Fluorescent Tracking Research Articles

Spectroscopic fluorescent tracking of a single molecule in a live cell with a dual-objective fluorescent reflection microscope

Single-molecule spectral imaging is a powerful technique for simultaneous study of biological samples labeled by multiple fluorophores. We have developed a robust prism-based single-molecule spectral tracking technique based on a dual-objective fluorescence–reflection system with tunable spectral dispersion (DOFR-SD). The system can capture the spatial position of a single molecule in real time, and rather than splitting photons into two channels its intrinsic spectral characteristics can also be captured without compromising the precision of the (x, y) position. Thus, the DOFR-SD technique is suitable for tracking the spectral variation of single particles in living cells and for multicolor coding.

Model-free 3D localization with precision estimates for brightfield-imaged particles.

Volumetric imaging and 3D particle tracking are becoming increasingly common and have a variety of microscopy applications including in situ fluorescent imaging, in-vitro single-molecule characterization, and analysis of colloidal systems. While recent interest has generated discussion of optimal schemes for localizing diffraction-limited fluorescent puncta, there have been relatively few published routines for tracking particles imaged with bright-field illumination. To address this, we outline a simple, look-up-table based 3D tracking strategy, which can be adapted to most commercially available wide-field microscopes, and present two image processing algorithms that together yield high-precision localization and return estimates of statistical accuracy. Under bright-field illumination, a particle's depth can be determined based on the size and shape of its diffractive pattern due to Mie scattering. Contrary to typical "super-resolution" fluorescence tracking routines, which typically fit a diffraction-limited spot to a model point-spread-function, the lateral (XY) tracking routine relies on symmetry to locate a particle without prior knowledge of the form of the particle. At low noise levels (signal:noise > 1000), the symmetry routine estimates particle positions with accuracy better than 0.01 pixel. Depth localization is accomplished by matching images of particles to those in a pre-recorded look-up-table. The routine presented here optimally interpolates between LUT entries with better than 0.05 step accuracy. Both routines are tolerant of high levels of image noise, yielding sub-pixel/step accuracy with signal-to-noise ratios as small as 1, and, by design, return confidence intervals indicating the expected accuracy of each calculated position. The included implementations operate extremely quickly and are amenable to real-time analysis at frame rates exceeding several hundred frames per second.

Antiproliferative Ability and Fluorescence Tracking of α-Linolenic Acid-Loaded Microemulsion as Label-Free Delivery Carriers in MDA-MB-231 Cells.

In this work, the effects of α-linolenic acid (ALA) loaded in oil-in-water (O/W) and water-in-oil-in-water (W/O/W) microemulsions on cell viability, lactic dehydrogenase (LDH) viability, and reactive oxygen species (ROS) levels were examined using Cell Counting Kit-8 (CCK-8), an LDH assay kit, and a fluorescence microscope, respectively. The CCK-8 assay demonstrated that ALA inhibited MDA-MB-231 human breast cancer cell proliferation in a dose-dependent manner. Further, the results of LDH activity and ROS levels revealed that ALA-induced cancer cell damage was closely related to oxidative stress. Under the irradiation of ultraviolet light, the microemulsion without any added fluorescent dye would emit bright blue fluorescence, and the fluorescent images of the cells treated with ALA-loaded O/W and W/O/W microemulsions at different incubation times were taken, which exhibited long-term photostability and biocompatibility. In addition, the fluorescence mechanism of the microemulsion was explained by immobilizing surfactant molecules with aggregation-induced emission (AIE) properties at the water-oil interface through the microemulsion with a self-assembled structure. These findings showed the potential application of O/W and W/O/W microemulsions as the label-free delivery carriers in long-term imaging of living cells and real-time release monitoring of nutrients.

Movement patterns of an arboreal gecko in fragmented agricultural landscapes reveal matrix avoidance

AbstractAnimal movement through agricultural landscapes is critical for population persistence of species within fragmented native vegetation patches. However, perceived habitat quality and the structural changes between differing land uses within such landscapes can reduce an animal's willingness to move. Understanding when animal movement behaviour varies in response to differing habitat types is necessary for identifying barriers to movement between habitat patches. We quantified the homing success and fine‐scale movement behaviour of a patch‐dependent gecko, Gehyra versicolor, in remnant patches, three different matrix types (crop, pasture and linear plantings), and at varying distances from the edge using fluorescent powder tracking, radio‐telemetry and experimental displacement. We found displaced geckos in pasture environments orientated more strongly and moved farther into farmland after being released and, away from their home ranges in remnant patches. In contrast, we found strong homing ability of displaced animals in plantings and crop matrix types, with animals moving towards remnant patches and away from farmland. Importantly, from the 48 individuals radio‐tracked, none moved into farmland, including pastures, despite 16 individuals approaching edge habitat. Because radio‐tracked geckos did not move into pastures, or any other matrix type, movement further into pasture by displaced animals likely represents limited orientation capacity in pasture rather than preference for pasture. We conclude geckos behaviourally avoided the farmland, irrespective of the presence of complex habitat (e.g. linear plantings). Our findings suggest that, despite efforts to improve farmland quality by planting, farmland is not generally preferred compared to remnant native vegetation. Understanding habitat‐specific movement behaviour is crucial to effectively identifying barriers to animal movement and will improve our efforts to conserve regional populations of patch‐dependent species.

Simultaneous photobleaching molecular tagging velocimetry for density-stratified flows of suspensions

To gain insight into the roll wave instability in particulate flow systems, the spatiotemporal velocities of particle and liquid phases in a clear-fluid layer are simultaneously measured by means of a photobleaching molecular tagging velocimetry (PB-MTV). A grid pattern tag is generated by intermittent irradiation of two intense laser beams, which facilitates measurement of a wall vicinity flow induced by the Boycott effect. To cope with a plurality of tagline intersections, which largely deform in shear flows, a novel spatial correlation technique is developed with a template-matching method. A priori study using artificial images of tags in a temporally oscillating flow is performed to examine the effects of the signal to noise (S/N) ratio and the degree of unsteadiness on the accuracy of the velocimetry. An experimental validation of the present PB-MTV is also conducted on the statistical values of the streamwise velocity through comparison with a laser-induced fluorescence and particle tracking velocimetry (LIF-PTV). A series of experiments are carried out using a suspension, which consists of tap water and spherical hollow light particles, varying the particle diameter and the particle volume fraction. The liquid velocity fluctuation tends to increase with decrease in the inter-particle distance compared to the thickness of the clear-fluid layer. We conclude that the roll wave instability occurs only under the fluid-like condition, where the suspension behaves as a continuum, and is affected by the sharpness of flow stratification between the clear-fluid and particle-rich phases.

Optical Tracking of Nanometer-Scale Cellular Membrane Deformation Associated with Single Vesicle Release.

Exocytosis involves interactions between secretory vesicles and the plasma membrane. Studying the membrane response is thus critical to understand this important cellular process and to differentiate different mediator release patterns. Here we introduce a label-free optical imaging method to detect the vesicle-membrane-interaction-induced membrane deformation associated with single exocytosis in mast cells. We show that the plasma membrane expands by a few tens of nanometers accompanying each vesicle-release event, but the dynamics of the membrane deformation varies from cell to cell, which reflect different exocytosis processes. Combining the temporal and spatial information allows us to resolve complex vesicle-release processes, such as two vesicle-release events that occur closely in time and location. Simultaneous following a vesicle release with fluorescence and membrane deformation tracking further allows us to determine the propagation speed of the vesicle-release-induced membrane deformation along the cell surface, which has an average value of 5.2 ± 1.8 μm/s.

Porphyrin as Diagnostic and Therapeutic Agent.

