A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy.

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm(2)) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

Smart fluorescent proteins: innovation for barrier-free superresolution imaging in living cells.

During the past decade, several novel fluorescence microscopy techniques have emerged that achieve incredible spatial and temporal resolution beyond the diffraction limit. These microscopy techniques depend on altered optical setups, unique fluorescent probes, or post-imaging analysis. Many of these techniques also depend strictly on the use of unique fluorescent proteins (FPs) with special photoswitching properties. These photoswitchable FPs are capable of switching between two states in response to light. All localization precision and patterned illumination techniques-such as photo-activation localization microscopy, stochastic optical reconstruction microscopy, reversible saturable optically linear transitions, and saturated structured illumination microscopy-take advantage of these inherent switching properties to achieve superior spatial resolution. This review provides extensive analysis of the positive and negative aspects of photoswitchable FPs, highlighting their application in diffraction-unlimited imaging and suggesting the most suitable fluorescent proteins for superresolution imaging.

Optically-assisted thermophoretic reversible assembly of colloidal particles and E. coli using graphene oxide microstructures.

Optically-assisted large-scale assembly of nanoparticles have been of recent interest owing to their potential in applications to assemble and manipulate colloidal particles and biological entities. In the recent years, plasmonic heating has been the most popular mechanism to achieve temperature hotspots needed for extended assembly and aggregation. In this work, we present an alternative route to achieving strong thermal gradients that can lead to non-equilibrium transport and assembly of matter. We utilize the excellent photothermal properties of graphene oxide to form a large-scale assembly of silica beads. The formation of the assembly using this scheme is rapid and reversible. Our experiments show that it is possible to aggregate silica beads (average size 385 nm) by illuminating thin graphene oxide microplatelet by a 785 nm laser at low intensities of the order of 50-100 µW/µm2. We further extend the study to trapping and photoablation of E. coli bacteria using graphene oxide. We attribute this aggregation process to optically driven thermophoretic forces. This scheme of large-scale assembly is promising for the study of assembly of matter under non-equilibrium processes, rapid concentration tool for spectroscopic studies such as surface-enhanced Raman scattering and for biological applications.

Dose-dependent in-vivo toxicity assessment of silver nanoparticle in Wistar rats.

This study aims to suggest the limits of silver nanoparticle (AgNP) uses for medicinal purpose and was performed to explore the effect of various doses of silver nanoparticle in rats. Four different doses of AgNP (4, 10, 20, and 40 mg/kg) were injected intravenously. For safety evaluation of injected AgNP, body weight, organ coefficient, whole blood count, and biochemistry panel assay for liver function enzyme (AST, ALT, ALP, and GGTP), comet assay, ROS, and histological parameter were performed; 10-12 week old animals were randomly divided into groups of six individuals each for control, and doses of 40, 20, 10, and 4 mg/kg AgNP injected. Significant changes were observed (p < 0.01) in hematological parameters (WBC count, platelets counts, haemoglobin, and RBC count) in the 40 and 20 mg/kg groups. The changes were non-significant in the other groups (4 and 10 mg/kg group). In the 40 mg/kg group, a significant increase was also found in liver function enzymes like ALT and AST (p < 0.01), ALP (p < 0.01), GGTP (p < 0.01), and bilirubin (p < 0.01). ROS in blood serum increased in the high dose group. Tail migration in single cell gel electrophoresis in the 40, 20, 10, 4 mg/kg, and control groups was 34.9, 29.5, 17.8, 5.8, and 0.0 µm, respectively, which indicated damage in the DNA strand in the high dose group. EDXRF showed a ∼ 10-times increase in silver concentration in the 40 mg/kg group and TEM image also showed particle deposition in the 40 mg/kg group. This study indicates that the AgNP in doses (< 10 mg/kg) is safe for biomedical application and has no side-effects, but its high dose (> 20 mg/kg) is toxic.

Synergistic Antibacterial Potential and Cell Surface Topology Study of Carbon Nanodots and Tetracycline Against E. coli.

