time. This review summarizes the development of rhodamine fluorophores since their discovery and provides strategies for modulating their absorption and emission spectra to generate specific bathochromic-shifts. We also explain how larger Stokes shifts and dual-emissions can be obtained from hybrid rhodamine dyes. These hybrid fluorophores can be classified into various categories based on structural features including the alkylation of amidogens, the substitution of the O atom of xanthene, and hybridization with other fluorophores.

A reversible and highly selective two-photon fluorescent "on-off-on" probe for biological Cu2+ detection.

A two-photon active probe for physiological copper (Cu2+) detection is expected to play an important role in monitoring biological metabolism. Herein, a novel Schiff base derivative (E)-2,2'-((4-((4-(diethylamino)-2-hydroxybenzylidene)amino)phenyl)azanediyl)bis(ethan-1-ol) (L) with remarkable two-photon activity was developed and synthetically investigated. L presents high selectivity and sensitivity for Cu2+ sensing in ethanol/HEPES buffer (v/v, 1 : 1), which is accompanied by the fluorescence switching "off" and subsequently "on" with the addition of EDTA. The mechanism for the detection of Cu2+ is further analyzed using 1H NMR titration, mass spectra and theoretical calculations. Furthermore, since the probe L possesses good photophysical properties, excellent biocompatibility and low cytotoxicity, it is successfully applied to track Cu2+ in the cellular endoplasmic reticulum by two-photon fluorescence imaging, showing its potential value for practical applications in biological systems.

LTCC Packaged Ring Oscillator Based Sensor for Evaluation of Cell Proliferation.

A complementary metal-oxide-semiconductor (CMOS) chip biosensor was developed for cell viability monitoring based on an array of capacitance sensors utilizing a ring oscillator. The chip was packaged in a low temperature co-fired ceramic (LTCC) module with a flip chip bonding technique. A microcontroller operates the chip, while the whole measurement system was controlled by PC. The developed biosensor was applied for measurement of the proliferation stage of adherent cells where the sensor response depends on the ratio between healthy, viable and multiplying cells, which adhere onto the chip surface, and necrotic or apoptotic cells, which detach from the chip surface. This change in cellular adhesion caused a change in the effective permittivity in the vicinity of the sensor element, which was sensed as a change in oscillation frequency of the ring oscillator. The sensor was tested with human lung epithelial cells (BEAS-2B) during cell addition, proliferation and migration, and finally detachment induced by trypsin protease treatment. The difference in sensor response with and without cells was measured as a frequency shift in the scale of 1.1 MHz from the base frequency of 57.2 MHz. Moreover, the number of cells in the sensor vicinity was directly proportional to the frequency shift.

Response strategies and biological applications of organic fluorescent thermometry: cell- and mitochondrion-level detection.

Temperature homeostasis is critical for cells to perform their physiological functions. Among the diverse methods for temperature detection, fluorescent temperature probes stand out as a proven and effective tool, especially for monitoring temperature in cells and suborganelles, with a specific emphasis on mitochondria. The utilization of these probes provides a new opportunity to enhance our understanding of the mechanisms and interconnections underlying various physiological activities related to temperature homeostasis. However, the complexity and variability of cells and suborganelles necessitate fluorescent temperature probes with high resolution and sensitivity. To meet the demanding requirements for intracellular/subcellular temperature detection, several strategies have been developed, offering a range of options to address this challenge. This review examines four fundamental temperature-response strategies employed by small molecule and polymer probes, including intramolecular rotation, polarity sensitivity, Förster resonance energy transfer, and structural changes. The primary emphasis was placed on elucidating molecular design and biological applications specific to each type of probe. Furthermore, this review provides an insightful discussion on factors that may affect fluorescent thermometry, providing valuable perspectives for future development in the field. Finally, the review concludes by presenting cutting-edge response strategies and research insights for mitigating biases in temperature sensing.

Highly water-dispersible surface-modified Gd(2)O(3) nanoparticles for potential dual-modal bioimaging.

