Real-time nanoscopy by using blinking enhanced quantum dots

Superresolution optical microscopy (nanoscopy) is of current interest in many biological fields. Superresolution optical fluctuation imaging, which utilizes higher-order cumulant of fluorescence temporal fluctuations, is an excellent method for nanoscopy, as it requires neither complicated optics nor illuminations. However, it does need an impractical number of images for real-time observation. Here, we achieved real-time nanoscopy by modifying superresolution optical fluctuation imaging and enhancing the fluctuation of quantum dots. Our developed quantum dots have higher blinking than commercially available ones. The fluctuation of the blinking improved the resolution when using a variance calculation for each pixel instead of a cumulant calculation. This enabled us to obtain microscopic images with 90-nm and 80-ms spatial-temporal resolution by using a conventional fluorescence microscope without any optics or devices.     

Wet electron microscopy with quantum dots

Wet electron microscopy (EM) is a new imaging method with the potential to allow higher spatial resolution of samples. In contrast to most EM methods, it requires little time to perform and does not require complicated equipment or difficult steps. We used this method on a common murine macrophage cell line, IC-21, in combination with various stains and preparations, to collect high resolution images of the actin cytoskeleton. Most importantly, we demonstrated the use of quantum dots in conjunction with this technique to perform light/electron correlation microscopy. We found that wet EM is a useful tool that fits into a niche between the simplicity of light microscopy and the high spatial resolution of EM.     

Advanced procedures for labeling of antibodies with quantum dots

Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs’ advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15 nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs’ amines with the sulfhydryl groups issued from the reduced Abs’ disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD–Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy–light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD–Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.     

Water-soluble quantum dots for biomedical applications

Semiconductor nanocrystals are 1-10nm inorganic particles with unique size-dependent optical and electrical properties due to quantum confinement (so they are also called quantum dots). Quantum dots are new types of fluorescent materials for biological labeling with high quantum efficiency, long-term photostability, narrow emission, and continuous absorption spectra. Here, we discuss the recent development in making water-soluble quantum dots and related cytotoxicity for biomedical applications.     

Facile labeling of lipoglycans with quantum dots

Bacterial endotoxins or lipopolysaccharides (LPS) are among the most potent activators of the innate immune system, yet mechanisms of their action and in particular the role of glycans remain elusive. Efficient non-invasive labeling strategies are necessary for studying interactions of LPS glycans with biological systems. Here we report a new method for labeling LPS and other lipoglycans with luminescent quantum dots. The labeling is achieved by partitioning of hydrophobic quantum dots into the core of various LPS aggregates without disturbing the native LPS structure. The biofunctionality of the LPS-Qdot conjugates is demonstrated by the labeling of mouse monocytes. This simple method should find broad applicability in studies concerned with visualization of LPS biodistribution and identification of LPS binding agents.     

Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry

Bioconjugated quantum dots (QDs) provide a new class of biological labels for evaluating biomolecular signatures (biomarkers) on intact cells and tissue specimens. In particular, the use of multicolor QD probes in immunohistochemistry is considered one of the most important and clinically relevant applications. At present, however, clinical applications of QD-based immunohistochemistry have achieved only limited success. A major bottleneck is the lack of robust protocols to define the key parameters and steps. Here, we describe our recent experience, preliminary results and detailed protocols for QD-antibody conjugation, tissue specimen preparation, multicolor QD staining, image processing and biomarker quantification. The results demonstrate that bioconjugated QDs can be used for multiplexed profiling of molecular biomarkers, and ultimately for correlation with disease progression and response to therapy. In general, QD bioconjugation is completed within 1 day, and multiplexed molecular profiling takes 1-3 days depending on the number of biomarkers and QD probes used.     

Chitosan encapsulated quantum dots platform for leukemia detection

We report results of the studies relating to electrophoretic deposition of nanostructured composite of chitosan (CS)-cadmium-telluride quantum dots (CdTe-QDs) onto indium-tin-oxide coated glass substrate. The high resolution transmission electron microscopic studies of the nanocomposite reveal molecular level coating of the CdTe-QDs with CS molecules in the colloidal dispersion medium. This novel composite platform has been explored to fabricate an electrochemical DNA biosensor for detection of chronic myelogenous leukemia (CML) by immobilizing amine terminated oligonucleotide probe sequence containing 22 base pairs, identified from BCR-ABL fusion gene. The results of differential pulse voltammetry reveal that this nucleic acid sensor can detect as low as 2.56 pM concentration of complementary target DNA with a response time of 60s. Further, the response characteristics show that this fabricated bioelectrode has a shelf life of about 6 weeks and can be used for about 5-6 times. The results of experiments conducted using clinical patient samples reveal that this sensor can be used to distinguish CML positive and the negative control samples.     

Bioconjugation of InGaP quantum dots for molecular sensing

Fluorescence-based molecular sensing and cellular imaging are commonly carried out with the application of organic dyes. Quantum dots (QDs) are now recognized as better tools because they are brighter, size tunable, and more photostable than dyes. Most of the proposed QD-based biosensing systems involve elements of known toxicity. The present work reports the functionalization of biocompatible InGaP/ZnS core-shell QDs with anti-bovine serum albumin (anti-BSA) to exploit them as fluorescent probes for antigen detection. Successful bioconjugation was characterized with the absorption and emission spectra showing blue shifts of around 40 and 30 nm, respectively. Gel electrophoresis and particle size distribution studies further confirmed the mass increment of QDs after their functionalization with anti-BSA. Surface plasmon resonance spectrometry has been used to study the affinity of QD-(anti-BSA) probes for bovine serum albumin (BSA). Photoluminescence quenching of the developed probe is observed in the presence of BSA.     

New method for measuring the distances between the nitroxide spin label and paramagnetic metal ions in macromolecules

A new method of estimation of the distance RLM between the nitroxide spin label (NSL) and the paramagnetic metal ions (PMI), such as Co2+, Ni2+, Cu2+, Mn2+, VO2+, Cr3+, Fe3+ is suggested. The influence of the longitudinal relaxation time T1 of the PMI on the line shape of the NSL at 77 degrees K has been studied. It was found that the efficiency of the dipole-dipole interaction between NSL and PMI depends strongly on the T1 value of the PMI. Measurements of the RLM for 4 spin-labelled proteins (haemoglobin, nitrogenase, cytochrome P450 and Ca2+-dependent ATPase) by three various methods have proved the correctness of the new method and also its simplicity.     

Horseradish peroxidase-driven fluorescent labeling of nanotubes with quantum dots

We describe the first enzyme-driven technique for fluorescent labeling of single-walled carbon nanotubes (SWNTs). The labeling was performed via enzymatic biotinylation of nanotubes in the tyramide-horseradish peroxidase (HRP) reaction. Both direct and indirect fuorescent labeling of SWNTs was achieved using either biotinyl tyramide or fluorescently tagged tyramides. Biotinylated SWNTs later reacted with streptavidin-conjugated fluorophores. Linking semiconductor nanocrystals, quantum dots (Q-dots), to the surface of nanotubes resulted in their fluorescent visualization, whereas conventional fluorophores bound to SWNTs directly or through biotin-streptavidin linkage, were completely quenched. Enzymatic biotinylation permits fluorescent visualization of carbon nanotubes, which could be useful for a number of biomedical applications. In addition, other organic molecules such as proteins, antibodies, or DNA can be conjugated to biotinylated SWNTs using this approach.     

Intracellular imaging of targeted proteins labeled with quantum dots

We developed a new method for imaging the movement of targeted proteins in living cancer cells with photostable and bright quantum dots (QDs). QDs were conjugated with various molecules and proteins, such as phalloidin, anti-tubulin antibody and kinesin. These bioconjugated QDs were mixed with a transfection reagent and successfully internalized into living cells. The movements of individual QDs were tracked for long periods of time. Phalloidin conjugated QDs bound to actin filaments and showed almost no movement. In contrast, anti-tubulin antibody conjugated QDs bound to microtubules and revealed dynamic movement of microtubules. Kinesin showed an interesting behavior whereby kinesin came to be almost paused briefly for a few seconds and then moved once again. This is in direct contrast to the smoothly continuous movement of kinesin in an in vitro assay. The maximum velocity of kinesin in cells was faster than that in the in vitro assay. These results suggest that intracellular movement of kinesin is different from that in the in vitro assay. This newly described method will be a powerful tool for investigating the functions of proteins in living cells.     

Preparation of peptide-conjugated quantum dots for tumor vasculature-targeted imaging

To take full advantage of the unique optical properties of quantum dots (QDs) and expedite future near-infrared fluorescence (NIRF) imaging applications, QDs need to be effectively, specifically and reliably directed to a specific organ or disease site after systemic administration. Recently, we reported the use of peptide-conjugated QDs for non-invasive NIRF imaging of tumor vasculature markers in small animal models. In this protocol, we describe the detailed procedure for the preparation of such peptide-conjugated QDs using commercially available PEG-coated QDs and arginine-glycine-aspartic acid (RGD) peptides. Conjugation of the thiolated RGD peptide to the QDs was achieved through a heterobifunctional linker, 4-maleimidobutyric acid N-succinimidyl ester. Competitive cell binding assay, using (125)I-echistatin as the radioligand, and live cell staining were carried out to confirm the successful attachment of the RGD peptides to the QD surface before in vivo imaging of tumor-bearing mice. In general, QD conjugation and in vitro validation of the peptide-conjugated QDs can be accomplished within 1-2 d; in vivo imaging will take another 1-2 d depending on the experimental design.     

Functionalized silicon quantum dots tailored for targeted siRNA delivery

For RNA interference (RNAi) mediated silencing of the ABCB1 gene in Caco-2 cells biocompatible luminescent silicon quantum dots (SiQDs) were developed to serve as self-tracking transfection tool for ABCB1 siRNA. While the 2-3 nm sized SiQD core exhibits green luminescence, the QD surfaces are completely saturated with covalently linked 2-vinylpyridine that may electrostatically bind siRNA. For down-regulating P-glycoprotein (Pgp) expression of the ABCB1 gene the SiQDs were complexed with siRNA. The cellular uptake and allocation of SiQD-siRNA complexes in Caco-2 cells were monitored using confocal laser scanning microscopy and transmission electron microscopy. The release of siRNA to the cytoplasm was verified through real-time PCR quantification of the reduced ABCB1 mRNA level. Additional evidence was obtained from time-resolved in situ fluorescence spectroscopic monitoring of the Pgp efflux dynamics in transfected Caco-2 cells which yielded significantly reduced transporter efficiencies for the Pgp substrate Rhodamine 123.     

Synthesis of functionalized amphiphilic polymers for coating quantum dots

Quantum dots (QDs) need to be attached to other chemical species if they are to be used as biomarkers, therapeutic agents or sensors. These materials also need to disperse well in water and have well-defined functional groups on their surfaces. QDs are most often synthesized in the presence of ligands such as trioctylphosphine oxide, which render the nanoparticle surfaces hydrophobic. We present a complete protocol for the synthesis and water solubilization of hydrophobic CdSe/ZnS QDs using designer amphiphilic polymeric coatings. The method is based on functionalization of an anhydride polymer backbone with nucleophilic agents. Small functional groups, bulky cyclic compounds and polymeric chains can be integrated into the coating prior to solubilization. We describe the preparation of acetylene- and azide-functionalized QDs for 'click' chemistry. The method is universal and applicable to any type of nanoparticle stabilized with hydrophobic ligands able to interact with the alkyl chains in the coating in water.     

Imaging the lateral diffusion of membrane molecules with quantum dots

This protocol describes a sensitive approach to tracking the motion of membrane molecules such as lipids and proteins with molecular resolution in live cells. This technique makes use of fluorescent semiconductor nanocrystals, quantum dots (QDs), as a probe to detect membrane molecules of interest. The photostability and brightness of QDs allow them to be tracked at a single particle level for longer periods than previous fluorophores, such as fluorescent proteins and organic dyes. QDs are bound to the extracellular part of the object to be followed, and their movements can be recorded with a fluorescence microscope equipped with a spectral lamp and a sensitive cooled charge-coupled device camera. The experimental procedure described for neurons below takes about 45 min. This technique is applicable to various cultured cells.