Research Papers

Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation—a case study for fluorescein

[+] Author Affiliations
Kevin Braeckmans, Barbara G. Stubbe, Katrien Remaut, Joseph Demeester, Stefaan C. De Smedt

Ghent University, Laboratory of General Biochemistry and Physical Pharmacy, Harelbekestraat 72, 9000 Ghent, Belgium

J. Biomed. Opt. 11(4), 044013 (August 25, 2006). doi:10.1117/1.2337531
History: Received December 23, 2005; Revised March 16, 2006; Accepted April 19, 2006; Published August 25, 2006; Online August 25, 2006
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In this study we examine the implications of excitation saturation on fluorescence recovery after photobleaching (FRAP) experiments. In particular we present both experimental and theoretical evidence that fluorescein, one of the most frequently used fluorophores in FRAP, does not always comply with the basic assumptions that are made in many FRAP models: an invariant bleaching illumination intensity distribution (BID) in combination with first-order photobleaching kinetics. High light intensity levels, which are typical for the photobleaching phase of FRAP experiments, can cause excitation saturation of fluorescein in the excited triplet state. We show by experiments and computer simulations that under such saturating conditions the higher-order diffraction maxima of the BID substantially contribute to the photobleaching process and can no longer be neglected. As a result, the bleached regions are larger than expected theoretically from the FRAP models. Although this effect is not always directly evident from the FRAP experiments, neglecting it may shift the calculated diffusion coefficient by as much as over one order of magnitude. We present a discussion on the implications of this saturation effect on various types of FRAP models.

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© 2006 Society of Photo-Optical Instrumentation Engineers

Citation

Kevin Braeckmans ; Barbara G. Stubbe ; Katrien Remaut ; Joseph Demeester and Stefaan C. De Smedt
"Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation—a case study for fluorescein", J. Biomed. Opt. 11(4), 044013 (August 25, 2006). ; http://dx.doi.org/10.1117/1.2337531


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