Gregor Jung

Highly Fluorescent Silver Nanoclusters Stabilized by Glutathione: A Promising Fluorescent Label for Bioimaging

AbstractA panel of silver nanoclusters (Ag NCs) protected by glutathione has been produced by collecting them on a plastic surface during an interfacial etching process. Blue-green, yellow and red emitting Ag NCs with size smaller than 2 nm exhibited distinct fluorescence properties (both emission and lifetime). In particular, the yellow emitting Ag NCs were found to reach a very high quantum yield of over 60% with a monoexponential fluorescence lifetime. These labels show no bleaching and high photostability over time and a high stability for a wide range of pH values. Cytotoxicity tests demonstrated the viability in the presence of of these luminescent probes even at high concentration (1 mg/mL). Cell studies confirmed the uptake of Ag NCs in epithelial lung cancer cells by an endocytotic process. These results show the high potential of fluorescent noble metal nanoclusters for biolabeling and imaging as alternatives to the standard fluorescent probes such as quantum dots or organic dyes.

NIR-emitting fluorescent gold nanoclusters doped in silica nanoparticles

In this paper we describe the synthesis and the optimisation of a new family of fluorescent core–shell nanoparticle using protein-stabilised gold nanoclusters. Fluorescent gold nanoclusters (<2 nm) entrapped in bovine serum albumin (BSA) protein were loaded in a 100 nm-silica nanoparticle with an optimal concentration of 3% (w/w). These nanoparticles kept the fluorescence properties of the metal clusters with a high Stokes shift and an emission in the near infrared region (λ = 670 nm). They were fully characterized and showed a high monodispersity and stability over more than 5 months. Steady-state fluorescence and lifetime measurements indicate the role of silica as a protective host to improve the photostability and the chemical stability of the fluorescent compound. This new label was taken up in tumor lung cells and tracked by a confocal microscope to show the great potential of this biolabel for sensing and imaging.

Comparative Photostability Studies of BODIPY and Fluorescein Dyes by Using Fluorescence Correlation Spectroscopy

In single-molecule applications, the photostability of fluorescent molecules is a key parameter. We apply fluorescence correlation spectroscopy to compare the photostability of four fluorescein and four borondipyrromethene (BODIPY) dyes of similar structure but different triplet yields. The latter class of dyes are more stable. In the kinetic analysis the, diffusion and photobleaching are treated as competitive processes. Corrections, which account for saturation and for experimental artefacts, are achieved solely by using experimental data. Photobleaching is found to occur mainly through the first excited singlet state S(1), in contrast to previous findings.

Confocal microscopy of single molecules of the green fluorescent protein

Single molecule detection has been extended into life sciences by use of strongly fluorescent labels. The green fluorescent protein (GFP) as a self-fluorescent biomolecule has attracted considerable attention. Here, single molecules of the GFP-mutant Glu222Gln are immobilized in a polyvinylalcohol matrix and detected by confocal fluorescence microscopy. Although this mutant stabilizes one of both conformers of the wild-type GFP, the investigation of its fluorescence dynamics reveals strong signal fluctuations. This fluorescence behaviour is—at least partly—caused by reversible photochemical changes of the protein framework, that can relax into the fluorescent state on different timescales. Thus, this protein appears particularly appropriate for studying the microheterogeneity of the macromolecule GFP on a single molecule level.

Nanoparticles of anionic starch and cationic cyclodextrin derivatives for the targeted delivery of drugs

Starch was oxidized with TEMPO for the synthesis of water-soluble copolymers of glucuronic acid and glucose. The carboxylate groups of these copolymers were conjugated with pteroic acid as cell-specific ligand for targeting to cancer cells. Stable spherical nanoparticles (NPs) were formulated mixing aqueous solutions of the anionic copolymers and of a cationic thioether of β-cyclodextrin (β-CD). Particle size distributions of NPs were investigated with DLS as the function of the charge ratio of the constituents. The smallest and most uniform particles with a diameter of about 130 nm were generated at a charge ratio of anion/cation close to 1, preferably 1.2. Stabilities and particle size distributions of these starch NPs were very satisfactory. The starch/β-CD NPs could be loaded with hydrophobic guest molecules like 1,4-dihydroxyanthraquinone (DHA), which served as a model for the important class of anthracycline antibiotics used in cancer therapy.

