Dresden 2006 – scientific programme

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AKB: Biologische Physik

AKB 30: Poster Session I

AKB 30.6: Poster

Monday, March 27, 2006, 15:30–18:00, P1

Single molecule fluorescence imaging of the photoconverting fluorescent protein Kaede — •Stephan P. Schäfer¹, Eugene P. Petrov¹, Petra S. Dittrich², and Petra Schwille¹ — ¹Institute for Biophysics, Tatzberg 47, 01307 Dresden — ²Department of Miniaturization, Institute for Analytical Sciences, 44013 Dortmund

We investigated the photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature using single molecule wide-field fluorescence microscopy. Based on a highly sensitive low-noise CCD, fluorescence emission of single molecules was detected in two color channels (No-dqgreen/redNo-dq) as function of time. In order to address the noise present in the low-intensity images (i.e. read out noise, photonic shot noise, fluorescence background noise), an interactive MATLAB-based analysis algorithm was developed (incl. spectral channel selection, background reduction, spot labeling, fitting and classification). 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): First, the green-to-red photoconversion and second, the photoactivation of green fluorescence without emission of red fluorescence. Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation and partial revival of green fluorescence. The significantly lower green-to-red conversion ratio as compared to bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel.