This PDF file contains the front matter associated with SPIE Proceedings Volume 7575, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

In the last few years, quantum confinement effects in semiconductor nanocrystals (quantum dots - QDs) have attracted a significant amount of interest due to their new optical properties and also because of their potential applications in biological systems. In this work, cadmium sulphide (CdS) nanoparticles were synthesized in aqueous medium and passivated with Cd(OH)2. Polyphosphate ions were used in order to avoid particle aggregation. After the passivation step, CdS/Cd(OH)2 quantum dots were coated with silica. Silica coating has been extensively investigated concerning its properties in biocompatibilizing QDs to biological systems. Silica coated core-shell CdS/Cd(OH)2 water soluble QDs optical properties were studied by absorption, excitation and emission spectroscopies, while their morphological characterization was carried out by transmission electron microscopy.

We create large gold domains (up to 15 nm) exclusively on one side of CdS or CdSe/CdS quantum rods by photoreduction of gold ions under anaerobic conditions. Electrons generated in the semiconductor by UV stimulation migrate to one tip where they reduce gold ions. Large gold domains eventually form; these support efficient plasmon oscillations with a light scattering cross section large enough to visualize single hybrid particles in a dark-field microscope during growth in real time.

We introduce a general approach to make colorful fluorescent gold nanoclusters which are protected by dihydrolipoic acid, mercaptoundecanoic acid and polyethylenimine. The fluorescent gold nanoclusters can perform a variety of bioconjugation processes such as PEGylation, biotinylation as well as forming complex nanobioconjugates with streptavidins. The brightening effects under proper surface modification are also reported. The clusters have a decent quantum yield, high colloidal stability, and can be readily conjugated with biological molecules. Nonspecific uptake by human aortic endothelial cells is demonstrated.

ZnSe quantum dots are wide-band gap materials suitable for UV-blue-emitting applications like laser diode or biolabeling probes. Initially, an organometallic approach have been proposed for preparing these quantum dots with high quantum yield (QY), crystallinity and monodispersity. Although, the chemicals used in this procedures are toxic, expensive, pyrophoric and even explosive. Aiming to develop a more convenient and applicable methodology we propose an alternative methods to synthesize blue-emitting ZnSe/ZnS nanocrystals in aqueous media. This work presents a new methodology using chemometric analysis for improving the quantum yield of these blue probes under aqueous colloidal synthesis. The structural characterization performed by X-Ray diffraction and transmission electronic microscopy shows that this material is in the strong confinement regime with average diameter of 2.5 nm with zinc blend crystalline structure. The optical properties of the colloids demonstrate a large Stoke´s shift between the absorption band (360 nm) and the emission peak (420 nm) with a half maximum at full width of 30 nm and a maximum quantum yield of 43%.

This works deals with the use of quantum chemical calculations in order to investigate the interaction between cadmium telluride quantum dot (CdTe) and the following functionalizers: mecapto acetic acid (MAA), mercaptopropionic acid (MPA) and cysteine (CYS). This interaction between the quantum dot and each functionalizer was quantified by an energy value resulting from theoretical calculations. Interesting features relating this theoretical energy and the experimental results related to stability of these systems were found. The passivation of the CdTe core generating a CdS shell was also considered in the calculations.

Fluorescence resonance energy transfer (FRET) between a quantum dot (QD) and the pH-sensitive fluorescent protein mOrange has been used to develop a fluorescent pH-indicator that is bright and photostable enough for applications in fluorescence imaging, including the tracking of molecules through endocytic pathways. As the molar extinction coefficient of mOrange increases with pH, the ratio of the mOrange emission to the QD emission (F_A/F_D) increases sharply, producing greater than 10-fold increases in the F_A/F_D ratio between pH 4.5 and 7.5. This probe has been thoroughly characterized and it intracellular imaging potential explored.

The efficient use of luminescent semiconductor quantum dots (QDs) as Förster Resonance Energy Transfer (FRET) acceptors can be accomplished with terbium complexes (TCs) as donors. TCs exhibit long excited state lifetimes (in the millisecond range) up to 10⁵ times longer than typical QD lifetimes. When FRET occurs from TCs to QDs the measured TC luminescence decay times decrease (FRET quenching), whereas the QD decay times increase (FRET sensitization). Due to the large difference between the TC and QD excited state lifetimes the FRET formalism can be applied to both the TC donors as well as the QD acceptors. This is a big advantage because the FRET information from one experiment can be received from both sides of the FRET pair allowing for the use of different detection channels and wavelengths for donor and acceptor. Thus, a multiplexing format becomes possible with one single donor (TC) and several different acceptors (different QDs). In this contribution we show the theoretical background for simultaneously applying FRET to donor and acceptor and give an example with a commercially available TC-QD donor-acceptor pair.

We present the use of luminescent terbium complexes (LTCs) as FRET donors and luminescent semiconductor quantum dots (QDs) as FRET acceptors for spectroscopic ruler measurements. The LTCs were labeled to polyhistidine-appended peptides which self-assembled onto three different QDs allowing optical multiplexed measurements. Forster distances were in the range of 60-75 Å and FRET efficiencies of up to 97 % were realized. Time-resolved analysis allowed the determination of donor-acceptor separation distances. The results suggests the efficient use of our LTC-to-QD FRET systems for multiplexed optical size determination, molecular ruler measurements and multi-parameter diagnostics.

A novel type of smart hybrid materials based on the in situ immobilization of quantum dots (QDs) on a responsive microgel template was prepared and investigated. Firstly, a temperature and pH dual responsive hybrid microgel was developed through the in-situ immobilization of CdS QDs in the interior of a copolymer microgel of poly(Nisopropylacrylamide- acrylamide-acrylic acid) [p(NIPAM-AAm-AA)]. The amino groups of the pAAm segments in the microgels are designed to sequester the precursor Cd²⁺ ions for in situ formation of CdS QDs in the interior of the microgels and stabilize the CdS QDs embedded in the microgels. We demonstrated that the carboxyl groups on the p(NIPAM-AAm-AA)-CdS hybrid microgels can be used for further coupling with 3-aminophenyl boronic acid for optical glucose sensing. The glucose concentration change can induce a reversible swelling/shrinkage of the hybrid microgels, which can further modify the physicochemical environment of the QDs immobilized inside the microgels, resulting in a reversible quenching/antiquenching in photoluminescence (PL). The method is extendable to other QDs with different emission wavelengths and other targeting ligands, thus it is possible to develop multifunctional hybrid micro-/nano-gels for additional important biomedical applications.

Highly sensitive, miniature biosensors are desired for the development of new techniques for biological and environmental analyte sensing. One potential approach uses the detection of optical resonances, known as Whispering Gallery Modes (WGMs), from quantum dot embedded polystyrene microspheres. These modes arise from the total internal reflection of the quantum dot emission light within the high index polystyrene microsphere, to produce narrow spectral peaks, which are sensitive to refractive changes in the immediate vicinity of the microsphere surface. The high refractometric sensitivity of the WGMs in these microspheres offers potential for remote detection of molecules adsorbed onto or bound to the microsphere surface without the need for direct coupling of the light via an optical fiber. The sensitivity of these modes has been shown to exceed the theoretical sensitivity of a homogeneous microsphere, using a Mie theory model. This enhancement is believed to be due to the embedded layer of quantum dots at the surface of the microspheres. A model was developed to demonstrate that the embedded QDs could be modeled as a high index outer layer to explain the observed WGM spectra and explore the sensitivity of the modes. In this work, we extend this idea to multiple layers to model the effects of protein adsorption or binding to the surface. The theoretical results are shown to provide a close fit to our previous experimental results.

Single molecule tracking in three dimensions (3D) in a live cell environment promises to reveal important new insights into cell biological mechanisms. However, classical microscopy techniques suffer from poor depth discrimination which severely limits single molecule tracking in 3D with high temporal and spatial resolution. We introduced a novel imaging modality, multifocal plane microscopy (MUM) for the study of subcellular dynamics in 3D. We have shown that MUM provides a powerful approach with which single molecules can be tracked in 3D in live cells. MUM allows for the simultaneous imaging at different focal planes, thereby ensuring that trajectories can be imaged continuously at high temporal resolution. A critical requirement for 3D single molecule tracking as well as localization based 3D super-resolution imaging is high 3D localization accuracy. MUM overcomes the depth discrimination problem of classical microscopy based approaches and supports high accuracy 3D localization of singe molecule/particles. In this way, MUM opens the way for high precision 3D single molecule tracking and 3D super-resolution imaging within a live cell environment. We have used MUM to reveal complex intracellular pathways that could not be imaged with classical approaches. In particular we have tracked quantum dot labeled antibody molecules in the exo/endocytic pathway from the cell interior to the plasma membrane at the single molecule level. Here, we present a brief review of these results.

