Zhao et al. reply:

Thank you for your comment1 on our recent paper on single-molecule imaging of transcription factor binding using reflected light-sheet microscopy (RLSM)2. Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) have both been extremely useful in the study of in vivo dynamics of a wide range of biomolecules in mammalian cells, and it is certainly not our intention to discredit their utility. However, we maintain that the single-molecule tracking (SMT) approach made possible by our RLSM technique does possess the advantage of being able to directly measure transcription factor dynamics without the need for additional calibrations or corrections that are commonly associated with FRAP and other techniques3. Owing to difficulties in analysis, the values reported on glucocorticoid receptor residence time using FRAP and FCS techniques differ in previous publications4,5,6. It is encouraging to know that now, after continuous improvement of analysis procedures over the years7, a consistent set of values has been obtained for the residence time and bound fraction of glucocorticoid receptor (as well as p53) by these three rather different techniques1,6. Therefore, it is foreseeable that SMT, FRAP and FCS, as a set of self-consistent and complementary techniques, will generate new insights into the in vivo dynamics of transcription factors as well as other key molecular players in mammalian nuclei for years to come.