Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 27
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nature ; 579(7797): 41-50, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132689

RESUMO

Tremendous progress in nanotechnology has enabled advances in the use of luminescent nanomaterials in imaging, sensing and photonic devices. This translational process relies on controlling the photophysical properties of the building block, that is, single luminescent nanoparticles. In this Review, we highlight the importance of single-particle spectroscopy in revealing the diverse optical properties and functionalities of nanomaterials, and compare it with ensemble fluorescence spectroscopy. The information provided by this technique has guided materials science in tailoring the synthesis of nanomaterials to achieve optical uniformity and to develop novel applications. We discuss the opportunities and challenges that arise from pushing the resolution limit, integrating measurement and manipulation modalities, and establishing the relationship between the structure and functionality of single nanoparticles.

2.
Nano Lett ; 19(3): 1695-1700, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30721068

RESUMO

The mechanisms of exciton generation and recombination in semiconductor nanocrystals are crucial to the understanding of their photophysics and for their application in nearly all fields. While many studies have been focused on type-I heterojunction nanocrystals, the photophysics of type-II nanorods, where the hole is located in the core and the electron is located in the shell of the nanorod, remain largely unexplored. In this work, by scanning single nanorods through the focal spot of radially and azimuthally polarized laser beams and by comparing the measured excitation patterns with a theoretical model, we determine the dimensionality of the excitation transition dipole of single type-II nanorods. Additionally, by recording defocused patterns of the emission of the same particles, we measure their emission transition dipoles. The combination of these techniques allows us to unambiguously deduce the dimensionality and orientation of both excitation and emission transition dipoles of single type-II semiconductor nanorods. The results show that in contrast to previously studied quantum emitters, the particles possess a 3D degenerate excitation and a fixed linear emission transition dipole.

3.
Sci Rep ; 8(1): 15542, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341334

RESUMO

Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeeded, via rational mutagenesis, to obtain variants that formed either tetramers or monomers. We compare two approaches: one is based on the structural similarity between SAASoti and Kaede, which helped us to identify a single point mutation (V127T) at the protein's hydrophobic interface that leads to monomerization. The other is based on a chemical modification of amino groups of SAASoti with succinic anhydride, which converts the protein aggregates into monomers. Mass-spectrometric analysis helped us to identify that the modification of a single ε-amino group of lysine K145 in the strongly charged interface AB was sufficient to convert the protein into its tetrameric form. Furthermore, site-directed mutagenesis was used to generate mutants that proved to be either monomeric or tetrameric, both capable of rapid green-to-red photoconversion. This allows SAASoti to be used as a photoconvertible fluorescent marker for in vivo cell studies.


Assuntos
Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutagênese Sítio-Dirigida , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Aminoácidos/genética , Proteínas Luminescentes/química , Espectrometria de Massas , Proteínas Recombinantes/química
4.
J Chem Phys ; 148(20): 204201, 2018 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-29865842

RESUMO

Our paper presents the first theoretical and experimental study using single-molecule Metal-Induced Energy Transfer (smMIET) for localizing single fluorescent molecules in three dimensions. Metal-Induced Energy Transfer describes the resonant energy transfer from the excited state of a fluorescent emitter to surface plasmons in a metal nanostructure. This energy transfer is strongly distance-dependent and can be used to localize an emitter along one dimension. We have used Metal-Induced Energy Transfer in the past for localizing fluorescent emitters with nanometer accuracy along the optical axis of a microscope. The combination of smMIET with single-molecule localization based super-resolution microscopy that provides nanometer lateral localization accuracy offers the prospect of achieving isotropic nanometer localization accuracy in all three spatial dimensions. We give a thorough theoretical explanation and analysis of smMIET, describe its experimental requirements, also in its combination with lateral single-molecule localization techniques, and present first proof-of-principle experiments using dye molecules immobilized on top of a silica spacer, and of dye molecules embedded in thin polymer films.

