RESUMO
Single-molecule (SM) fluorescence microscopy was used to investigate the photochromic fluorescent system spiropyran-merocyanine (SP â MC) interacting with gold nanoparticles (AuNPs). We observe a significant increase in the brightness of the emissive MC form, in the duration of its ON time, and in the total number of emitted photons. The spatial distribution of SMs with improved photophysical performance was obtained with 40 nm precision relative to the nearest AuNP. We demonstrate that even photochromic systems with poor photochemical performance for SM can become suitable for long time monitoring and high performance microscopy by interaction with metallic NP.
Assuntos
Benzopiranos/química , Corantes Fluorescentes/química , Ouro/química , Indóis/química , Nanopartículas Metálicas/química , Nitrocompostos/química , Fluorescência , Microscopia de FluorescênciaRESUMO
The morphological features of α-synuclein (AS) amyloid aggregation in vitro and in cells were elucidated at the nanoscale by far-field subdiffraction fluorescence localization microscopy. Labeling AS with rhodamine spiroamide probes allowed us to image AS fibrillar structures by fluorescence stochastic nanoscopy with an enhanced resolution at least 10-fold higher than that achieved with conventional, diffraction-limited techniques. The implementation of dual-color detection, combined with atomic force microscopy, revealed the propagation of individual fibrils in vitro. In cells, labeled protein appeared as amyloid aggregates of spheroidal morphology and subdiffraction sizes compatible with in vitro supramolecular intermediates perceived independently by atomic force microscopy and cryo-electron tomography. We estimated the number of monomeric protein units present in these minute structures. This approach is ideally suited for the investigation of the molecular mechanisms of amyloid formation both in vitro and in the cellular milieu.
Assuntos
Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Nanoestruturas/química , Multimerização Proteica , alfa-Sinucleína/química , Cor , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Estrutura Secundária de Proteína , Rodaminas/química , alfa-Sinucleína/metabolismoRESUMO
The behavior of a fluorophore near a gold nanoparticle is rationalized by a theoretical description of the parameters that modify the fluorescence emission: nanoparticle-fluorophore distance, fluorescence quantum yield (φ(0)), and fluorophore absorption and emission spectra, to find optimum conditions for designing fluorophore-nanoparticle probes. The theoretical maximum gain in brightness of the nanoparticle-fluorophore system with respect to the isolated molecule increases almost inversely proportional to φ(0). The brightness enhancement in imaging experiments in vitro was assessed by using Au-SiO(2) core-shell nanoparticles deposited on glass. A ~13-fold emission brightness enhancement for weakly fluorescent molecules was observed. A significant increase in fluorophore photostability, rendering longer imaging times, was obtained for fluorophores interacting with gold nanoparticles incorporated by endocytosis in cells. Our results illustrate a way to increase imaging times and to study molecules in the vicinity of a metallic nanoparticle after photobleaching of background fluorescence.
Assuntos
Corantes Fluorescentes/química , Ouro/química , Nanopartículas Metálicas/química , Microscopia de Fluorescência/métodos , Animais , Células Cultivadas , Melanóforos/citologia , Dióxido de Silício/química , Xenopus laevisRESUMO
Quantum dots multifunctionalized with the amyloid protein alpha-synuclein act at nanomolar concentrations as very potent inducers of the aggregation of micromolar-millimolar bulk concentrations of the protein in vitro and in cells. Fibrillation in live cells, a process diagnostic of Parkinson's disease, is accelerated up to 15-fold with only approximately 100 nanoparticles. The combination with a tetracysteine-tagged form of alpha-synuclein specific for fluorogenic biarsenicals constitutes a very sensitive system for studying pathological amyloid formation in cells.