Mutagenic Stabilization of the Photocycle Intermediate of Green Fluorescent Protein (GFP)

The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited‐state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild‐type(wt)‐GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.

Two-color fluorescence correlation spectroscopy of one chromophore: Application to the E222Q mutant of the green fluorescent protein

Fluorescence correlation spectroscopy (FCS) is an important method for investigations of diffusion processes as well as of photophysical properties of fluorescing molecules. It has lately been applied in studies of the photodynamics of the green fluorescent protein (GFP). In this case FCS yields valuable information about the population of dark, non-fluorescing states of the molecule. For three-level systems rate constants into and out of the dark state can easily be determined with FCS. This task however becomes significantly more complex for molecules that possess several dark states. Here we present two-color FCS with simultaneous two-color excitation as a method that also yields spectroscopic information about the dark states. This makes the complete analysis of a molecular four-level system possible. The analysis of the GFP mutant E222Q is given as an example of two-color FCS that is readily applicable to other molecules with photoconvertible dark states. For E222Q we determine all the rate constants...

Visualization of Cu2+ uptake and release in plant cells by fluorescence lifetime imaging microscopy

A principal objective in life sciences is the visualization of biochemical processes. Fluorescence‐based techniques are widely used to demonstrate transport of relevant substances across cellular membranes. In this paper we report a novel noninvasive, real‐time fluorescence lifetime imaging microscopy method for visualizing uptake and release of divalent copper ions (Cu2+) in vivo. For this purpose, we employed a green fluorescent protein (GFP) form able to change its fluorescence lifetime upon Cu2+ binding. We demonstrate that this technique is selective for Cu2+. We show the reversible decrease of the fluorescence lifetime of GFP from 2.2 to 1.6 ns in Escherichia coli and from 1.8 to 1.3 ns in root cells of Arabidopsis after the addition of Cu2+. Cu2+ uptake of epidermal tobacco cells leads to a drop of the GFP lifetime from 2.5 to 2.2 ns. In summary, the spatially resolved visualization of Cu2+ distribution in vivo is demonstrated in prokaryote and eukaryote cells.

Cellular delivery of polynucleotides by cationic cyclodextrin polyrotaxanes

Cationic polyrotaxanes, obtained by temperature activated threading of cationic cyclodextrin derivatives onto water-soluble cationic polymers (ionenes), form metastable nanometric polyplexes with pDNA and combinations of siRNA with pDNA. Because of their low toxicity, the polyrotaxane polyplexes constitute a very interesting system for the transfection of polynucleotides into mammalian cells. The complexation of Cy3-labeled siRNA within the polyplexes was demonstrated by fluorescence correlation spectroscopy. The uptake of the polyplexes (red) was imaged by confocal fluorescence microscopy using the A549 cell line as a model (blue: nuclei, green: membranes). The results prove the potential of polyrotaxanes for further investigations involving knocking down genes of therapeutic interest.

Ultrafast Photoconversion of the Green Fluorescent Protein Studied by Accumulative Femtosecond Spectroscopy

The irreversible photoconversion of T203V green fluorescent protein (GFP) via decarboxylation is studied under femtosecond excitation using an accumulative product detection method that allows us to measure small conversion efficiencies of down to DeltaOD = 10(-7) absorbance change per pulse. Power studies with 800- and 400-nm pulse excitation reveal that excitation to higher states of the neutral form of the GFP chromophore induces photoconversion very efficiently. The singly excited neutral chromophore is a resonant intermediate of the two-step excitation process that leads to efficient photoconversion. We determine the dynamics of this two-step process by separating the excitation step of the neutral chromophore from the further excitation step to the reactive state in a time-resolved two-color experiment. The dynamics show that a further excitation to the very reactive higher excited state is only possible from the initially excited neutral chromophore and not from the fluorescent intermediate state. For applications of GFP in two-photon fluorescence microscopy, the found photochemical behavior implies that the high intensity conditions used in microscopy can lead to photoconversion easily and care has to be taken to avoid unwanted photoconversion.