5.
Mol Biol Cell ; 2018 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-29444956

RESUMO

We report a novel method, dual color axial nanometric localization by Metal Induced Energy Transfer (dcMIET), and combine it with Förster Resonant Energy Transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells (hMSCs). Our approach is based on Fluorescence-Lifetime-Imaging Microscopy (FLIM), and allows for precise determination of the 3D architecture of stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell-matrix mechanics. In addition to resolving nanometric structural details along the z-axis, we use FRET to gain precise information on the distance between actin and vinculin at focal adhesions.

6.
J Phys Chem Lett ; 8(23): 5751-5757, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29125299

RESUMO

This study focuses on the mechanism of fluorescence blinking of single carbon nanodots, which is one of their key but less understood properties. The results of our single-particle fluorescence study show that the mechanism of carbon nanodots blinking has remarkable similarities with that of semiconductor quantum dots. In particular, the temporal behavior of carbon nanodot blinking follows a power law both at room and at cryogenic temperatures. Our experimental data suggest that static quenching via Dexter-type electron transfer between surface groups of a nanoparticle plays a major role in the transition of carbon nanodots to off or gray states, whereas the transition back to on states is governed by an electron tunneling from the particle's core. These findings advance our understanding of the complex mechanism of carbon nanodots emission, which is one of the key steps for their application in fluorescence imaging.

7.
ACS Nano ; 11(12): 11839-11846, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-28921961

RESUMO

The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs), which are embedded in the nuclear envelope, control transport of macromolecules between the two compartments. Here, using dual-color metal-induced energy transfer (MIET), we determine the axial distance between Lap2ß and Nup358 as markers for the inner nuclear membrane and the cytoplasmic side of the NPC, respectively. Using MIET imaging, we reconstruct the 3D profile of the nuclear envelope over the whole basal area, with an axial resolution of a few nanometers. This result demonstrates that optical microscopy can achieve nanometer axial resolution in biological samples and without recourse to complex interferometric approaches.

8.
Nano Lett ; 17(5): 3320-3326, 2017 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-28440076

RESUMO

The biological process of the epithelial-to-mesenchymal transition (EMT) allows epithelial cells to enhance their migratory and invasive behavior and plays a key role in embryogenesis, fibrosis, wound healing, and metastasis. Among the multiple biochemical changes from an epithelial to a mesenchymal phenotype, the alteration of cellular dynamics in cell-cell as well as cell-substrate contacts is crucial. To determine these variations over the whole time scale of the EMT, we measure the cell-substrate distance of epithelial NMuMG cells during EMT using our newly established metal-induced energy transfer (MIET) microscopy, which allows one to achieve nanometer axial resolution. We show that, in the very first hours of the transition, the cell-substrate distance increases substantially, but later in the process after reaching the mesenchymal state, this distance is reduced again to the level of untreated cells. These findings relate to a change in the number of adhesion points and will help to better understand remodeling processes associated with wound healing, embryonic development, cancer progression, or tissue regeneration.


Assuntos
Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Nanoestruturas/química , Animais , Comunicação Celular , Linhagem Celular , Movimento Celular , Adesões Focais , Mesoderma/citologia , Camundongos
9.
J Phys Chem Lett ; 8(7): 1472-1475, 2017 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-28296418

RESUMO

Precise knowledge of the quantum yield is important for many fluorescence-spectroscopic techniques, for example, for Förster resonance energy transfer. However, to measure it for emitters in a complex environment and at low concentrations is far from being trivial. Using a plasmonic nanocavity, we measure the absolute quantum yield value of lipid-conjugated dyes incorporated into a supported lipid bilayer. We show that for both hydrophobic and hydrophilic molecules the quantum yield of dyes inside the lipid bilayer strongly differs from its value in aqueous solution. This finding is of particular importance for all fluorescence-spectroscopic studies involving lipid bilayers, such as protein-protein or protein-lipid interactions in membranes or direct fluorescence-spectroscopic measurements of membrane physical properties.

10.
Nano Lett ; 16(7): 4312-6, 2016 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-27243936

RESUMO

Photobleaching of fluorophores is one of the key problems in fluorescence microscopy. Overcoming the limitation of the maximum number of photons, which can be detected from a single emitter, would allow one to enhance the signal-to-noise ratio and thus the temporal and spatial resolution in fluorescence imaging. It would be a breakthrough for many applications of fluorescence spectroscopy, which are unachievable up to now. So far, the only approach for diminishing the effect of photobleaching has been to enhance the photostability of an emitter. Here, we present a fundamentally new solution for increasing the number of photons emitted by a fluorophore. We show that, by exposing a single SiO2 nanoparticle to UV illumination, one can create new luminescent centers within this particle. By analogy with nanodiamonds, SiO2 nanoparticles can possess luminescent defects in their regular SiO2 structure. However, due to the much weaker chemical bonds, it is possible to generate new defects in SiO2 nanostructures using UV light. This allows for the reactivation of the nanoparticle's fluorescence after its photobleaching.

11.
Nano Lett ; 16(1): 237-42, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26605640

RESUMO

Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.


Assuntos
Carbono/química , Imagem Molecular , Nanopartículas/química , Núcleo Celular/ultraestrutura , Corantes Fluorescentes/química , Microtúbulos/ultraestrutura , Pontos Quânticos/química
12.
Mol Biol Cell ; 26(22): 4087-99, 2015 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-26337388

RESUMO

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non-small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Família de Proteínas EGF/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/enzimologia , Animais , Células CHO , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Transformação Celular Neoplásica , Cricetulus , Família de Proteínas EGF/genética , Receptores ErbB/genética , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Microscopia Confocal , Mutação , Fosforilação , Agregados Proteicos , Inibidores de Proteínas Quinases , Multimerização Proteica , Transdução de Sinais
13.
Phys Chem Chem Phys ; 17(22): 14994-5000, 2015 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-25986857

RESUMO

We present the results of a comprehensive photoluminescence study of defect centres in single SiO2 nanoparticles. We show that the photo-physical properties of the luminescent centres strongly resemble those of single dye molecules. However, these properties exhibit a large variability from particle to particle due to the different local chemical environment around each centre of each particle. This variability provides new insight into the complex photo-physics of single quantum emitters embedded into a random chemical environment. Moreover, a better understanding of the fundamental mechanism of the photoluminescence of defect centres in SiO2 structure is paramount for their application as white-light sources, non-toxic labels for bio-imaging, or for combining them with metallic and semiconductor nanostructures.

14.
Nano Lett ; 14(10): 5656-61, 2014 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-25247753

RESUMO

Inorganic carbon nanomaterials, also called carbon nanodots, exhibit a strong photoluminescence with unusual properties and, thus, have been the focus of intense research. Nonetheless, the origin of their photoluminescence is still unclear and the subject of scientific debates. Here, we present a single particle comprehensive study of carbon nanodot photoluminescence, which combines emission and lifetime spectroscopy, defocused emission dipole imaging, azimuthally polarized excitation dipole scanning, nanocavity-based quantum yield measurements, high resolution transmission electron microscopy, and atomic force microscopy. We find that photoluminescent carbon nanodots behave as electric dipoles, both in absorption and emission, and that their emission originates from the recombination of photogenerated charges on defect centers involving a strong coupling between the electronic transition and collective vibrations of the lattice structure.

15.
Chemphyschem ; 15(4): 705-11, 2014 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-24478241

RESUMO

We present a new concept for measuring distance values of single molecules from a surface with nanometer accuracy using the energy transfer from the excited molecule to surface plasmons of a metal film. We measure the fluorescence lifetime of individual dye molecules deposited on a dielectric spacer as a function of a spacer thickness. By using our theoretical model, we convert the lifetime values into the axial distance of individual molecules. Similar to Förster resonance energy transfer (FRET), this allows emitters to be localized with nanometer accuracy, but in contrast to FRET the distance range at which efficient energy transfer takes place is an order of magnitude larger. Our technique can be potentially used as a tool for measuring intramolecular distances of biomolecules and complexes.