Heterogeneous nanostructures that possess multiple properties as a result of their differing constituent materials have attracted significant interest in the last few years. In particular, fluorescent-magnetic nanostructures have potential applications in imaging, separations, and single molecule tracking as a result of their fluorescent and magnetic properties. Here we report the synthesis of fluorescent-magnetic nanocomposites composed of fluorescent semiconductor quantum dots or graphitic carbon nanoparticles and magnetic iron oxide nanoparticles. We have developed synthetic strategies using either micellular or polymer encapsulation, yielding composites from ~10 - 100s of nms. Composites maintain the fluorescent and magnetic properties of their constituent materials. These composites can be used for in vitro and in vivo imaging using fluorescent or magnetic (e.g., MRI) modalities. Additionally, we describe the manipulation of these composites using magnetic instrumentation. In particular, we have designed a magnetic needle that can be used to manipulate nanocomposites. Particles as small as 30 nm can be manipulated while simultaneous observed through their fluorescent property. Single particle status can be confirmed through quantum dot blinking, demonstrating the potential of these composites for single molecule tracking.

In the present study we describe sandwich design hybridization probes consisting of magnetic particles (MP) and quantum dots (QD) with target DNA, and their application in the detection of avian influenza virus (H5N1) sequences. Hybridization of 25-, 40-, and 100-mer target DNA with both probes was analyzed and quantified by flow cytometry and fluorescence microscopy on the scale of single particles. The following steps were used in the assay: (i) target selection by MP probes and (ii) target detection by QD probes. Hybridization efficiency between MP conjugated probes and target DNA hybrids was controlled by a fluorescent dye specific for nucleic acids. Fluorescence was detected by flow cytometry to distinguish differences in oligo sequences as short as 25-mer capturing in target DNA and by gel-electrophoresis in the case of QD probes. This report shows that effective manipulation and control of micro- and nanoparticles in hybridization assays is possible.

Quantum dots (QDs) are a promising class of fluorescent probes that can be conjugated to a variety of specific cell antibodies. For this reason, simple, cheap and reproducible routes of QDs´s syntheses are the main goal of many researches in this field. The main objective of this work was to demonstrate the ability of QDs as biolabels for flow cell cytometry analysis. We have synthesized biocompatible water soluble CdS/Cd(OH)2 and CdTe/CdS QDs and applied them as fluorescent labels of hematologic cells. CdTe/CdS QDs was prepared using using a simple aqueous route with mercaptoacetic acid and mercaptopropionic acid as stabilizing agents. The resulting CdTe/CdS QDs can target biological membrane proteins and can also be internalized by cells. We applied the CdTe/CdS QDs as biolabels of human lymphocytes and compared the results obtained for lymphocytes treated and non-treated with permeabilizing agents for cell membranes. Permeabilized cells present higher fluorescence pattern than non permeabilized ones. We associated antibody A to the CdS/Cd(OH)2 QDs to label type A red blood cell (RBC). In this case, the O erythrocytes were used as the negative control. The results demonstrate that QDs were successfully functionalized with antibody A. There was a specific binding of QDs-antibody A to RBC membrane antigen only for A RBCs. We have also monitored QDs-hematologic cell interaction by using fluorescence microscopy. Our method shows that QDs can be conjugated to a variety of specific cell antibodies and can become a potential, highly efficient and low cost diagnostic tool for flow cell cytometry, very compatible with the lasers and filters used in this kind of equipments.

Efficient intracellular delivery of quantum dots (QDs) in living cells and elucidating the mechanism of the delivery are essential for advancing the applications of QDs to in vivo imaging and in vivo photodynamic therapy. Here, we demonstrate that clathrin-mediated endocytosis is the most dominant pathway for the delivery of peptide-conjugated QDs. We selected an insect neuropeptide, allatostatin (AST1), conjugated with CdSe-ZnS QDs, and investigated the delivery of the conjugate in living cells. We evaluated the contributions of clathrin-mediated endocytosis, receptormediated endocytosis, and charge-based cell penetration to the delivery of QD605-AST1 conjugates by flow cytometry and fluorescence video microscopy. The delivery was suppressed by ~57% in inhibiting phosphoinositide 3-kinase with wortmannin, which blocks the formation of clathrin-coated vesicles, and by ~45% in incubating the cells at 4°C. Also, we identified clathrin-mediated endocytosis by two-color experiment to find colocalization of QD560-labeled clathrin heavy-chain antibody and QD605-AST1. We further observed reduction of the galanin receptor-mediated delivery of QD605-AST1 by ~8% in blocking the cells with a galanin antagonist, and reduction of charge-based cell penetration delivery by ~30% in removing the positive charge in the peptide from arginine and suppressing the cell-surface negative charge from glycosaminoglycan.

