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Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede.
Microsc Res Tech 2006; 69(3):210-9MR

Abstract

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.

Authors+Show Affiliations

Institute of Biophysics/BIOTEC, TU Dresden, Tatzberg 47-51, D-01307 Dresden, Germany.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16538628

Citation

Schäfer, S P., et al. "Single Molecule Fluorescence Imaging of the Photoinduced Conversion and Bleaching Behavior of the Fluorescent Protein Kaede." Microscopy Research and Technique, vol. 69, no. 3, 2006, pp. 210-9.
Schäfer SP, Dittrich PS, Petrov EP, et al. Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede. Microsc Res Tech. 2006;69(3):210-9.
Schäfer, S. P., Dittrich, P. S., Petrov, E. P., & Schwille, P. (2006). Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede. Microscopy Research and Technique, 69(3), pp. 210-9.
Schäfer SP, et al. Single Molecule Fluorescence Imaging of the Photoinduced Conversion and Bleaching Behavior of the Fluorescent Protein Kaede. Microsc Res Tech. 2006;69(3):210-9. PubMed PMID: 16538628.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede. AU - Schäfer,S P, AU - Dittrich,P S, AU - Petrov,E P, AU - Schwille,P, PY - 2006/3/16/pubmed PY - 2006/6/9/medline PY - 2006/3/16/entrez SP - 210 EP - 9 JF - Microscopy research and technique JO - Microsc. Res. Tech. VL - 69 IS - 3 N2 - Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins. SN - 1059-910X UR - https://www.unboundmedicine.com/medline/citation/16538628/Single_molecule_fluorescence_imaging_of_the_photoinduced_conversion_and_bleaching_behavior_of_the_fluorescent_protein_Kaede_ L2 - https://doi.org/10.1002/jemt.20283 DB - PRIME DP - Unbound Medicine ER -