16.
Chem Soc Rev ; 43(4): 1263-86, 2014 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-24365864

RESUMO

While single-molecule fluorescence from emitters with high quantum efficiencies such as organic dye molecules can easily be detected by modern apparatus, many less efficient emission processes such as Raman scattering and metal luminescence require dramatic enhancement to exceed the single-particle detection limit. This enhancement can be achieved using resonant optical systems such as plasmonic particles or nanoantennas, the study of which has led to substantial progress in understanding the interaction of quantum emitters with their electromagnetic environment. This review is focused on the advances in measurement techniques and potential applications enabled by a deeper understanding of fundamental optical interaction processes occurring between single quantum systems on the nanoscale. While the affected phenomena are numerous, including molecular fluorescence and also exciton luminescence and Raman scattering, the interaction itself can often be described from a unified point of view. Starting from a single underlying model, this work elucidates the dramatic enhancement potential of plasmonic tips and nanoparticles and also the more deterministic influence of a Fabry-Pérot microresonator. With the extensive knowledge of the radiative behavior of a quantum system, insight can be gained into nonradiative factors as well, such as energy transfer phenomena or spatial and chemical configurations in single molecules.

17.
J Phys Chem Lett ; 5(7): 1198-202, 2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-26274471

RESUMO

Using a metal nanocavity, we measure absolute values of the photoluminescence quantum yield in a mixture of different types of chromophores (dye molecules and semiconductor nanocrystals). We show that measurements can be performed in an attoliter volume, both in liquid and solid phases, even if both types of chromophores absorb and emit light in the same spectral range. The method is based on recording photoluminescence decay curves of the chromophore mixture as a function of the cavity length. Changing the distance between the cavity mirrors modifies the local density of states of the electromagnetic field and thus, the radiative transition rate of the enclosed emitters. By extracting individual decay components, corresponding to the different types of the emitters, we determine their quantum yield values separately and simultaneously. The nanocavity-based method opens up new perspectives for studying quantum emitters in complex photophysical systems, for instance, multichromophoric thin films, fluorescent proteins, or dyes incorporated into a lipid bilayer.

18.
Nano Lett ; 13(3): 1348-51, 2013 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-23438062

RESUMO

Using our nanocavity-based method for precise determination of the absolute quantum yield values, we measure the quantum yield of each of the three different chromophores types with strongly overlapping absorption spectra in a multicolor solution, which is impossible to do by any other existing technique. By measuring the modification of the radiative rate of the chromophores at different cavity lengths, we extract the quantum yield of CdSe/ZnS semiconductor nanocrystals emitting at three different wavelengths and excited by a single wavelength in the multicolor solution. Using several detection channels, we show that only one measurement is needed for determining the quantum yield values of all the types of chromophores in the sample. The obtained results show a very good agreement with the values commonly observed using other techniques. The nanocavity-based method opens up new opportunities of determining fluorescence quantum yields in complex systems and can be used for a big variety of fluorescence studies where multichromophore samples are involved.

19.
Chemphyschem ; 14(3): 505-13, 2013 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-23335303

RESUMO

We present a new method for determining absolute values of quantum yield of luminescent emitters, which is based on the modification of the radiative transition of emitters within a tunable metallic nanocavity. The method presented is easy to set up and works without any calibration. It will thus be useful for all applications where absolute and calibration-free measurements of luminescence quantum yields are needed. Moreover, it requires only a minute amount of low-concentration fluorophore solution. We give a detailed description of the theory and data evaluation of the nanocavity measurements, and report experimental results for several common dyes in aqueous solution.

20.
Phys Rev Lett ; 108(16): 163002, 2012 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-22680716

RESUMO

We investigate the impact of rotational diffusion on the electrodynamic coupling of fluorescent dye molecules (oscillating electric dipoles) to a tunable planar metallic nanocavity. Fast rotational diffusion of the molecules leads to a rapidly fluctuating mode density of the electromagnetic field along the molecules' dipole axis, which significantly changes their coupling to the field as compared to the opposite limit of fixed dipole orientation. We derive a theoretical treatment of the problem and present experimental results for rhodamine 6G molecules in cavities filled with low and high viscosity liquids. The derived theory and presented experimental method is a powerful tool for determining absolute quantum yield values of fluorescence.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA