Quantum Dots For Imaging Research Articles

Wavelength-dependent three-dimensional single-molecule superlocalization imaging for yoctomole detection of thyroid-stimulating hormone on a quantum dot nanobiosensor

A wavelength-dependent three-dimensional (3D) superlocalization imaging method on gold nanoislands (GNIs) chip was developed as a supersensitive single-molecule thyroid-stimulating hormone (TSH) nanobiosensor. Scattered and fluorescent signals from gold nanoislands on the substrate and quantum dots (QDs) nanoprobes were simultaneously isolated and acquired within an evanescent field layer generated by total internal reflection (TIR) of incident light using a dual-view device. The 3D TIR fluorescence images of TSH-bound QDs on the GNIs were obtained using z-axis optical sectioning at 10 nm intervals before/after immunoreaction to identify the optimal conditions for detection. The localized centroid position of QD nanoprobes and GNI were distinguished at a subdiffraction limit resolution using 3D Gaussian fitting to the point spread function. The QD TSH nanobiosensor using wavelength-dependent 3D TIR fluorescence-based single-molecule localization microscopy (3D TIRF-SLM) imaging technique showed an excellent detection limit of 90 yoctomoles (∼54 molecules) and a wide linear dynamic range of 1.14 zmol/L − 100 pmol/L for TSH. The detection sensitivity was about 4.4 × 109 times higher than conventional enzyme-linked immunosorbent assay and could successfully quantify TSH in human serum. The wavelength-dependent 3D TIRF-SLM technique may emerge as a reliable platform for ultrahigh-sensitive nanobiosensors at the single-molecule level and early diagnosis with quantification of disease-related ultra-trace biomolecules.

Single-virus tracking with quantum dots in live cells.

Single-virus tracking (SVT) offers the opportunity to monitor the journey of individual viruses in real time and to explore the interactions between viral and cellular structures in live cells, which can assist in characterizing the complex infection process and revealing the associated dynamic mechanisms. However, the low brightness and poor photostability of conventional fluorescent tags (e.g., organic dyes and fluorescent proteins) greatly limit the development of the SVT technique, and challenges remain in performing multicolor SVT over long periods of time. Owing to the outstanding photostability, high brightness and narrow emission with tunable color range of quantum dots (QDs), QD-based SVT (QSVT) enables us to follow the fate of individual viruses interacting with different cellular structures at the single-virus level for milliseconds to hours, providing more accurate and detailed information regarding viral infection in live cells. So far, the QSVT technique has yielded spectacular achievements in uncovering the mechanisms associated with virus entry, trafficking and egress. Here, we provide a detailed protocol for QSVT implementation using the viruses that we have previously studied systematically as an example. The specific procedures for performing QSVT experiments in live cells are described, including virus preparation, the QD labeling strategies, imaging approaches, image processing and data analysis. The protocol takes 1-2 weeks from the preparation of viruses and cellular specimens to image acquisition, and 1 d for image processing and data analysis.

Osmotic-Enhanced-Photoelectrochemical Hydrogen Production Based on Nanofluidics

Osmotic-Enhanced-Photoelectrochemical Hydrogen Production Based on Nanofluidics

Multifunctional MnCuInSe/ZnS quantum dots for bioimaging and photodynamic therapy.

In this work, manganese (Mn)-doped CuInSe quantum dots (QDs) with a ZnS passivation layer (MnCuInSe/ZnS) have been synthesized via a one-pot microwave-assisted hydrothermal reaction using glutathione (GSH) as a stabilizer. The MnCuInSe/ZnS core-shell QDs combine magnetic resonance imaging (MRI), excitation-dependent red emission, and reactive oxygen radical generation functions, in which regulation of Mn2+ incorporation leads to synergistic imaging and therapeutic modalities. The MnCuInSe/ZnS QDs exhibit high colloidal and photochemical stability in simulated media and at different pH values. An r2/r1 ratio of 9.99 was calculated from MRI studies suggesting their potential application as dual-modal imaging agents. Based on in vitro tests on Hela, B16, and HepG2 cell lines, it is apparent that MnCuInSe/ZnS QDs impose no significant cytotoxicity in the dark, while they can efficiently generate singlet oxygen radicals for photodynamic therapy of cancers, killing more than 80% of B16 cells within 5min of laser irradiation (671nm, 1W cm-2). Furthermore, in vitro fluorescence imaging and cellular internalization of QDs are examined to visualize cellular uptake and in situ ROS generation. Therefore, this research exemplifies a new set of multifunctional chalcogenide QDs for theranostic applications.

Aggregation-Induced Fluorogens in Bio-Detection, Tumor Imaging, and Therapy: A Review

Aggregation-Induced Fluorogens in Bio-Detection, Tumor Imaging, and Therapy: A Review

Labeling Neuronal Proteins with Quantum Dots for Single-Molecule Imaging.

Single-molecule imaging has illuminated dynamics and kinetics of neuronal proteins in their native membranes helping us understand their effective roles in the brain. Here, we describe how nanometer-sized fluorescent semiconductors called quantum dots (QD) can be used to label neuronal proteins in a single QD imaging format. We detail two generalizable protocols accompanied by experimental considerations giving the user options in approach tailored to the materials and equipment available. These protocols can be modified for experiments to verify target specificity, as well as single molecule analysis such as single particle tracking and protein clustering.

Pushing the limits of detection for proteins secreted from single cells using quantum dots.

Single cell analysis methods are increasingly being utilized to investigate how individual cells process information and respond to diverse stimuli. Soluble proteins play a critical role in controlling cell populations and tissues, but directly monitoring secretion is technically challenging. Microfabricated well arrays have been developed to assess secretion at the single cell level, but these systems are limited by low detection sensitivity. Semiconductor quantum dots (QD) exhibit remarkably bright and photostable luminescence signal, but to date they have not been evaluated in single cell secretion studies using microfabricated well arrays. Here, we used QDs in a sandwich immunoassay to detect secretion of the soluble cytokine tumor necrosis factor-α (TNF-α) from single cells. To enhance detection sensitivity, we employed two different strategies. First, we used a unique single QD imaging approach, which provided a detection threshold (180 attomolar) that was >100-fold lower than previously reported results using QDs. We also amplified QD binding to each captured TNF-α molecule using the bioorthogonal cycloaddition reaction between trans-cyclooctene and tetrazine, which further lowered detection threshold to 60 attomolar. This is 6 orders of magnitude more sensitive than organic fluorophores that have been used for single cell secretion studies, and far surpasses single molecule resolution within sub-picoliter microwells that are used to assess single cell secretion. Finally, single cell secretion studies were performed using phorbol 12-myristate 13-acetate (PMA) differentiated and lipopolysaccharide (LPS) activated U-937 cells. TNF-α secretion was detected from 3-fold more single cells using the QD-based method in comparison to rhodamine, which was accomplished by extending sensitivity into the range of ∼2 to 10 000 molecules captured per microwell. In future work, we will apply this technique to assess immune cell secretion dynamics under diverse stimuli and disease settings. We will also incorporate multiplexing capabilities to evaluate the secretome at the resolution of single molecules.

Fluorescent quantum dots for microbial imaging

Quantum dots (QDs), with several unique optical and chemical features, are becoming desirable fluorescent tags for the biological applications that require long-term and highly sensitive imaging. Besides, the conjugation of various functional biomolecules to QDs has enabled wide applications of QDs in biological imaging. This review focuses on the following four types of QDs: semiconductor quantum dots (semiconductor QDs), carbon nanodots (CDs), silicon quantum dots (SiQDs), and polymer dots (Pdots), and summarizes the recent advancements of using these QDs in imaging microorganisms including viruses, bacteria, and fungi. We hope that this review will promote the development of new fluorescent QDs for microbial imaging and extend the applications of QD-based imaging techniques in cell biology and beyond.

