A Stroboscopic Approach for Fast Photoactivation-Localization Microscopy with Dronpa Mutants.
The photophysical properties and photoswitching scheme of the reversible photoswitchable green fluorescent protein-like fluorescent proteins Dronpa-2 and Dronpa-3 were investigated by means of ensemble and single-molecule fluorescence spectroscopy and compared to those of the precursor protein Dronp...
| Publicado en: | Journal of the American Chemical Society Vol. 129; no. 45; pp. 13970 - 13978 |
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| Autores principales: | , , , , , , , |
| Formato: | Artículo |
| Publicado: |
American Chemical Society
11/14/2007
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| Materias: | |
| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=hlh&AN=27630745&site=ehost-live header: @attributes: shortDbName: hlh uiTerm: 27630745 longDbName: Humanities International Complete uiTag: AN controlInfo: bkinfo: jinfo: jid: 00027863 ACS jtl: Journal of the American Chemical Society issn: 00027863 maglogo: N pubinfo: dt: 11/14/2007 vid: 129 iid: 45 pid: 997 pub: American Chemical Society artinfo: ui: 27630745 10.1021/ja074704l ppf: 13970 ppct: 8 formats: tig: atl: A Stroboscopic Approach for Fast Photoactivation-Localization Microscopy with Dronpa Mutants. aug: au: Flors, Cristina Hotta, Jun-ichi Uji-i, Hiroshi Dedecker, Peter Ando, Ryoko Mizuno, Hideaki Miyawaki, Atsushi Hofkens, Johan affil: Department of Chemistry and Institute for Nanoscale Physics and Chemistry (INPAC), Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium Laboratory for Cell Function and Dynamics, Brain Science institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan su: Stroboscopes Green fluorescent protein Fluorescence spectroscopy Fluorescent polymers Optical diffraction sug: subj: Stroboscopes Green fluorescent protein Fluorescence spectroscopy Fluorescent polymers Optical diffraction ab: The photophysical properties and photoswitching scheme of the reversible photoswitchable green fluorescent protein-like fluorescent proteins Dronpa-2 and Dronpa-3 were investigated by means of ensemble and single-molecule fluorescence spectroscopy and compared to those of the precursor protein Dronpa. The faster response to light and the faster dark recovery of the new mutants observed in bulk also hold at the single-molecule level. Analysis of the single-molecule traces allows us to extract the efficiencies and rate constants of the pathways involved in the forward and backward switching, and we find important differences when comparing the mutants to Dronpa. We rationalize our results in terms of a higher conformational freedom of the chromophore in the protein environment provided by the β-can. This thorough understanding of the photophysical parameters has allowed us to optimize the acquisition parameters for camera-based sub-diffraction-limit imaging with these photochromic proteins. We show that Dronpa and its mutants are useful for fast photoactivation-localization microscopy (PALM) using common wide-field microscopy equipment, as individual fluorescent proteins can be localized several times. We provide a new approach to achieve fast PALM by introducing simultaneous two-color stroboscopic illumination. pubtype: Academic Journal doctype: Article src: R language: English refInfo: copyright: @attributes: flag: Y dt: @attributes: year: 2007 holdings: @attributes: islocal: N |
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