Photoswitching-Free FRAP Analysis with a Genetically Encoded Fluorescent Tag
Figure 5
Artifacts in FRAP arising from alteration in the temporal sampling rate at the intentional photobleach.
H2B spot FRAP was performed as in Fig. 2C, but now the photobleach profile immediately after the bleach was measured. With a GFP tag, the photobleach profile appears to extend a distance of up to 6 µm for a 0.5 µm radius bleach spot and could not be fitted with the conventional model for a photobleach profile [3] (A). This is an artifact due to an alteration in temporal sampling rates at the time of the photobleach imposed by our microscope software [9]. This effect is not observed with a TMR-HaloTag, where instead the photobleach profile returns to the normalized intensity of one within 3 um from the center of the photobleach and could be well fitted with the conventional model for a photobleach profile (B).