The synthesis and application of porphyrins has seen a huge shift towards research in porphyrin bio-molecular based systems in the past decade. The preferential localization of porphyrins in tumors, as well as their ability to generate reactive singlet oxygen and low dark toxicities has resulted in their use in therapeutic applications such as photodynamic therapy. However, their inherent lack of bio-distribution due to water insolubility has shifted research into porphyrin-nanomaterial conjugated systems to address this challenge. This has broadened their bio-applications, viz. bio-sensors, fluorescence tracking, in vivo magnetic resonance imaging (MRI), and positron emission tomography (PET)/CT imaging to photo-immuno-therapy just to highlight a few. This paper reviews the unique theranostic role of porphyrins in disease diagnosis and therapy. The review highlights porphyrin conjugated systems and their applications. The review ends by bringing current challenges and future perspectives of porphyrin based conjugated systems and their respective applications into light.

3D particle tracking using a dual-objective fluorescent reflection system with spherical aberration

It is often difficult to implement complex microscopy systems without spherical aberration. Herein, we developed a novel, robust, three-dimensional (3D), bifocal plane, single-particle tracking technique, based on a dual-objective fluorescent reflection system with spherical aberration (DOFR–SA). It can simultaneously image a pair of focused and defocused planes containing fluorescent particles with a single camera instead of splitting photons into two channels. Based on the 3D DOFR–SA, the desired position accuracy along the z-axis was achieved without compromising the precisions of the (x, y ) positions, even with limited number of photons from a single molecule. Accordingly, this method was applied to fluorescent particle tracking in biofluids and living cells with high-spatial and temporal precisions.

Doped Graphene Quantum Dots for Intracellular Multicolor Imaging and Cancer Detection.

Despite significant advances of nanomedicine, the issues of biocompatibility, accumulation-derived toxicity, and the lack of sensing and in vivo imaging capabilities hamper the translation of most nanocarriers into clinic. To address this, we utilize nitrogen, boron/nitrogen, and sulfur-doped graphene quantum dots (GQDs) as fully biocompatible multifunctional platforms allowing for multicolor visible/near-IR imaging and cancer-sensing. These GQDs are scalably produced in one-step synthesis from a single biocompatible glucosamine precursor, are water-soluble, show no cytotoxicity at high concentrations of 1 mg/mL, and demonstrate substantial degradation at 36 h in biological environments as verified by TEM imaging. Because of their small sizes, GQDs exhibit efficient internalization maximized at 12 h followed by further degradation/excretion. Their high-yield intrinsic fluorescence in blue/green and near-infrared allows for multicolor in vitro imaging on its own or in combination with other fluorophores, and offers the capabilities for in vivo near-IR fluorescence tracking. Additionally, nitrogen- and sulfur-doped GQDs exhibit pH-dependent fluorescence response that is successfully utilized as a sensing mechanism for acidic extracellular environments of cancer cells. It allows for the deterministic, ratiometric spectral discrimination between cancerous (HeLa and MCF-7 cell) versus healthy (HEK-293 cell) environments with substantial intensity ratios of 1.6 to 8. These results suggest fully biocompatible GQDs developed in this work as multifunctional candidates for in vitro delivery of active agents, multicolor visible/near-IR fluorescence imaging, and pH-sensing of cancerous environments.

Characterization of Single-Protein Dynamics in Polymer-Cushioned Lipid Bilayers Derived from Cell Plasma Membranes.

Native cell-membrane-derived supported lipid bilayers (SLBs) are an emerging platform with broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is the proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact between the bilayer membrane and the supporting substrate, and thus, the mobility of the transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins with distinct structures, i.e., a GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescent protein (GFP), and their dynamics were measured by fluorescent single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length on protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1000 to 5000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were augmented with the polymer length, whereas the diffusion coefficient of the GPI-anchored protein remained almost identical for different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm2/s approximately) for the cushioned membrane with the longest polymer length (molecular weight of 5000), indicating that at the microscopic length scale, the SLBs were fully suspended from the substrate by the polymer cushion. Transient confinements were observed for all three proteins, and increasing the polymer length reduced the tendency of transient confinement. The measured dynamics of membrane proteins were found to be nearly unchanged after the depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer-cushioned plasma membrane bilayers that are potentially useful for the future developments of membrane-based biosensors and analytical assays.

Electronic Energy Transfer in a Single Donor–Acceptor Pair with Triplet–Triplet Absorption

Forster resonance energy transfer (FRET) is examined in a single donor (D)–acceptor (A) pair whose molecules have both singlet and triplet states. Two cases are considered: (i) the excitation of the D–А pair occurs with the participation of a singlet band of the donor, and (ii) the excitation also involves the participation of an additional triplet–triplet absorption. The triplet state of the donor is shown to have no effect on the expression for the FRET efficiency, but it generates off intervals in the fluorescence track of both the donor and the acceptor. The triplet state of the acceptor strongly affects the expression for the FRET efficiency, making the complete energy transfer impossible and generating off intervals in the acceptor fluorescence track. It is shown that, if the light can excite not only the singlet transition in the donor, but also the triplet–triplet absorption band, then a weak luminescence appears in off intervals, which is orders of magnitude weaker than that in on intervals.

Synthesis of Thermosensitive Conjugated Triblock Copolymers by Sequential Click Couplings for Drug Delivery and Cell Imaging.

The elegant integration of an excellent light-emitting segment and a biorelevant signal-responsive moiety could generate advanced polymeric delivery systems with simultaneously favorable diagnostic and therapeutic functions with respect to cancer theranostics. Although polymeric delivery systems based on fluorescent polyfluorene (PF) or thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) have been extensively developed, the preparation of a ternary polymer formulation composed of a PF block, a PNIPAAm sequence, and a hydrophilic moiety remains rarely explored likely because of the difficulty in integrating different synthesis strategies for polymer synthesis. To this end, herein we reported the design and controlled synthesis of a PF- and PNIPAAm-based amphiphilic triblock copolymer, PF11-b-PNIPAAm120-b-poly(oligo(ethylene glycol) monomethyl ether methacrylate)17 (PF11-b-PNIPAAm120-b-POEGMA17), with a well-defined structure by a strategy of sequential click couplings between Suzuki-coupling-generated PF and atom-transfer radical polymerization (ATRP)-produced PNIPAAm and POEGMA. The as-prepared triblock copolymers can self-assemble into micelles with a core-shell-corona (CSC) structure that is composed of an inner hydrophobic core of the PF moiety for fluorescent tracking and drug encapsulation, a thermosensitive middle shell of PNIPAAm block for thermomodulated drug loading and release, and a hydrophilic outer corona of the POEGMA segment for micelle stabilization. Interestingly, the doxorubicin (DOX)-loaded micelles prepared at 25 °C had a greater drug loading capacity than the analogues fabricated at 37 °C due to the better stability of the former formulation, leading to its higher in vitro cytotoxicity in HeLa cells. Together with the integration of a localized hyperthermia-triggered drug release profile and efficiently intracellular trafficking of the nanocarriers by monitoring the fluorescence of the PF moiety, this formulation demonstrates a great potential for cancer theranostics.

Synergistic Effects of Novel Superparamagnetic/Upconversion HA Material and Ti/Magnet Implant on Biological Performance and Long-Term In Vivo Tracking.