To realize their full potential as intracellular imaging and sensing reagents, robust and efficient methods for the targeted cellular delivery of luminescent semiconductor quantum dots (QDs) must be developed. We have previously shown that QDs decorated with histidine-terminated polyarginine cell-penetrating peptides (CPP) are rapidly and specifically internalized via endocytosis by several mammalian cell lines with no cytotoxicity. Here we demonstrate the long-term intracellular stability and fate of these QD-peptide conjugates. We found that the QD-peptide conjugates remain sequestered within endolysosomal vesicles for up to three days after delivery. However, the CPP appeared to remain stably associated with the QD within these acidic vesicles over this time period. Hence, we explored a number of techniques to either actively deliver QDs directly to the cytosol or to facilitate the endosomal release of endocytosed QDs to the cytosol. Active methods (e.g., electroporation) delivered only modest amounts of QDs to the cytosol that appeared to form aggregates. Delivery of QDs using polymer-based transfection reagents resulted primarily in the endosomal sequestration of the QDs, although one commercial polymer tested delivered QDs to the cytosol but only after several days in culture and with a considerable degree of polymer-induced toxicity. Finally, a modular, amphiphilic peptide containing functionalities designed for cell penetration and vesicular membrane interaction demonstrated the ability to deliver QDs in a well-dispersed manner to the cytosol. This peptide mediated rapid QD uptake followed by a slower efficient endosomal release of the QDs to the cytosol that peaked at 48 hours post-delivery. Importantly, this QD-peptide conjugate elicited minimal cytotoxicity in two cell lines tested. A more detailed understanding of the mechanism of the peptide's uptake and endosomal escape attributes will lead to the design of further QD conjugates for targeted imaging and sensing applications.

Indium phosphide (InP) nanocrystals show similar absorbance and emission spectra to CdTe quantum dots, but unlike particles containing cadmium, may potentially be used in in vivo applications. However, the particles are more challenging to make water-soluble, show broader emission spectra than most quantum dots, and their behavior in living cells is largely unknown. In this work we solubilize InP nanocrystals with simple thiols (mercaptopropionic acid) and conjugate them to the neurotransmitter dopamine or the protein transferrin. Degree of uptake and labeling patterns of QDs alone, QD-dopamine, and QD-transferrin are compared in different cell lines and toxicity is evaluated using the sulforhodamine B (SRB) assay.

Cerium-doped lanthanum fluoride colloidal nanocrystals offer a way to improve radiation therapy through the enhanced absorption of high-energy photons. Lanthanum fluoride nanocrystals doped with 10% cerium and capped with oleic acid were synthesized in anhydrous methanol as platelets 3-6 nm in diameter and 1-3 nm thick. The nanocrystals were characterized by transmission electron microscopy and photoluminescence spectroscopy. Previously synthesized lanthanum fluoride nanocrystals doped with 10% cerium and capped with hydroxyl were used in radiation dose enhancement experiments that involved an incoming gamma flux from a 137Cs source and a FOX assay to measure absorbed energy. Possibility for lanthanide ions to be released into solution under gamma irradiation and to interfere with the assay was shown after the results were compared with the outcome of a similar previous experiment with the Fricke dosimeter solution. Finally, increased cell mortality of S. cerevisiae under gamma irradiation was observed in the presence of hydroxyl-capped lanthanum fluoride nanocrystals in the solution.

Semiconductor quantum dots (QDs) are widely used in different fields of bioscience and biotechnology due to their unique optical properties. QDs can be used as fluorescent markers for optical detection and monitoring of deeply located tumors in vivo after specific labeling achieved by conjugating of QDs with targeting molecules. In this work the possibilities of intravital tumor labeling with QDs and subsequent in vivo tumor imaging were estimated. The experiments were run on immunodeficient nu/nu mice bearing human breast carcinoma SKBR-3, overexpressing surface protein HER2/neu. We used quantum dots Qdot 705 ITK (Invitrogen, USA) linked to anti-HER2/neu 4D5 scFv antibody. Antibody scFv fragments as a targeting agent for directed delivery of fluorophores possess significant advantages over full-size antibodies due to their small size, lower cross-reactivity and immunogenicity. QDs were bound to 4D5 scFv by barnase-barstar system (bn-bst) analogous to the streptavidin-biotidin system. Whole-body images were obtained using diffuse fluorescence tomography (DFT) setup with low-frequency modulation and transilluminative configuration of scanning, created at the Institute of Applied Physics of RAS (Russia). DFT-results were confirmed ex vivo by confocal microscopy. We report the results of in vivo whole-body tumor imaging with QDs complexes as contrasting agents. Intravital images of QDs-labeled tumors were obtained using specific tumor cells targeting and fluorescence transilluminative imaging method, while "passive" QD-labeling failed to mark effectively the tumor.

