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. 2012 Aug 9;7(8):e42854. doi: 10.1371/journal.pone.0042854

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© 2012 Wu et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Captured images from a FRAP experiment in an NIH 3T3 fibroblast expressing GFP-VASP. The box shows the bleach spot. Scale bar, 1 µm. (B) Observed recovery with only slight apparent bleaching of cytoplasmic free molecules (see D) due to image capture. The excitation laser power was 4%. The solid curve is the fitting of the data to the model in Eq 9. The immobile fraction = 0.026 was estimated from a separate experiment (solid circles) in the same focal adhesion as described in the text. The value of was determined from the fluorescence values before and immediately after the bleach, = 1.3 s. The fitting yielded the parameters = 0.0902, = 0.0014, = 0.17 s⁻¹. (C) Observed recovery in the same focal adhesion from a second FRAP experiment with apparent bleaching of the free molecules. The excitation laser intensity was increased to 10%. The fluorescence is observed to go through a peak and then decrease due to bleaching caused by image capture. The fitting of the data to the model gave the parameters = 0.13, = 0.009, = 0.18 s⁻¹. The value of is very close to that estimated from the fitting in (B) thus validating the model. Solid triangles in (B) and (C) indicate the normalized initial intensity before the photobleaching. (D) Fluorescent intensity profile in the cytoplasm (free molecules) in experiment (B), which shows there is no detectable photobleaching of cytoplasmic molecules. (E) Fluorescent intensity profile of the cytoplasm (free molecules) in experiment (C) showing a clear decrease in the concentration due to pronounced bleaching. Fitting of the cytoplasmic intensity to Eq. 3 yields the bleaching parameter = 0.011, which is close to the value determined from the fitting in (C). The model for the cytoplasmic intensity was fit to 70 of the 80 seconds for which the data was collected (corresponding to 53 measurements); the first 10 seconds showed a significant deviation possibly due to deviations in focus.

Inline graphic — (A) Captured images from a FRAP experiment in an NIH 3T3 fibroblast expressing GFP-VASP. The box shows the bleach spot. Scale bar, 1 µm. (B) Observed recovery with only slight apparent bleaching of cytoplasmic free molecules (see D) due to image capture. The excitation laser power was 4%. The solid curve is the fitting of the data to the model in Eq 9. The immobile fraction = 0.026 was estimated from a separate experiment (solid circles) in the same focal adhesion as described in the text. The value of was determined from the fluorescence values before and immediately after the bleach, = 1.3 s. The fitting yielded the parameters = 0.0902, = 0.0014, = 0.17 s⁻¹. (C) Observed recovery in the same focal adhesion from a second FRAP experiment with apparent bleaching of the free molecules. The excitation laser intensity was increased to 10%. The fluorescence is observed to go through a peak and then decrease due to bleaching caused by image capture. The fitting of the data to the model gave the parameters = 0.13, = 0.009, = 0.18 s⁻¹. The value of is very close to that estimated from the fitting in (B) thus validating the model. Solid triangles in (B) and (C) indicate the normalized initial intensity before the photobleaching. (D) Fluorescent intensity profile in the cytoplasm (free molecules) in experiment (B), which shows there is no detectable photobleaching of cytoplasmic molecules. (E) Fluorescent intensity profile of the cytoplasm (free molecules) in experiment (C) showing a clear decrease in the concentration due to pronounced bleaching. Fitting of the cytoplasmic intensity to Eq. 3 yields the bleaching parameter = 0.011, which is close to the value determined from the fitting in (C). The model for the cytoplasmic intensity was fit to 70 of the 80 seconds for which the data was collected (corresponding to 53 measurements); the first 10 seconds showed a significant deviation possibly due to deviations in focus.