To solve the clinical challenges presented by the long-term tracking of implanted hydroxyapatite (HA) bone repair material and to investigate the synergistic effects of superparamagnetic HA and a static magnetic field (SMF) on the promotion of osteogenesis, herein a new type of superparamagnetic/upconversion-generating HA material (HYH-Fe) is developed via a two-step doping method, as well as a specially-designed titanium implant with a built-in magnet to provide a local static magnetic field in vivo. The results show that the prepared HYH-Fe material maintains the crystal structure of HA and exhibits good cytocompatibility. The combined use of the superparamagnetic HYH-Fe material and SMF can effectively and synergistically promote osteogenesis/osteointegration surrounding the Ti implants. In addition, the HYH-Fe material exhibits distinct advantages in terms of both long-term fluorescence tracking and microcomputed tomography (micro-CT) tracking. The new material and tracking strategy in this study provide scientific feasibility and will have important clinical value for long-term tracking and evaluation of implanted materials and the bone repair effect.

Analysis of SPICE microbeam size using fluorescent nuclear track detector (FNTD)

Single Particle Irradiation system to Cell (SPICE) facility at the National Institute of Radiological Sciences (NIRS) provides a magnetically focused 3.4 MeV proton microbeam. Throughout the radiobiological studies using SPICE over the years, we have realized that biological effect can be affected by the highly localized dose distribution of the microbeam and results may differ from that of broad beam irradiation. Previously, we have reported that the beam size of the SPICE microbeam was approximately 2 µm in diameter; the proton traversal was identified as etch pits produced on plastic nuclear track detectors, CR-39. However, this widely used method cannot distinguish individual proton tracks of highly focused microbeam due to the overlapping of etch pits, which was major limitation for beam size analysis as well as estimation of localized dose distribution in the cells. To overcome this limitation, we used fluorescent nuclear track detectors (FNTD; Al2O3: C, Mg) to investigate the beam size and its proton-number dependence (7–1000 protons per position). Detail analysis of Z(optical axis)-stacks of fluorescent images of the FNTD with a confocal laser scanning microscope clearly demonstrated that FNTDs has better spatial resolution and detection sensitivity compared to the conventional CR-39 detectors. The overall beam size was evaluated as elliptical shape or rectangular shape and determined to be in the range of 2.0 × 1.5–3.1 × 2.4 μm, depending on the number of protons per position. This result suggests that out-focused fraction of the incident protons contributes the broadening of the beam size. Throughout the present study, the FNTD irradiation and related image processing technology have demonstrated the feasibility of high-resolution measurement of the microbeam size over a wide range of the microbeam intensity.

Conjugated carbon quantum dots: Potent nano-antibiotic for intracellular pathogens

A common theme in the persistence of microbial infections involves intracellular survival of microbial pathogens within host cells where they stay sheltered from attack by antimicrobial agents. In order to improve antimicrobial access inside host cells, we developed nanoparticles intracellular delivery of antibiotics. Using an intracellular infection model with the periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), we demonstrated significantly enhanced intracellular microbicidal activity with the standard antibiotic metronidazole (MET) through its conjugation onto 1–5 nm biocompatible nano-carrier, carbon quantum dot, which was derived from chlorophyll (cCQD). The conjugated cCQD-MET were rapidly internalized into the cultured cells, reaching almost 90% uptake within 3 h of the challenge. Our results consistently showed enhanced antimicrobial activity of the conjugate compared to MET alone. Even at concentrations as low as 0.26 µM, the conjugate showed 72% enhancement compared to the drug alone, resulting in significantly increased inhibition of intracellular P. gingivalis at lower antibiotic dosages. We achieved a high drug payload (80% w/w) on cCQD without affecting the potency of metronidazole as determined by cytotoxicity assays, cellular uptake of metronidazole, P. gingivalis invasion and elimination assays. The synthesized cCQD also displayed high fluorescence with 56% quantum yield at an absorbance peak of 380 nm and an emission peak of 480 nm, thus, allowing for fluorescence tracking and quantification of the drug intracellularly. A similar strategy may be used to repurpose other antibiotics for the treatment of intracellular bacterial infections.

(Invited) Biocompatible Doped Graphene Quantum Dots Enabling Visible/Near-Infrared Imaging, Drug Delivery and Cancer Detection

Despite significant advances of nanomedicine, the issues of biocompatibility, accumulation-derived toxicity and the lack of sensing and in vivo imaging capabilities hamper the translation of many nanocarriers into clinic. To address these needs we develop and test the feasibility of new nitrogen, sulfur or metal-doped graphene quantum dots (GQDs) as fully biocompatible multifunctional platforms enabling theranostic image-guided approaches both in vitro and in vivo. These GQDs scalably produced in one-step synthesis from a single biocompatible carbon-rich precursor form 3-5 nm few-layer structures functionalized with oxygen-containing groups, however, showing high crystallinity and lattice spacing indicative of a graphitic platform. They are water-soluble, show no cytotoxicity at high concentrations of 1 mg/mL, and demonstrate substantial degradation at 36h in biological environments as verified by TEM imaging. Due to their small sizes, GQDs exhibit efficient internalization maximized at 12 h followed by further degradation/excretion and enable active agent delivery. Their intrinsic fluorescence in blue/green with up to 62% quantum yield and near-infrared allows for multicolor in vitro imaging on its own or in combination with other fluorophores and offers the potential capabilities for in vivo near-IR fluorescence tracking. Additionally, several GQD types exhibit pH-dependent fluorescence response that is successfully utilized as a sensing mechanism for acidic extracellular environments of cancer cells. It allows for deterministic, ratiometric discrimination between cancerous (HeLa and MCF-7 cell) and healthy (HEK-293 cell) environments with substantial spectral peak intensity ratios of 1.6 to 8 in healthy versus cancer cells. Such spectral technique, less prone to quenching discrepancies, provides a useful tool for pH-sensing of microenvironments. These results suggest fully biocompatible doped GQDs as multifunctional candidates for in vitro delivery of active agents, multicolor visible/near-IR fluorescence imaging, and pH-sensing of cancerous environments.

Understanding Corona Exchange Dynamics on Single-Walled Carbon Nanotubes with Multiplexed Fluorescence Monitoring

Adsorption of polymers on single-walled carbon nanotubes (SWCNTs) has enabled developments in molecular sensing, in vivo imaging, gene and drug delivery, and chirality sorting applications. Noncovalent functionalization offers a modification route that preserves the pristine atomic structure, thus retaining the intrinsic near-infrared fluorescence of the SWCNTs for sensing or imaging functions, and offers a reversible binding mode for cargo delivery or chirality separation processes. However, noncovalent adsorption is an inherently dynamic process, where exchange occurs between molecules in the bulk solution and molecules on the surface, into what is known as the ‘corona phase’. In the case of polymers on SWCNTs, the nature, strength, and kinetics of both the polymer binding and unbinding processes are important contributors to the success of polymer-SWCNT based technologies. Understanding this binding process is especially important for intended uses of functionalized SWCNTs in biological environments, where native biomolecules compete with the original polymer to occupy the SWCNT surface. Binding of proteins and other biomolecules to the SWCNT not only disrupts the intended functionality of the nanoparticle, but further leads to potentially adverse biocompatibility outcomes. We present an assay to study the process of corona exchange dynamics between solution-phase and corona-phase polymers on SWCNTs. Real-time tracking of polymer and protein adsorption and desorption events are conducted with systematic variation of the molecular entities and solution conditions. This assay exploits the quenching property of fluorophores in close proximity to the SWCNT surface to monitor ligand binding and unbinding events. Binding profiles are thus extracted from the experimental assay and used to inform a kinetic model of the system. This study encompasses exchange of SWCNT-adsorbed ssDNA and either ssDNA, surfactant, or protein added to the bulk solution with multiplexed fluorescence tracking of all entities. Moreover, the kinetics of these binding profiles are validated against an orthogonal and more ubiquitously-used platform monitoring solvatochromic shifting of the near-infrared SWCNT spectrum as a proxy for SWCNT corona perturbations (Beyene et al., Nano Letters, 2018; Heller et al., PNAS, 2011; Jena et al., ACS Appl. Mater. Interfaces, 2017). The work presented herein develops an understanding of the fundamental corona exchange mechanism, contextualizes the nature of the ligand exchange process versus SWCNT solvatochromic shifting, and provides insight into performance of these designed SWCNT-based systems in biologically relevant, protein-rich conditions.

