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Titolo:
Visualising individual green fluorescent proteins with a near field optical microscope
Autore:
Garcia-Parajo, MF; Veerman, JA; Segers-Nolten, GMJ; de Grooth, BG; Greve, J; van Hulst, NF;
Indirizzi:
Univ Twente, Dept Appl Phys, Appl Opt Grp, NL-7500 AE Enschede, Netherlands Univ Twente Enschede Netherlands NL-7500 AE 500 AE Enschede, Netherlands Univ Twente, MESA Res Inst, NL-7500 AE Enschede, Netherlands Univ Twente Enschede Netherlands NL-7500 AE 500 AE Enschede, Netherlands
Titolo Testata:
CYTOMETRY
fascicolo: 3, volume: 36, anno: 1999,
pagine: 239 - 246
SICI:
0196-4763(19990701)36:3<239:VIGFPW>2.0.ZU;2-9
Fonte:
ISI
Lingua:
ENG
Soggetto:
SINGLE MOLECULES; FORCE MICROSCOPY; BEHAVIOR; DNA;
Keywords:
single molecule detection (SMD); green fluorescent proteins (GFP); near field scanning optical microscopy (NSOM);
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
32
Recensione:
Indirizzi per estratti:
Indirizzo: Garcia-Parajo, MF UnivtherlandsDept Appl Phys, Appl Opt Grp, POB 217, NL-7500 AE Enschede, Ne Univ Twente POB 217 Enschede Netherlands NL-7500 AE Ne
Citazione:
M.F. Garcia-Parajo et al., "Visualising individual green fluorescent proteins with a near field optical microscope", CYTOMETRY, 36(3), 1999, pp. 239-246

Abstract

The use of the green fluorescence protein (GFP) as an individual marker for applications in molecular biology requires detailed understanding of its photophysical and photodynamical properties. We investigated individual S65T mutants of GFP both on a glass surface and embedded in a water-pore gel. An aperture-type near field scanning optical microscope (NSOM) with two polarisation detection channels was applied to afford high spatial (approximate to 70 nm) and temporal (0.5 ms) resolution. Shear-force and near field fluorescence imaging were performed simultaneously, allowing direct correlation between topographic and optical features. Polarisation data showed that the emission dipole moment of the proteins is fixed in space within both the barrel structure of the protein and the gel matrix used for spatial confinement of the proteins. The photophysical behaviour of the S65T-GFP mutantswas monitored in time, with 500-mu s real-time resolution and continuous imaging for periods of more than 2 h. Our results show the reversible on-offbehaviour on a time scale that spans from 10(-4) to 10(3) s. Even a process generally identified as "bleaching" turns out to be reversible if a sufficient long observation time is allowed. As such, the photodynamics of individual GFPs appear to be much more complex than the properties deduced from ensemble-averaged measurements. Cytometry 36:239-246, 1999. (C) 1999 Wiley-Liss, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 04/12/20 alle ore 13:26:41