Quantum Dot Conjugates in Functional Imaging and Highly Sensitive Biochemical Assays

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Surfactants as Quantum Dots used in Bio-Imaging

Quantum Dots (QDs) are semiconductor nanocrystals, which arose a new class for chemical analysis, molecular imaging, and biomedical diagnostics as fluorescent labels. There are traditional ways to synthesize QD. In QD micelle formation takes place, with hydrophobic tails and hydrophilic head. The core and the ligand play a key role in defining the properties of quantum dots. Colloidal Synthesis, Plasma Synthesis and Green Synthesis methods are followed to synthesize QD. The green synthesis method would be helpful in near future because of the use of oil and micro-organisms as raw material. In this paper, the size, shape of the QD, presence of ligand and other factors affecting the properties of QDs are discussed. Also, the toxic effects in vitro and in vivo imaging of QDs are highlighted along with the preventive measures to subjugate the difficulty. QDs overcome the limitations of organic dyes and fluorescent protein because of their ability to tune the size of fluorescence emission and generating any specific wavelength from UV through near infrared. QDs may provide the new class of biological values that can overcome the limitations of organic dyes and fluorescent protein. These properties are apposite for imaging and for multiplexed biomedical diagnostics at very high sensitivity. Hence, QDs are growing interest in medical field as an imaging tool of diseased tissues and organs.

Advances in quantum dot-mediated siRNA delivery

Nano-sized quantum dots (QDs) exhibit uniquely optical properties that are tunable with different sizes and shapes. QDs can emit narrow symmetric bands under a wide excitation range, possess anti-photobleaching stability, and be bio-functionalized on the large surface area. Therefore, QDs are attractive vectors for imaging-guided therapy. Small-interfering RNA (siRNAs)-based therapeutics hold great potential to target a large part of the currently undruggable genes, but overcoming the lipid bilayer to deliver siRNA into cells has remained a major challenge to solve for widespread development of siRNA therapeutics. In this mini-review, we focus on theranostic QD/siRNA assembles for enhancing delivery of siRNA and facilitating evaluation of therapeutic efficacy via imaging of QDs, with special attention to carbonaceous QDs for delivery of siRNA.

Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots

The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. How local transport depends on the heterogeneous organization and dynamics of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for active and passive intracellular transport. Rapid imaging of non-functionalized QDs reveals two populations with a 100-fold difference in diffusion constant, with the faster fraction increasing upon actin depolymerization. When nanobody-functionalized QDs are targeted to different kinesin motor proteins, their trajectories do not display strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 displays subtle directional fluctuations, because the subset of microtubules used by this motor undergoes prominent undulations. Using actin-targeting agents reveals that F-actin suppresses most microtubule shape remodelling, rather than promoting it. These results demonstrate how the spatial heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics.

Enhanced cell membrane enrichment and subsequent cellular internalization of quantum dots via cell surface engineering: illuminating plasma membranes with quantum dots.

Efficient cellular uptake of nanoparticles is crucial for modulating the cell behaviors as well as dictating the cell fate. In this work, by using two commercial reagents (the membrane modification reagent "cholesterol-PEG-biotin" and the avidin-modified quantum dots (QDs) "QD-avidin"), we achieved the enhanced plasma membrane enrichment and endocytosis of fluorescent QDs in cancer cells through cell surface engineering. The QD-cell interaction involved two stages: adsorption and internalization. After incubation with cholesterol-PEG2k-biotin, the cell membrane was engineered with biotin groups that would actively recruit QD-avidin to the cell surface within 1 min. This fast adsorption process could realize high quality and photostable plasma membrane imaging, which is simple, low-cost and generally applicable as compared with the previously reported membrane protein/receptor labeling-based QD imaging. After that, the QDs attached on the cell surface underwent the internalization process and 12 h later, almost all the QDs were internalized through endocytosis. Notably, we found that the internalization of QDs was not via common endocytosis pathways (such as clathrin- or caveolae-mediated endocytosis or macropinocytosis) but more likely via lipid raft-dependent endocytosis. In contrast, without cell surface engineering, the QD-avidin showed negligible cellular uptake. The results demonstrate that cell surface engineering is an efficient strategy to image the plasma membrane and increase cellular uptake of nanoparticles, and will be potentially applied to enhance the efficacy of nanomedicines when therapeutic nanoparticles are used.