Multifunctional Gene Carriers Labeled by Perylene Diimide Derivative as Fluorescent Probe for Tracking Gene Delivery.

Multifunctional carriers with both gene transfection property and fluorescent tracking function have attracted significant attention in recent years. Herein, a kind of perylene diimide derivative (PDI-C10C8) is conjugated onto the polyethylenimine-g-poly(lactide-co-glycolide)-g-polyethylenimine (PLGA-PEI) polymer to obtain fluorescent multifunctional polymer and micelles (abbreviated as MP). Then, the REDV-G-TAT-G-NLS (TP-G) peptide sequence is grafted onto this MP to obtain multifunctional micelles labeled by perylene diimide derivative (MP-TP-G). These micelles exhibit enhanced photobleaching stability compared with the reference Cy5-labeled micelles, and the fluorescent images of cellular uptake show bright red emission without any background noise. Confocal laser scanning microscope (CLSM) experiments show that gene complexes can deliver gene into nucleus. MP-TP-G carriers do not enter into the cell nucleus, which proves that the nuclear localization signal sequence may not exert its nucleus accumulation ability via conjugating to the amphiphilic polymers. The high transfection efficiency and the enhanced photobleaching stability, combined with the ability to monitor the detailed process of cellular uptake and gene delivery, make these multifunctional micelles have great potential application for endothelialization of artificial blood vessels and gene delivery process study.

Bioactive carbon dots direct the osteogenic differentiation of human bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) have been the focus of bone regeneration due to their excellent osteogenic potential and abundant source. However, the high-cost and low-efficiency differentiation of BMSCs into functional osteoblasts limits their clinical application. It is desirable to develop bioactive materials to integrate efficient differentiation and traceable properties in a biocompatible manner for MSC-based therapy. In this study, a new kind of bioactive carbon dot (CD) was facilely fabricated through a one-step hydrothermal method from adenosine and aspirin. These bioactive CDs were cytocompatible and biosafe with the capability of long-term fluorescent tracking of human bone marrow mesenchymal stem cells (hBMSCs). Notably, the presence of bioactive CDs triggered and directed a series of events that followed the temporal pattern of osteogenic differentiation through the promotion of osteogenic transcription and enhancement of matrix mineralization. Moreover, cells with bioactive CDs exhibited more effective osteogenic differentiation behavior than cells treated with either adenosine or aspirin alone. Overall, these findings clearly showed that adenosine and aspirin-based CDs can direct osteogenic differentiation of hBMSCs in the absence of any external osteoinductive factors. The unique properties of bioactive CDs could provide insight into their potential for achieving efficient and safe MSC-based therapy.

Luminescent properties of LiF crystals for fluorescent imaging of nuclear particles tracks

Fluorescent nuclear track detectors (FNTD) based on LiF crystals were used for imaging ionizing particle tracks (including high-energy iron ions), by exploiting photoluminescence of radiation induced F2 and F3+ color centers. The relation between photoluminescence emission spectrum of LiF and quality of FNTD images was investigated. It was found that in the applied experimental conditions, the emission within the green band (530 nm, F3+) is very weak compared to the red band (670 nm, F2). The optimum signal to noise ratio was achieved with setting the measuring window at wavelengths longer than 570 nm. Good quality track images in the green spectral range were obtained only for the highest ionization density: for stopping iron ions.Investigations of the bleaching phenomenon revealed a strong decrease of the PL intensity after long excitation times, in the range of several minutes. The amount of this decrease depends on the used excitation power. In the conditions which were typically applied during FNTD measurements the intensity loss was negligible.