Increasing drugs and antibiotic resistance against pathogenic bacteria create the necessity to explore novel biocompatible antibacterial materials. This study investigated the antibacterial effect of carbon dot (C-dot) against E. coli and suggested an effective synergistic dose of tetracycline with C-dot, using mathematical modeling of antibacterial data. Colony count and growth curve studies clearly show an enhanced antibacterial activity against E. coli synergistically treated with C-dot and tetracycline, even at a concentration ten times lower than the minimum inhibitory concentration (MIC). The Richards model-fit of growth curve clearly showed an increase in doubling time, reduction in growth rate, and early stationary phase in the synergistic treatment with 42% reduction in the growth rate (µm) compared to the control. Morphological studies of E. coli synergistically treated with C-dot + tetracycline showed cell damage and deposition of C-dots on the bacterial cell membrane in scanning electron microscopy imaging. We further validated the topological changes, cell surface roughness, and significant changes in the height profile (ΔZ) with the control and treated E. coli cells viewed under an atomic force microscope. We confirmed that the effective antibacterial doses of C-dot and tetracycline were much lower than the MIC in a synergistic treatment.

Growing tool-kit of photosensitizers for clinical and non-clinical applications.

Photosensitizers are photosensitive molecules utilized in clinical and non-clinical applications by taking advantage of light-mediated reactive oxygen generation, which triggers local and systemic cellular toxicity. Photosensitizers are used for diverse biological applications such as spatio-temporal inactivation of a protein in a living system by chromophore-assisted light inactivation, localized cell photoablation, photodynamic and immuno-photodynamic therapy, and correlative light-electron microscopy imaging. Substantial efforts have been made to develop several genetically encoded, chemically synthesized, and nanotechnologically driven photosensitizers for successful implementation in redox biology applications. Genetically encoded photosensitizers (GEPS) or reactive oxygen species (ROS) generating proteins have the advantage of using them in the living system since they can be manipulated by genetic engineering with a variety of target-specific genes for the precise spatio-temporal control of ROS generation. The GEPS variety is limited but is expanding with a variety of newly emerging GEPS proteins. Apart from GEPS, a large variety of chemically- and nanotechnologically-empowered photosensitizers have been developed with a major focus on photodynamic therapy-based cancer treatment alone or in combination with pre-existing treatment methods. Recently, immuno-photodynamic therapy has emerged as an effective cancer treatment method using smartly designed photosensitizers to initiate and engage the patient's immune system so as to empower the photosensitizing effect. In this review, we have discussed various types of photosensitizers, their clinical and non-clinical applications, and implementation toward intelligent efficacy, ROS efficiency, and target specificity in biological systems.

Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells.

The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23∼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells.

A guide to use photocontrollable fluorescent proteins and synthetic smart fluorophores for nanoscopy.

Recent advances in nanoscopy, which breaks the diffraction barrier and can visualize structures smaller than the diffraction limit in cells, have encouraged biologists to investigate cellular processes at molecular resolution. Since nanoscopy depends not only on special optics but also on 'smart' photophysical properties of photocontrollable fluorescent probes, including photoactivatability, photoswitchability and repeated blinking, it is important for biologists to understand the advantages and disadvantages of fluorescent probes and to choose appropriate ones for their specific requirements. Here, we summarize the characteristics of currently available fluorescent probes based on both proteins and synthetic compounds applicable to nanoscopy and provide a guideline for selecting optimal probes for specific applications.

Bio-distribution and toxicity assessment of intravenously injected anti-HER2 antibody conjugated CdSe/ZnS quantum dots in Wistar rats.

Anti-HER2 antibody conjugated with quantum dots (anti-HER2ab-QDs) is a very recent fluorescent nanoprobe for HER2+ve breast cancer imaging. In this study we investigated in-vivo toxicity of anti-HER2ab conjugated CdSe/ZnS QDs in Wistar rats. For toxicity evaluation of injected QDs sample, body weight, organ coefficient, complete blood count (CBC), biochemistry panel assay (AST, ALT, ALP, and GGTP), comet assay, reactive oxygen species, histology, and apoptosis were determined. Wistar rat (8-10 weeks old) were randomly divided into 4 treatment groups (n = 6). CBC and biochemistry panel assay showed nonsignificant changes in the anti-HER2ab-QDs treated group but these changes were significant (P < 0.05) in QDs treated group. No tissue damage, inflammation, lesions, and QDs deposition were found in histology and TEM images of the anti-HER2ab-QDs treated group. Apoptosis in liver and kidney was not found in the anti-HER2ab-QDs treated group. Animals treated with nonconjugated QDs showed comet formation and apoptosis. Cadmium deposition was confirmed in the QDs treated group compared with the anti-HER2ab-QDs treated group. The QDs concentration (500 nM) used for this study is suitable for in-vivo imaging. The combine data of this study support the biocompatibility of anti-HER2ab-QDs for breast cancer imaging, suggesting that the antibody coating assists in controlling any possible adverse effect of quantum dots.

Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.