(A, B) Integrated green intensity (I.I.) of IBs in the green channel and (B) mean green cytoplasmic intensity of mHtt(72Q)-mEos2–expressing cells after photoconversion was measured over the 4.5-h time course. Values were normalized to the initial value immediately following photoconversion. The graph shows mean intensity value ± SEM for 17 cells with IBs. Intensity values corrected for photobleaching (circles) and uncorrected for photobleaching (squares) are shown. The correction for photobleaching assumes that all green mHtt(72Q)-mEos2 present in the cell were exposed to every imaging cycle that occurred during the 4.5-h time course, that is, that all green mHtt(72Q)-mEos2 molecules were present for the entire time course. Therefore, the intensity values corrected for photobleaching are maximum possible intensity values. (C) Normalized intensity values for cellular green mHtt(72Q)-mEos2 when imaged 10 times as rapidly as possible; error bars indicate SEM. After 10 rapid cycles of imaging, the green cytoplasmic intensity is 0.54 ± 0.04 A.U. If no new mHtt(72Q)-mEos2 was synthesized, bleaching due to 28 rounds of imaging would be expected to cause the average cytoplasmic intensity to drop to ∼3.5% of its original value at most, considering only fluorescence loss due to cell division. However, it drops only slightly, to 84% of the original value. From the uncorrected intensity values, it is clear that the green mHtt(72Q)-mEos2 present in the cell is a mixed population which contains a substantial amount of newly synthesized green mHtt(72Q)-mEos2. Therefore, the true value lies between the corrected and uncorrected values.