Water-dispersible and luminescent gadolinium oxide (GO) nanoparticles (NPs) were designed and synthesized for potential dual-modal biological imaging. They were obtained by capping gadolinium oxide nanoparticles with a fluorescent glycol-based conjugated carboxylate (HL). The obtained nanoparticles (GO-L) show long-term colloidal stability and intense blue fluorescence. In addition, L can sensitize the luminescence of europium(III) through the so-called antenna effect. Thus, to extend the spectral ranges of emission, europium was introduced into L-modified gadolinium oxide nanoparticles. The obtained EuIII-doped particles (Eu:GO-L) can provide visible red emission, which is more intensive than that without L capping. The average diameter of the monodisperse modified oxide cores is about 4 nm. The average hydrodynamic diameter of the L-modified nanoparticles was estimated to be about 13 nm. The nanoparticles show effective longitudinal water proton relaxivity. The relaxivity values obtained for GO-L and Eu:GO-L were r1=6.4 and 6.3 s−1 mM−1 with r2/r1 ratios close to unity at 1.4 T. Longitudinal proton relaxivities of these nanoparticles are higher than those of positive contrast agents based on gadolinium complexes such as Gd-DOTA, which are commonly used for clinical magnetic resonance imaging. Moreover, these particles are suitable for cellular imaging and show good biocompatibility.

Preparation of amyloid-like fibrils containing magnetic iron oxide nanoparticles: effect of protein aggregation on proton relaxivity.

A method to prepare amyloid-like fibrils functionalized with magnetic nanoparticles has been developed. The amyloid-like fibrils are prepared in a two step procedure, where insulin and magnetic nanoparticles are mixed simply by grinding in the solid state, resulting in a water soluble hybrid material. When the hybrid material is heated in aqueous acid, the insulin/nanoparticle hybrid material self assembles to form amyloid-like fibrils incorporating the magnetic nanoparticles. This results in magnetically labeled amyloid-like fibrils which has been characterized by Transmission Electron Microscopy (TEM) and electron tomography. The influence of the aggregation process on proton relaxivity is investigated. The prepared materials have potential uses in a range of bio-imaging applications.

Effects of gadolinium oxide nanoparticles on the oxidative burst from human neutrophil granulocytes.

We have previously shown that gadolinium oxide (Gd(2)O(3)) nanoparticles are promising candidates to be used as contrast agents in magnetic resonance (MR) imaging applications. In this study, these nanoparticles were investigated in a cellular system, as possible probes for visualization and targeting intended for bioimaging applications. We evaluated the impact of the presence of Gd(2)O(3) nanoparticles on the production of reactive oxygen species (ROS) from human neutrophils, by means of luminol-dependent chemiluminescence. Three sets of Gd(2)O(3) nanoparticles were studied, i.e. as synthesized, dialyzed and both PEG-functionalized and dialyzed Gd(2)O(3) nanoparticles. In addition, neutrophil morphology was evaluated by fluorescent staining of the actin cytoskeleton and fluorescence microscopy. We show that surface modification of these nanoparticles with polyethylene glycol (PEG) is essential in order to increase their biocompatibility. We observed that the as synthesized nanoparticles markedly decreased the ROS production from neutrophils challenged with prey (opsonized yeast particles) compared to controls without nanoparticles. After functionalization and dialysis, more moderate inhibitory effects were observed at a corresponding concentration of gadolinium. At lower gadolinium concentration the response was similar to that of the control cells. We suggest that the diethylene glycol (DEG) present in the as synthesized nanoparticle preparation is responsible for the inhibitory effects on the neutrophil oxidative burst. Indeed, in the present study we also show that even a low concentration of DEG, 0.3%, severely inhibits neutrophil function. In summary, the low cellular response upon PEG-functionalized Gd(2)O(3) nanoparticle exposure indicates that these nanoparticles are promising candidates for MR-imaging purposes.

Cerium Oxide Nanoparticles with Entrapped Gadolinium for High T 1 Relaxivity and ROS-Scavenging Purposes.

Gadolinium chelates are employed worldwide today as clinical contrast agents for magnetic resonance imaging. Until now, the commonly used linear contrast agents based on the rare-earth element gadolinium have been considered safe and well-tolerated. Recently, concerns regarding this type of contrast agent have been reported, which is why there is an urgent need to develop the next generation of stable contrast agents with enhanced spin-lattice relaxation, as measured by improved T 1 relaxivity at lower doses. Here, we show that by the integration of gadolinium ions in cerium oxide nanoparticles, a stable crystalline 5 nm sized nanoparticulate system with a homogeneous gadolinium ion distribution is obtained. These cerium oxide nanoparticles with entrapped gadolinium deliver strong T 1 relaxivity per gadolinium ion (T 1 relaxivity, r 1 = 12.0 mM-1 s-1) with the potential to act as scavengers of reactive oxygen species (ROS). The presence of Ce3+ sites and oxygen vacancies at the surface plays a critical role in providing the antioxidant properties. The characterization of radial distribution of Ce3+ and Ce4+ oxidation states indicated a higher concentration of Ce3+ at the nanoparticle surfaces. Additionally, we investigated the ROS-scavenging capabilities of pure gadolinium-containing cerium oxide nanoparticles by bioluminescent imaging in vivo, where inhibitory effects on ROS activity are shown.

ON THE POSSIBILITY TO RESOLVE GADOLINIUM- AND CERIUM-BASED CONTRAST AGENTS FROM THEIR CT NUMBERS IN DUAL-ENERGY COMPUTED TOMOGRAPHY.

Cerium oxide nanoparticles with integrated gadolinium have been proved to be useful as contrast agents in magnetic resonance imaging. Of question is their performance in dual-energy computed tomography. The aims of this work are to determine (1) the relation between the computed tomography number and the concentration of the I, Gd or Ce contrast agent and (2) under what conditions it is possible to resolve the type of contrast agent. Hounsfield values of iodoacetic acid, gadolinium acetate and cerium acetate dissolved in water at molar concentrations of 10, 50 and 100 mM were measured in a water phantom using the Siemens SOMATOM Definition Force scanner; gadolinium- and cerium acetate were used as substitutes for the gadolinium-integrated cerium oxide nanoparticles. The relation between the molar concentration of the I, Gd or Ce contrast agent and the Hounsfield value was linear. Concentrations had to be sufficiently high to resolve the contrast agents.

Protein interaction, monocyte toxicity and immunogenic properties of cerium oxide crystals with 5% or 14% gadolinium, cobalt oxide and iron oxide nanoparticles - an interdisciplinary approach.

Metal oxide nanoparticles are widely used in both consumer products and medical applications, but the knowledge regarding exposure-related health effects is limited. However, it is challenging to investigate nanoparticle interaction processes with biological systems. The overall aim of this project was to improve the possibility to predict exposure-related health effects of metal oxide nanoparticles through interdisciplinary collaboration by combining workflows from the pharmaceutical industry, nanomaterial sciences, and occupational medicine. Specific aims were to investigate nanoparticle-protein interactions and possible adverse immune reactions. Four different metal oxide nanoparticles; CeOx nanocrystals with 5% or 14% Gd, Co3O4, and Fe2O3, were characterized by dynamic light scattering and high-resolution transmission electron microscopy. Nanoparticle-binding proteins were identified and screened for HLA-binding peptides in silico. Monocyte interaction with nanoparticle-protein complexes was assessed in vitro. Herein, for the first time, immunogenic properties of nanoparticle-binding proteins have been characterized. The present study indicates that especially Co3O4-protein complexes can induce both 'danger signals', verified by the production of inflammatory cytokines and simultaneously bind autologous proteins, which can be presented as immunogenic epitopes by MHC class II. The clinical relevance of these findings should be further evaluated to investigate the role of metal oxide nanoparticles in the development of autoimmune disease. The general workflow identified experimental difficulties, such as nanoparticle aggregate formation and a lack of protein-free buffers suitable for particle characterization, protein analyses, as well as for cell studies. This confirms the importance of future interdisciplinary collaborations.

Selective colorimetric detection of copper (II) by a protein-based nanoprobe.

In this work, we report a novel protein-based nanoprobe (PNP) that can be employed for quantitative analysis of Cu2+ in pure water medium and real samples. Structurally, the proposed nanoprobe comprises a biofriendly protein (hen egg-white lysozyme (HEWL)) and a Cu2+-specific chromogenic agent, where HEWL acts as a nanocarrier encapsulating a structurally tailored rhodamine B derivate. The resulting PNP exhibits a hydrodynamic diameter of ~ 106 nm and efficiently disperses in water, enabling the detection of Cu2+ in pure aqueous systems without the aid of any organic co-solvents. The high sensitivity and selectivity of PNP allow the colorimetric detection of Cu2+ in the presence of other metal interferents with a low detection limit of 160 nM. The satisfying recovery of trace level Cu2+ in environmental samples demonstrate the great potential of employing PNP for the determination of Cu2+ in actual applications. Most importantly, the simple co-grinding method employing proteins and chromogenic agents provides a novel strategy to generate sensing systems that are useful detection of pollutants in aqueous samples.

Impact of Amine Additives on Perovskite Precursor Aging: A Case Study of Light-Emitting Diodes.

Amines are widely employed as additives for improving the performance of metal halide perovskite optoelectronic devices. However, amines are well-known for their high chemical reactivity, the impact of which has yet to receive enough attention from the perovskite light-emitting diode community. Here, by investigating an unusual positive aging effect of CH3NH3I/CsI/PbI2 precursor solutions as an example, we reveal that amines gradually undergo N-formylation in perovskite precursors over time. This reaction is initialized by hydrolysis of dimethylformamide in the acidic chemical environment. Further investigations suggest that the reaction products collectively impact perovskite crystallization and eventually lead to significantly enhanced external quantum efficiency values, increasing from ∼2% for fresh solutions to ≳12% for aged ones. While this case study provides a positive aging effect, a negative aging effect is possible in other perovksite systems. Our findings pave the way for more reliable and reproducible device fabrication and call for further attention to underlying chemical reactions within the perovskite inks once amine additives are included.

Nanoscale Ln(III)-carboxylate coordination polymers (Ln = Gd, Eu, Yb): temperature-controlled guest encapsulation and light harvesting.

We report the self-assembly of stable nanoscale coordination polymers (NCPs), which exhibit temperature-controlled guest encapsulation and release, as well as an efficient light-harvesting property. NCPs are obtained by coordination-directed organization of pi-conjugated dicarboxylate (L1) and lanthanide metal ions Gd(III), Eu(III), and Yb(III) in a DMF system. Guest molecules trans-4-styryl-1-methylpyridiniumiodide (D1) and methylene blue (D2) can be encapsulated into NCPs, and the loading amounts can be controlled by changing reaction temperatures. Small angle X-ray diffraction (SAXRD) results reveal that the self-assembled discus-like NCPs exhibit long-range ordered structures, which remain unchanged after guest encapsulations. Experimental results reveal that the negatively charged local environment around the metal connector is the driving force for the encapsulation of cationic guests. The D1 molecules encapsulated in NCPs at 140 degrees C can be released gradually at room temperature in DMF. Guest-loaded NCPs exhibit efficient light harvesting with energy transfer from the framework to the guest D1 molecule, which is studied by photoluminescence and fluorescence lifetime decays. This coordination-directed encapsulation approach is general and should be extended to the fabrication of a wide range of multifunctional nanomaterials.

Synthesis and characterization of PEGylated Gd2O3 nanoparticles for MRI contrast enhancement.

Recently, much attention has been given to the development of biofunctionalized nanoparticles with magnetic properties for novel biomedical imaging. Guided, smart, targeting nanoparticulate magnetic resonance imaging (MRI) contrast agents inducing high MRI signal will be valuable tools for future tissue specific imaging and investigation of molecular and cellular events. In this study, we report a new design of functionalized ultrasmall rare earth based nanoparticles to be used as a positive contrast agent in MRI. The relaxivity is compared to commercially available Gd based chelates. The synthesis, PEGylation, and dialysis of small (3-5 nm) gadolinium oxide (DEG-Gd(2)O(3)) nanoparticles are presented. The chemical and physical properties of the nanomaterial were investigated with Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, and dynamic light scattering. Neutrophil activation after exposure to this nanomaterial was studied by means of fluorescence microscopy. The proton relaxation times as a function of dialysis time and functionalization were measured at 1.5 T. A capping procedure introducing stabilizing properties was designed and verified, and the dialysis effects were evaluated. A higher proton relaxivity was obtained for as-synthesized diethylene glycol (DEG)-Gd(2)O(3) nanoparticles compared to commercial Gd-DTPA. A slight decrease of the relaxivity for as-synthesized DEG-Gd(2)O(3) nanoparticles as a function of dialysis time was observed. The results for functionalized nanoparticles showed a considerable relaxivity increase for particles dialyzed extensively with r(1) and r(2) values approximately 4 times the corresponding values for Gd-DTPA. The microscopy study showed that PEGylated nanoparticles do not activate neutrophils in contrast to uncapped Gd(2)O(3). Finally, the nanoparticles are equipped with Rhodamine to show that our PEGylated nanoparticles are available for further coupling chemistry, and thus prepared for targeting purposes. The long term goal is to design a powerful, directed contrast agent for MRI examinations with specific targeting possibilities and with properties inducing local contrast, that is, an extremely high MR signal at the cellular and molecular level.

Rapid detection of mercury (II) ions and water content by a new rhodamine B-based fluorescent chemosensor.

A rhodamine B-based sensor (RS) was designed and synthesized by a combination of the spirolacton rhodamine B (fluorophore) and multidentate chelates (ionophore) with high affinity towards Hg2+. In the presence of Hg2+, the resulting red-orange fluorescence (under UV light) and naked eye red color of RS are supposed to be used for quantitative and qualitative measurement of Hg2+. Further fluorescent titration and analysis demonstrate that RS can selectively detect Hg2+ within 1 s with a low limit of detection (LOD) of 16 nM in acetonitrile media, meanwhile, the association constant (Ka) was calculated to be 0.32 × 105 M-1. More importantly, the resultant complex (RSHg) of RS and Hg2+ has also been successfully applied to detect limited water content in acetonitrile solution.