Multicolor biomass based carbon nanodots for bacterial imaging

Biomass-based carbon nanodots(CNDs) are becoming promising fluorescent materials due to their superior optical properties and excellent biocompatibility. However, most fluorescent CNDs are prepared under high temperatures with artificial chemicals as precursors. In this work, multicolor biomass-based CNDs have been prepared by employing natural biomass as precursors through an ultrasonic-assisted method at room temperature. The multicolor biomass-based CNDs can be prepared within 10 min, and cav...     

Single nanoparticle imaging and characterization of different phospholipid-encapsulated quantum dot micelles

Phospholipid quantum dot (QD) micelles have been extensively used as fluorescent tags in single nanoparticle imaging for biomedical imaging. In this work, the microscopic structures and photophysical properties of the phospholipid QD micelles were studied at the single nanoparticle level. Two commonly used types of phospholipid QD micelles were prepared and tested both on a solid-phase surface and in liquid phase, including 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-encapsulated QD micelles (DSPE-QDMs) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-encapsulated QD micelles (PEG-DSPE-QDMs). Their fluorescence intensities and diffusion trajectories were determined by a total internal reflection fluorescence-based single nanoparticle imaging platform and comparatively analyzed carefully. It was demonstrated that DSPE-QDMs possessed a comparably wider intensity distribution and lower diffusion coefficient than that of PEG-DSPE-QDMs. PEG-DSPE-QDMs exhibited an obvious fluorescent intermittence. The results suggested that for most of the DSPE-QDMs, more than one QD were encapsulated in a single micelle. On the other hand, only one QD was embedded in a single micelle of PEG-DSPE-QDMs for most of the cases. Such variances suggested that phospholipids play a key role in the fabrication of the QD micelles. This work provides a useful foundation for their further biomedical applications.     

Quantum dot capped magnetite nanorings as high performance nanoprobe for multiphoton fluorescence and magnetic resonance imaging

In the present study, quantum dot (QD) capped magnetite nanorings (NRs) with a high luminescence and magnetic vortex core have been successfully developed as a new class of magnetic-fluorescent nanoprobe. Through electrostatic interaction, cationic polyethylenimine (PEI) capped QD have been firmly graft into negatively charged magnetite NRs modified with citric acid on the surface. The obtained biocompatible multicolor QD capped magnetite NRs exhibit a much stronger magnetic resonance (MR) T2* effect where the r2* relaxivity and r2*/r1 ratio are 4 times and 110 times respectively larger than those of a commercial superparamagnetic iron oxide. The multiphoton fluorescence imaging and cell uptake of QD capped magnetite NRs are also demonstrated using MGH bladder cancer cells. In particular, these QD capped magnetite NRs can escape from endosomes and be released into the cytoplasm. The obtained results from these exploratory experiments suggest that the cell-penetrating QD capped magnetite NRs could be an excellent dual-modality nanoprobe for intracellular imaging and therapeutic applications. This work has shown great potential of the magnetic vortex core based multifunctional nanoparticle as a high performance nanoprobe for biomedical applications.     

A Fluorescent Polymeric Quantum Dot/Aptamer Superstructure and Its Application for Imaging of Cancer Cells

In this work, a novel polymeric quantum dot/aptamer superstructure with a highly intense fluorescence was fabricated by a molecular engineering strategy and successfully applied to fluorescence imaging of cancer cells. The polymeric superstructure, which is composed of both multiple cell‐based aptamers and a high ratio of quantum dot (QD)‐labeled DNA, exploits the target recognition capability of the aptamer, an enhanced cell internalization through multivalent effects, and cellular disruption by the polymeric conjugate. Importantly, the polymeric superstructure exhibits an increasingly enhanced fluorescence with recording time and is thus suitable for long‐term fluorescent cellular imaging. The unique and excellent fluorescence property of the QD superstructure paves the way for developing polymeric QD superstructures that hold promise for applications such as in vivo imaging.     

