Minimizing the Impact of Photoswitching of Fluorescent Proteins on FRAP Analysis

Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, Maryland, USA.
Biophysical Journal (Impact Factor: 3.97). 04/2012; 102(7):1656-65. DOI: 10.1016/j.bpj.2012.02.029
Source: PubMed


Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring the mobility of fluorescently tagged proteins in living cells. Although FRAP presumes that high-intensity illumination causes only irreversible photobleaching, reversible photoswitching of many fluorescent molecules, including GFP, can also occur. Here, we show that this photoswitching is likely to contaminate many FRAPs of GFP, and worse, the size of its contribution can be up to 60% under different experimental conditions, making it difficult to compare FRAPs from different studies. We develop a procedure to correct FRAPs for photoswitching and apply it to FRAPs of the GFP-tagged histone H2B, which, depending on the precise photobleaching conditions exhibits apparent fast components ranging from 9-36% before correction and ∼1% after correction. We demonstrate how this ∼1% fast component of H2B-GFP can be used as a benchmark both to estimate the role of photoswitching in previous FRAP studies of TATA binding proteins (TBP) and also as a tool to minimize the contribution of photoswitching to tolerable levels in future FRAP experiments. In sum, we show how the impact of photoswitching on FRAP can be identified, minimized, and corrected.

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    • "Photoswitching dominates for low-power laser bleaching (Mueller et al. 2012); therefore, its role in FRAP measurements done with photosynthetic proteins using 1-s long bleach of high power is rather minimal. A higher laser power during bleaching period eliminates the effect of reversible chromophore photoswitching, but on the other hand, it increases the possibility of some other FRAP's artifacts such as photo-induced proteins crosslinking and membrane photodamage. "
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