Nanoimaging: photophysical and pharmaceutical characterization of poly-lactide-co-glycolide nanoparticles engineered with quantum dots

Quantum dots (QDs) and polymeric nanoparticles (NPs) are considered good binomials for the development of multifunctional nanomedicines for multimodal imaging. Fluorescent imaging of QDs can monitor the behavior of QD-labeled NPs in both cells and animals with high temporal and spatial resolutions. The comprehension of polymer interaction with the metallic QD surface must be considered to achieve a complete chemicophysical characterization of these systems and to describe the QD optical properties to be used for their unequivocal identification in the tissue. In this study, by comparing two different synthetic procedures to obtain polymeric nanoparticles labeled with QDs, we investigated whether their optical properties may change according to the formulation methods, as a consequence of the different polymeric environments. Atomic force microscopy, transmission electron microscopy, confocal and fluorescence lifetime imaging microscopy characterization demonstrated that NPs modified with QDs after the formulation process (post-NPs-QDs) conserved the photophysical features of the QD probe. In contrast, by using a polymer modified with QDs to formulate NPs (pre-NPs-QDs), a significant quenching of QD fluorescence and a blueshift in its emission spectra were observed. Our results suggest that the packaging of QDs into the polymeric matrix causes a modification of the QD optical properties: these effects must be characterized in depth and carefully considered when developing nanosystems for imaging and biological applications.

Fluorescent approach for visually observing quantum dot uptake in living organisms.

This study examines the in vivo uptake and internalization of fluorescent quantum dots (QDs) in Escherichia coli and Caenorhabditis elegans models. E. coli cells were directly exposed to QDs of different concentrations (up to 20 nM), and the uptake or sorption of QDs was monitored by flow cytometry. We observed a concentration-dependent increase in QD fluorescence with no changes in the forward or side scatter for any QD concentration, likely because the QDs are very small. Furthermore, QD uptake/adsorption did not significantly affect E. coli viability assessed by colony formation and size. QD-exposed E. coli were then fed to C. elegans to monitor the localization and effects of QDs. In our study, QDs had no observable effect on the viability or reproduction of C. elegans. We visualized QD incorporation and biodistribution by using confocal laser scanning microscopy (CLSM) with z-stacks, lambda scanning, and linear unmixing techniques, which allowed us to observe QDs in vivo and deconvolute QD fluorescence from autofluorescence. CLSM z-stacks with 10-μm depth revealed that the QDs exclusively localized to the gut and intestine with no transfer to other tissues. The combination of these techniques for in vivo imaging of QDs and other fluorescent nanoparticles will be a powerful tool for future studies examining the uptake and biodistribution of nanoparticles.

Preoperative Detection and Intraoperative Visualization of Brain Tumors for More Precise Surgery: A New Dual-Modality MRI and NIR Nanoprobe.

In clinical practice, it is difficult to identify tumor margins during brain surgery due to its inherent infiltrative character. Herein, a unique dual-modality nanoprobe (Gd-DOTA-Ag2S QDs, referred as Gd-Ag2S nanoprobe) is reported, which integrates advantages of the deep tissue penetration of enhanced magnetic resonance (MR) imaging of Gd and the high signal-to-noise ratio and high spatiotemporal resolution of fluorescence imaging in the second near-infrared window (NIR-II) of Ag2S quantum dots (QDs). Due to the abundant tumor angiogenesis and the enhanced permeability and retention effect in the tumor, a brain tumor (U87MG) in a mouse model is clearly delineated in situ with the help of the Gd assisted T1 MR imaging and the intraoperative resection of the tumor is precisely accomplished under the guidance of NIR-II fluorescence imaging of Ag2S QDs after intravenous injection of Gd-Ag2S nanoprobe. Additionally, no histologic changes are observed in the main organs of the mouse after administration of Gd-Ag2S nanoprobe for 1 month, indicating the high biocompatibility of the nanoprobe. We expect that such a novel "Detection and Operation" strategy based on Gd-Ag2S nanoprobe is promising in future clinical applications.

Quantum dot imaging in the second near-infrared optical window: studies on reflectance fluorescence imaging depths by effective fluence rate and multiple image acquisition.

Quantum dot (QD) imaging capability was investigated by the imaging depth at a near-infrared second optical window (SOW; 1000 to 1400 nm) using time-modulated pulsed laser excitations to control the effective fluence rate. Various media, such as liquid phantoms, tissues, and in vivo small animals, were used and the imaging depths were compared with our predicted values. The QD imaging depth under excitation of continuous 20 mW/cm(2) laser was determined to be 10.3 mm for 2 wt%hemoglobin phantom medium and 5.85 mm for 1 wt% intralipid phantom, which were extended by more than two times on increasing the effective fluence rate to 2000 mW/cm(2). Bovine liver and porcine skin tissues also showed similar enhancement in the contrast-to-noise ratio (CNR) values. A QD sample was inserted into the abdomen of a mouse.With a higher effective fluence rate, the CNR increased more than twofold and the QD sample became clearly visualized, which was completely undetectable under continuous excitation.Multiple acquisitions of QD images and averaging process pixel by pixel were performed to overcome the thermal noise issue of the detector in SOW, which yielded significant enhancement in the imaging capability, showing up to a 1.5 times increase in the CNR.

Use of quantum nanodot crystals as imaging probes for cereal proteins

Quantum dots (QDs) also known as fluorescent semiconductor nanocrystals have unique optical and chemical properties. Their properties make them excellent fluorescent probes for biological applications which enable long term high quality imaging with the ability to conjugate to multiple biological targets. There are many applications of QDs in the biomedical field; however no applications exist in imaging of food structures. In this paper a detailed protocol for the use of QDs in imaging of gluten network in flat bread and zein in corn extrudates by combining covalent bioconjugation principles to the rules of immunohistochemistry is presented. In this study water soluble CdSe/ZnS core/shell quantum dots containing carboxyl terminated groups were used. The quantum dots had a wavelength of emission of 620nm. Confocal laser scanning microscopy was used to observe the structure of cereal proteins tagged by QDs to visualize their location within the food matrix. Optical sections were collected within a sample size of 20μm and processed with Zen 2009 image analysis software (Carl Zeiss MicroImaging GmbH) to obtain three dimensional images. Results showed that QDs can be covalently conjugated to both gluten and zein. They provide long term bright stable imaging capability for wheat gluten with very little quenching after multiple laser excitations overtime.

Potential usage for in vivo lectin screening in live animals utilizing cell surface mimetic glyco-nanoparticles, phosphorylcholine-coated quantum dots (PC-QDs).

Utilizing glycosylated derivatives as a tag, we are able to explore novel counter-receptor of endogenous lectins or lectin-like molecules in vivo. We have established the standardized methodology including preparation of glycosylated derivatives and construction of a platform for tracing the molecules in vivo at first. Combined use of an aminooxy-terminated thiol derivative and a phosphorylcholine (PC) derivative provides quantum dots (QDs) with novel functions for the chemical ligation of ketone-functionalized compounds and the prevention of nonspecific protein adsorption concurrently. In order to track the derivatives in vivo, near-infrared (NIR) fluorescence imaging of QDs displaying various simple sugars (glyco-PC-QDs) after administration into the tail vein of the mouse can be performed. It has revealed that distinct long-term delocalization over 2 h can be observed depending on the species of glycans ligated to PC-QDs at least in the liver. Until today we have performed live animal imaging utilizing various kinds of sialyl glyco-PC-QDs. They are still retained stably in whole body after 2 h while they showed significantly different in vivo dynamics in the tissue distribution, suggesting that structure/sequence of the neighboring sugar residues in the individual sialyl oligosaccharides might influence the final organ-specific distribution, which should be equivalent to the distribution of sialic acid-recognizing lectins. Here we describe a standardized protocol using ligand-displayed PC-QDs for live cell/animal imaging by versatile NIR fluorescence photometry without influence of size-dependent accumulation/excretion pathway for nanoparticles (e.g., viruses)>10 nm in hydrodynamic diameter by the liver.

Automatic Morphological Measurement of the Quantum Dots Based on Marker-Controlled Watershed Algorithm

In the field of material growth, the quantum dot (QD) image analysis is a fundamental tool. The main challenge is that such study is used to be made by nonautomatic procedures which are time consuming and subjective. We aim to implement an algorithm of automatic analysis of the QDs images. In this frame, efficient QDs segmentation is prerequisite. In this paper, a fast and robust method for the visual segmentation of QDs image based on marker-controlled watershed transform is proposed. According to the foreground markers and the boundary of the coarse partition, the watershed transform is utilized to accurately separate QDs. A next process is then implemented to filter the possible attached substrates based on the area-height distribution of the extracted QDs. Finally, almost all the QDs can be accurately and robustly extracted and thus their properties can be measured. The experimental results show that the proposed approach gives a good tradeoff between the easy usability and efficiency, execution time, and segmentation quality.