Biomedical subjects

F Beltram

Publications and source records attributed to F Beltram.

17 recordsLinked to original sources

PubMed2005

Selective fluorescence recovery after bleaching of single E2GFP proteins induced by two-photon excitation.

We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.

Animals↗

PubMed2004

Multiphoton switching dynamics of single green fluorescent proteins.

Multi-photon driven photo-switching between dark and bright (fluorescent) states of a green fluorescent protein (GFP) mutant is demonstrated. A single-protein investigation shows the existence of two distinct bright states that display sharp two-photon cross-section bands peaked at 780 nm and at 870 nm. Fluorescence of these two species can be independently switched on and off. These results highlight a new photoconversion pathway for photochromic GFPs and can have significant applications in multi-photon confocal microscopy and in optical data-storage architectures.

Green Fluorescent Proteins↗

PubMed2004

Tailoring Josephson coupling through superconductivity-induced nonequilibrium.

The distinctive quasiparticle distribution existing under nonequilibrium in a superconductor-insulator-normal metal-insulator-superconductor mesoscopic line is proposed as a novel tool to control the supercurrent intensity in a long Josephson weak link. We present a description of this system in the framework of the diffusive-limit quasiclassical Green-function theory and take into account the effects of inelastic scattering with arbitrary strength. Supercurrent enhancement and suppression, including a marked transition to a pi junction, are striking features leading to a fully tunable structure.

Journal Article↗

PubMed2001

Resonant transport in Nb /GaAs /AlGaAs heterostructures: realization of the de Gennes-Saint-James model.

Resonant transport is demonstrated in a hybrid superconductor-semiconductor heterostructure junction grown by molecular beam epitaxy on GaAs. This heterostructure realizes the model system introduced by de Gennes and Saint-James in 1963 [P. G. de Gennes and D. Saint-James, Phys. Lett. 4, 151 (1963)]. At low temperatures a single marked resonance peak is shown superimposed to the characteristic Andreev-dominated subgap conductance. The observed magnetotransport properties are successfully analyzed within the random matrix theory of quantum transport, and ballistic effects are included by directly solving the Bogoliubov-de Gennes equations.

Journal Article↗

PubMed2001

Visualization of in vivo direct interaction between HIV-1 TAT and human cyclin T1 in specific subcellular compartments by fluorescence resonance energy transfer.

Human cyclin T1, a component of the P-TEFb kinase complex, was originally identified through its biochemical interaction with the Tat transactivator protein of human immunodeficiency virus type 1 (HIV-1). Current understanding suggests that binding of Tat to P-TEFb is required to promote efficient transcriptional elongation of viral RNAs. However, the dynamics and the subnuclear localization of this process are still largely unexplored in vivo. Here we exploit high resolution fluorescence resonance energy transfer (FRET) to visualize and quantitatively analyze the direct interaction between Tat and cyclin T1 inside the cells. We observed that cyclin T1 resides in specific subnuclear foci which are in close contact with nuclear speckles and that Tat determines its redistribution outside of these compartments. Consistent with this observation, strong FRET was observed between the two proteins both in the cytoplasm and in regions of the nucleus outside of cyclin T1 foci and overlapping with Tat localization. These results are consistent with a model by which Tat recruits cyclin T1 outside of the nuclear compartments where the protein resides to promote transcriptional activation.

Animals↗

PubMed2001

Coherent dynamics of photoexcited green fluorescent proteins.

The coherent dynamics of vibronic wave packets in the green fluorescent protein is reported. At room temperature the nonstationary dynamics following impulsive photoexcitation displays an oscillating optical transmissivity pattern with components at 67 fs (497 cm(-1)) and 59 fs (593 cm(-1)). Our results are complemented by ab initio calculations of the vibrational spectrum of the chromophore. This analysis shows the interplay between the dynamics of the aminoacidic structure and the electronic excitation in the primary optical events of green fluorescent proteins.

Green Fluorescent Proteins↗

PubMed2000

The enhanced green fluorescent protein as a tool for the analysis of protein dynamics and localization: local fluorescence study at the single-molecule level.

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein-protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64-->Leu, Ser-65-->Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.

Base Sequence↗

PubMed1996