Rewritable multicolor fluorescent patterns for multistate memory devices with high data storage capacity

We report a branched polyethyleneimine (BPEI)-quantum dot (QD) based rewritable fluorescent system with a multicolor recording mode, in which BPEI is both QD-multicolor patterning "writer" and data erasing "remover". This method could write distinct colors from size-tailored QDs to represent large numbers of logic states for high data storage capacity.     

Chemical analysis and cellular imaging with quantum dots

Quantum dots are tiny light-emitting particles on the nanometer scale. They are emerging as a new class of biological labels with properties and applications that are not available with traditional organic dyes and fluorescent proteins. Their novel properties such as improved brightness, resistance against photobleaching, and multicolor light emission, have opened new possibilities for ultrasensitive chemical analysis and cellular imaging. In this Research Highlight article , we discuss the unique optical properties of semiconductor quantum dots, surface chemistry and bioconjugation, current applications in bioanalytical chemistry and cell biology, and future research directions.     

水溶性量子点的制备及应用

高量子产率的水溶性量子点在众多领域如发光晶体、薄膜光激发器件,尤其是生物荧光标记中展现出巨大的潜在应用价值.量子点荧光明亮、稳定,激发光谱宽,发射光谱窄,且发射波长可通过改变材料的粒径大小和组成来调控,因而在生物样本尤其是活组织的多色成像中极为有用,能有效避免因样本自身发光和光散射导致的信号干扰.量子点的研究已成为一门新兴的交叉科学,是目前最有吸引力的研究领域之一.近年来,水溶性量子点作生物荧光标记物的研究取得了长足的进展.本文简述了量子点的结构特征及光学特性,重点综述了水溶性量子点的制备方法及应用研究进展,特别是它在生物、医学中作荧光探针的最新研究.     

Fluorescence imaging and point measurements of tissue: applications to the demarcation of malignant tumors and atherosclerotic lesions from normal tissue

The possibilities of using laser-induced fluorescence for tissue diagnostics are discussed. The tissue types investigated are malignant tumors and atherosclerotic lesions. Studies with natural autofluorescence as well as with fluorescent tumor markers are included in this paper. Fluorescence emission and decay data are presented for some tissue chromophores contributing to tissue autofluorescence. Optical spectroscopic characteristics of fluorescent malignant tumor markers are analyzed and instrumental designs for clinical applications are discussed. Images recorded with a multicolor fluorescence imaging system developed in Lund are presented.     

Tetrapeptide self-assembled multicolor fluorescent nanoparticles for bioimaging applications

The biocompatibility and biodegradability of peptide self-assembled materials makes them suitable for many biological applications, such as targeted drug delivery, bioimaging, and tracking of therapeutic agents. According to our previous research, self-assembled fluorescent peptide nanoparticles can overcome the intrinsic optical properties of peptides. However, monochromatic fluorescent nanomaterials have many limitations as luminescent agents in biomedical applications. Therefore, combining different fluorescent species into one nanostructure to prepare fluorescent nanoparticles with multiple emission wavelengths has become a very attractive research area in the bioimaging field. In this study, the tetrapeptide Trp-Trp-Trp-Trp (WWWW) was self-assembled into multicolor fluorescent nanoparticles (TPNPs). The results have demonstrated that TPNPs have the blue, green, red and near infrared (NIR) fluorescence emission wavelength. Moreover, TPNPs have shown excellent performance in multicolor bioimaging, biocompatibility, and photostability. The facile preparation and multicolor fluorescence features make TPNPs potentially useful in multiplex bioanalysis and diagnostics.     

Hybrid fluorescent nanoparticles from quantum dots coupled to cellulose nanocrystals

Carboxylated cellulose nanocrystals (CNCs) were decorated with CdSe/ZnS quantum dots (QDs) using a carbodiimide chemistry coupling approach. The one-step covalent modification was supported by nanoscale imaging, which showed QDs clustered on and around the CNCs after coupling. The QD–CNC hybrid nanoparticles remained colloidally stable in aqueous suspension and were fluorescent, exhibiting the broad excitation and narrow emission profile characteristic of the QDs. QD–CNCs in nanocomposite films imparted strong fluorescence within CNC-compatible matrices at relatively low loadings (0.15 nmol QDs/g of dry film), without altering the overall physical properties or self-assembly of the CNCs. The hybrid QD–CNCs may find applications in nanoparticle tracking, bio-imaging, optical/sensing devices, and anti-counterfeit technologies.     