Many studies have been done in order to verify the possible nanotoxicity of quantum dots in some cellular types. Protozoan pathogens as Trypanosoma cruzi, etiologic agent of Chagas¹ disease is transmitted to humans either by blood-sucking triatomine vectors, blood transfusion, organs transplantation or congenital transmission. The study of the life cycle, biochemical, genetics, morphology and others aspects of the T. cruzi is very important to better understand the interactions with its hosts and the disease evolution on humans. Quantum dot, nanocrystals, highly luminescent has been used as tool for experiments in in vitro and in vivo T. cruzi life cycle development in real time. We are now investigating the quantum dots toxicity on T. cruzi parasite cells using analytical methods. In vitro experiments were been done in order to test the interference of this nanoparticle on parasite development, morphology and viability (live-death). Ours previous results demonstrated that 72 hours after parasite incubation with 200 μM of CdTe altered the development of T. cruzi and induced cell death by necrosis in a rate of 34%. QDs labeling did not effect: (i) on parasite integrity, at least until 7 days; (ii) parasite cell dividing and (iii) parasite motility at a concentration of 2 μM CdTe. This fact confirms the low level of cytotoxicity of these QDs on this parasite cell. In summary our results is showing T. cruzi QDs labeling could be used for in vivo cellular studies in Chagas disease.

Nanoparticles, whose size is 1-100 nm, easily aggregate as their size becomes smaller. Therefore, it is difficult to produce solution in which nanoparticles are dispersed. We have, as a way to disperse aggregated particles, for example, a media-typed disperse machine. During the procedures, however, we have to deal with some complicating operations; separation of the media from the solution, the defacement of the media into the solution, and so on. Furthermore, it is not an effective method for particles whose size is less than 50 nm. We tried to find an easier and more effective method for producing solution in which we re-disperse aggregated nanoparticles to still smaller particles. The aggregated particles were put into a machine with a pinhole small needle valve, and they were re-dispersed by "sheering stress". The estimation of re-dispersion was carried out by the measurement of their size distribution and surface z-average. With the utility of the machine, the re-dispersions of aggregated particles were observed. Furthermore, the increase of the pressure and of the velocity of the flow caused the decrease of particle size, which makes the surface area larger and therefore the surface z-average larger. It become clear that it is possible to re-disperse aggregated nanoparticles by adding shearing stress. We can regulate shearing stress by controlling the pressure and flow, and therefore we can control the effectiveness and the yield.

The use of AgNP is becoming more and more widespread in biomedical field. But compared with the promising bactericidal function, other physiological effects of AgNP on cells are relatively scant. In this research, we propose quantitative phase microscopy (QPM) as a new method to study the degranulation, and AgNP-induced RBL-2H3 cell degranulation is studied as well. Firstly, HeLa cells as the cell control and PBS as the solvent control, we measured the cell volume and cross section profile (x-z plane) with QPM. The results showed that the volume and cross section profile changed only the RBL-2H3 cells exposed to calcium ionophore A23187, which demonstrates the validity of QPM in degranulation research. Secondly, 50μg/mL of AgNP was used instead of A23187, and the measurement of cell volume and cross section profile was carried out again. RBL-2H3 cell volume increased immediately after AgNP was added, and cross section profile showed that the cell surface became granulated, but HeLa cell was lack of that effect. Phase images obviously indicated the RBL-2H3 cell deformation. Thirdly, stained with Fluo-3/AM, intracellular calcium Ca²⁺]_i of single RBL-2H3 cell treated with AgNP was observed with fluorescent microscopy; incubated with AgNP for 20min, the supernatant of RBL-2H3 cells was collected and reacted with o-phthalaldehyde (OPA), then the fluorescent intensity of histamine-OPA complex was assayed with spectrofluorometer. The results of Ca²⁺]_i and histamine increase showed that degranulation of AgNP-induced RBL-2H3 cell occurred. So, the cell volume was used as a parameter of degranulation in our study and AgNP-induced RBL-2H3 cells degranulation was confirmed by the cell volume increment, cross section profile change, and [Ca²⁺]_i and histamine in supernatant increase.