Fluorescence correlation spectroscopy using quantum dots: advances, challenges and opportunities

Semiconductor nanocrystals (quantum dots) have been increasingly employed in measuring the dynamic behavior of biomacromolecules using fluorescence correlation spectroscopy. This poses a challenge, because quantum dots display their own dynamic behavior in the form of intermittent photoluminescence, also known as blinking. In this review, the manifestation of blinking in correlation spectroscopy will be explored, preceded by an examination of quantum dot blinking in general.     

Quantitative in vitro demonstration of two-photon photodynamic therapy using photofrin and visudyne

Photodynamic therapy (PDT), the combined action of a photosensitizer and light to produce a cytotoxic effect, is an approved therapy for a number of diseases. At present, clinical PDT treatments involve one-photon excitation of the photosensitizer. A major limitation is that damage may be caused to healthy tissues that have absorbed the drug and lie in the beam path. Two-photon excitation may minimize this collateral damage, as the probability of absorption increases with the square of the light intensity, enabling spatial confinement of the photosensitizer activation. A potential application is the treatment of the wet-form of age-related macular degeneration, the foremost cause of central vision loss in the elderly. Herein, the commercial photosensitizers Visudyne and Photofrin are used to demonstrate quantitative in vitro two-photon PDT. A uniform layer of endothelial cells (YPEN-1) was irradiated with a Ti:sapphire laser (300 fs, 865 nm, 90 MHz) using a confocal scanning microscope. Quantification of the two-photon PDT effect was achieved using the permeability stain Hoechst 33258 and a SYTOX Orange viability stain. Visudyne was found to be around seven times more effective as a two-photon photosensitizer than Photofrin under the conditions used, consistent with its higher two-photon absorption cross-section. We also demonstrate for the first time the quadratic intensity dependence of cellular two-photon PDT. This simple in vitro method for quantifying the efficacy of photosensitizers for two-photon excited PDT will be valuable to test specifically designed two-photon photosensitizers before proceeding to in vivo studies in preclinical animal models.     

The development of direct multicolour fluorescence cross-correlation spectroscopy: towards a new tool for tracking complex biomolecular events in real-time

Direct three-colour fluorescence cross-correlation spectroscopy can reveal interactions between three fluorescently labelled biomolecules, giving insight toward the complex events that constitute signal transduction pathways. Here we provide the optical and theoretical basis for this technology and demonstrate its ability to detect specific biological associations between nanoparticle-labelled DNA molecules.     

Stability of a microwave heated fluid Layer

When a time harmonic electromagnetic wave impinges on a slaba certain portion of the wave creates heat within the slab throughdipolar and ohmic heating. The electrical and thermal propertiesof the material dictate the dynamical nature of the heatingprocess, as well as the steady-state temperature profile. Thematerial considered here is a slab of fluid. We consider thecase where the fluid is bounded by thin rigid layers of transparentmaterial. The steady-state heating profile governs the typesof convective motions that can occur and also affects the stabilitycharacteristics of temperature, pressure and velocity perturbationsintroduced in the slab. The main objective here is to examinesuch stability characteristics, initially in the linear regime.Both rigid-rigid and rigid-free configurations are considered.     

Nonblinking plasmonic quantum dot assemblies for multiplex biological detection

Nonblinking nanosystems are prepared by layer-by-layer polyelectrolyte deposition, which precisely controls the stoichiometry and the distance between quantum dots (QDs) and gold nanoparticles (GNPs). Conjugation of biorecognition molecules to these nanobarcodes enables cell targeting and entry with prolonged retention and minimal toxicity.     

A sponge-like luminescent coordination framework via an Aufbau approach

A luminescent mixed-metal coordination network with sponge-like sorption features is formed by stepwise assembly.     

Microporous metal-organic frameworks formed in a stepwise manner from luminescent building blocks

A series of trivalent lanthanides (Sm, Eu, Gd, Tb, Dy) have been complexed to the dianionic ligand, 4,4'-disulfo-2,2'-bipyridine-N,N'-dioxide, L, in a 3:1 ratio to form trianionic complex building blocks. These units were then cross-linked into a network solid by addition of BaCl2 to form mixed-metal networks of formula {Ba2(H2O)4[LnL3(H2O)2](H2O)nCl}infinity, Ln = Sm3+ (1), Eu3+ (2), Gd3+ (3), Tb3+ (4), Dy3+ (5). The networks were isostructural and contained open channels which readily absorbed and desorbed water accompanied by a spongelike shrinkage and expansion of the host. CO2 sorption measurements confirmed microporosity giving a DR surface area of 718 m2/gm and an average pore size of 6.4 A. Ligand L sensitized all the lanthanide ions with the exception of Gd3+. Studying the series of Ln complexes allowed the determination of the triplet state energy of L which is itself a new ligand for sensitization purposes. The luminescent properties of the lanthanide building blocks were retained in the porous network solid. From the luminescence data, it was possible to attribute the spongelike properties of the network to the Ba2+ coordination sphere rather than the Ln3+ center. Networks were characterized by X-ray crystallography, PXRD, DSC/TGA, water vapor and gas sorption, and luminescence spectroscopy.     

Photobleaching kinetics of Verteporfin and Lemuteporfin in cells and optically trapped multilamellar vesicles using two-photon excitation

Verteporfin and Lemuteporfin are compared to examine the effect of their functional groups and therefore the localization in two-photon excitation (TPE) photodynamic therapy (PDT). We used singlet oxygen-related photobleaching of the sensitizers to assess TPE-induced singlet oxygen generation in multilamellar vesicles (MLVs) and U343 glioma cells under a variety of conditions. It was found that Lemuteporfin photobleached at a faster rate than Verteporfin in the majority of environments. Also, Verteporfin and Lemuteporfin exhibited different behaviors when in hypoxic environments relative to those in oxygenated MLVs. These differences are attributed to the sensitizer location in the membrane and their relative mobilities throughout membranes and cells.     

The Adsorption of L -Tryptophan to the H2O–CCl4 Interface, Monitored by Red-Edge Two Photon-Induced Polarized Fluorescence

The adsorption of L-tryptophan to the H₂O–CCl₄ interface has been studied using the novel method of two photon-induced fluorescence polarization. The process of two-photon absorption allows excitation of a very small volume of molecules near an interface. In the instance where molecules maintain a preferential orientation at the interface, interface-specific phenomena are observed as a dependence of the absorption and emission processes on the polarization of the light. In this way, an isotherm for the adsorption of L-tryptophan from the bulk aqueous phase to the H₂O–CCl₄ interface was determined. Associated with this process is a change in free energy of kJ/mol. By comparing the free energy of adsorption in this study to that at the H₂O–CH₃(CH₂)₆CH₂OH interface, we find that L-tryptophan binds more readily to the H₂O–CCl₄ interface. Our polarization data suggest that the plane of the indolyl chromophore of tryptophan is oriented in a more parallel than perpendicular fashion to the liquid–liquid interface. Additionally, we present a mechanism for the observed two-photon absorption, 20 nm to the red of the traditional onset of the transition into the first excited singlet state of tryptophan in aqueous solution. This mechanism involves simultaneous hot-band transitions into the ¹L_a state and direct excitation into the triplet manifold.     

Near-infrared two-photon excitation of protoporphyrin IX: photodynamics and photoproduct generation

The spectroscopy and photochemistry of protoporphyrin IX in ethanol and in Triton X-100 micelle solution have been examined using near-infrared two-photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. We have determined that the photochemistry subsequent to TPE is very similar to that found for one-photon excitation. Moreover, the photoproducts observed possess very intense TPE fluorescence spectra, which allows their detection at low relative concentrations.