Single-molecule measurements with a single quantum dot

Recent progress of quantum dot (QD) applications in single-molecule measurements are reviewed in this paper. Bright fluorescence and anti-photobleaching properties of QDs have explored the way to conduct long-time trajectory tracking of transmembrane proteins both in vitro and in vivo. Coupled with diversities of chemical and biochemical modifications of QD surfaces, their application fields are expanding to multidiscipline fields including imaging on the basis of a single molecule. Currently, molecular interactions and conformational changes on the QD surface can be detected at a single-molecule level. These expansions of application fields also involve toxicity problems in cells since most commercially available QDs consist of cadmium selenide or cadmium telluride, which are inherently toxic. For widespread applications of QDs including in vivo and therapeutic use in place of current organic fluorophore, cytotoxicity is discussed as well in this paper. 10.1002/tcr.20128.     

DNA‐Templated Assembly of a Heterobivalent Quantum Dot Nanoprobe For Extra‐ and Intracellular Dual‐Targeting and Imaging of Live Cancer Cells

Quantum dots (QDs) hold great promise for the molecular imaging of cancer because of their superior optical properties. Although cell‐surface biomarkers can be readily imaged with QDs, non‐invasive live‐cell imaging of critical intracellular cancer markers with QDs is a great challenge because of the difficulties in the automatic delivery of QD probes to the cytosol and the ambiguity of intracellular targeting signals. Herein, we report a new type of DNA‐templated heterobivalent QD nanoprobes with the ability to target and image two spatially isolated cancer markers (nucleolin and mRNA) present on the cell surface and in the cell cytosol. Bypassing endolysosomal sequestration, this type of QD nanoprobes undergo macropinocytosis following the nucleolin targeting and then translocate to the cytosol for mRNA targeting. Fluorescence resonance energy transfer (FRET) based confocal microscopy enables unambiguous signal deconvolution of mRNA‐targeted QD nanoprobes inside cancer cells.     

Development of a far-red to near-infrared fluorescence probe for calcium ion and its application to multicolor neuronal imaging

To improve optical imaging of Ca(2+) and to make available a distinct color window for multicolor imaging, we designed and synthesized CaSiR-1, a far-red to near-infrared fluorescence probe for Ca(2+), using Si-rhodamine (SiR) as the fluorophore and the well-known Ca(2+) chelator BAPTA. This wavelength region is advantageous, affording higher tissue penetration, lower background autofluorescence, and lower phototoxicity in comparison with the UV to visible range. CaSiR-1 has a high fluorescence off/on ratio of over 1000. We demonstrate its usefulness for multicolor fluorescence imaging of action potentials (visualized as increases in intracellular Ca(2+)) in brain slices loaded with sulforhodamine 101 (red color; specific for astrocytes) that were prepared from transgenic mice in which some neurons expressed green fluorescent protein.     

In vitro and in vivo imaging with quantum dots

Quantum dots (QDs), also named semiconductor nanocrystals, have initiated a new realm of bioscience by combining nanomaterials with biology, which will profoundly influence future biological and biomedical research. In this review, we describe the extraordinary optical properties of QDs and developments in methods for their synthesis. We focus on fluorescent imaging with QDs both in vitro and in vivo, and the cytotoxicity of QDs and potential barriers to their use in practical biomedical applications. Finally, we provide insights into improvements aimed at decreasing the cytotoxicity of QDs and the future outlook of QD applications in biomedical fields.

A FRET-Facilitated Photoswitching Using an Orange Fluorescent Protein with the Fast Photoconversion Kinetics

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via F?rster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells.     

Spatiotemporal multicolor labeling of individual cells using peptide-functionalized quantum dots and mixed delivery techniques

Multicolor fluorescent labeling of both intra- and extracellular structures is a powerful technique for simultaneous monitoring of multiple complex biochemical processes. This approach remains extremely challenging, however, as it often necessitates the combinatorial use of numerous targeting probes (e.g., antibodies), multistep bioconjugation chemistries, different delivery strategies (e.g., electroporation or transfection reagents), cellular fixation coupled with membrane permeabilization, and complex spectral deconvolution. Here, we present a nanoparticle-based fluorescence labeling strategy for the multicolor labeling of distinct subcellular compartments within live cells without the need for antibody conjugation or cellular fixation/permeabilization. This multipronged approach incorporates an array of delivery strategies, which localize semiconductor quantum dots (QDs) to various subcellular structures. QD uptake is implemented in a spaciotemporal manner by staggering the delivery of QD-peptide composites and exploiting various innate (peptide-mediated endocytosis, peptide-membrane interaction, polymer-based transfection) along with physical (microinjection) cellular delivery modalities to live cells growing in culture over a 4 day period. Imaging of the different intracellular labels is simplified by the unique photophysical characteristics of the QDs in combination with Fo?rster resonance energy transfer sensitization, which allow for multiple spectral windows to be accessed with one excitation wavelength. Using this overall approach, QDs were targeted to both early and late endosomes, the cellular cytosol, and the plasma membrane in live cells, ultimately allowing for simultaneous five-color fluorescent imaging.     

Encapsulation of magnetic and fluorescent nanoparticles in emulsion droplets

Oils containing both fluorescent semiconductor and magnetic oxide nanoparticles are used to produce oil in water emulsions. This technique produces oil droplets with homogeneous fluorescence and high magnetic nanoparticle concentrations. The optical properties of the oil droplets are studied as a function of the droplet sizes for various concentrations of fluorescent and magnetic nanoparticles. For all concentrations tested, we find a linear variation of the droplet fluorescent intensity as a function of the droplet volume. For a given size and a given quantum dot (QD) concentration, the droplet fluorescence intensity drops sharply as a function of the magnetic nanoparticle concentration. We show that this decrease is due mainly to the strong absorption cross section of the magnetic nanoparticles and to a lesser extent to the dynamic and static quenching of the QD fluorescence. The role of the iron oxide nanoparticle localization in the droplet (surface versus volume) is also discussed.     

Quantum dots as simultaneous acceptors and donors in time-gated Förster resonance energy transfer relays: characterization and biosensing

The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in F?rster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.     

ECHO probes: a concept of fluorescence control for practical nucleic acid sensing

An excitonic interaction caused by the H-aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence-controlled nucleic acid sensing. This critical review points out the recent advances in exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, which have a fluorescence-labeled nucleotide in which two molecules of thiazole orange or its derivatives are linked covalently. ECHO probes show absorption shift and emission switching depending on hybridization with the target nucleic acid. The hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible a variety of practical applications, such as multicolor RNA imaging in living cells and facile detection of gene polymorphism (144 references).     

Forming highly fluorescent near-infrared emitting PbS quantum dots in water using glutathione as surface-modifying molecule

We present a new facile procedure for transferring oil-soluble oleic acid-capped NIR-emitting PbS quantum dots (QDs) into water, using hydrophilic thiol ligands as the surface-modifying agents of the primary capping molecules (oleic acid). The influence of exchange of the primary capping molecules with five different types of thiol molecules is investigated. The results show that highly fluorescent water-soluble PbS QDs are obtained using glutathione as a surface-modifying agent (photoluminescence quantum yield (PL QY), >30%); significantly less fluorescent water-soluble QDs were obtained using l-cysteine (PL QY, ~5%); with other three thiol molecules, PbS QDs lose almost completely their fluorescence in aqueous solution. This striking difference among the five thiol molecules may be attributed to the difference in the molecular structure. Next, we explored systematically the conditions of QD water solubilization, storage stability, photostability and cytotoxicity and tested further the resulting water-soluble PbS QDs for the imaging of living animals. The preliminary results from these studies illustrate that our synthesis procedure is very facile and that the as-prepared water-soluble PbS QDs are stable and low-cytotoxic and will be an important potential probe in the imaging of living animals due to free carboxyl and amino groups on the external surface of the QDs.