We then used FCS to measure the diffusion characteristics of the mobile fractions of BZip domain, or HP1α labeled with mCerulean3. First, we determined the suitability of mCerulean3 as a fluorophore for FCS studies by analyzing the diffusion characteristics of both the purified mCerulean3, as well as the protein expressed in live cells, comparing the results to earlier reports for GFP and ECFP.40 Representative autocorrelation curves for mCerulean3 in solution and for the protein expressed in living cells are shown in Fig. 4(a), and the statistical analyses derived using Eq. (3) are shown in Table 2. Brownian (simple) diffusion provided the best fit for mCerulean3 in solution, and the diffusion coefficient for the purified mCerulean3 is the same (within experimental error) as that reported by others for purified EGFP and ECFP, and is consistent with a monomeric fluorophore.40 Moreover, when the diffusion coefficient was measured at different laser powers, no buildup of a triplet state for mCerulean3 was detected, which, if present could introduce anomalies in measurements of the diffusion coefficients.33 The results in Table 2 show the diffusion coefficients for purified mCerulean3 determined at laser powers ranging from 0.37 to 3.1 μW, demonstrating that there was no change in the measured diffusion coefficient at the laser powers used in live cells (less than 1.7 μW power at the objective). Next, autocorrelation curves for mCerulean3 expressed in living GHFT1 cells were acquired, and a representative one component simple anomalous diffusion fit for mCerulean3 in living cells () is shown in Fig. 4(a). The average diffusion coefficient was (Table 2), which is consistent with the rate determined for ECFP and GFP in the cytoplasm of living cells.40 This confirmed that mCerulean3, like ECFP and GFP, displays the diffusion characteristic of a monomer and therefore is suitable for FCS studies in live cells.