Fluorescence fluctuation spectroscopy: an invaluable microscopy tool for uncovering the biophysical rules for navigating the nuclear landscape.

Nuclear architecture is fundamental to the manner by which molecules traverse the nucleus. The nucleoplasm is a crowded environment where dynamic rearrangements in local chromatin compaction locally redefine the space accessible toward nuclear protein diffusion. Here, we review a suite of methods based on fluorescence fluctuation spectroscopy (FFS) and how they have been employed to interrogate chromatin organization, as well as the impact this structural framework has on nuclear protein target search. From first focusing on a set of studies that apply FFS to an inert fluorescent tracer diffusing inside the nucleus of a living cell, we demonstrate the capacity of this technology to measure the accessibility of the nucleoplasm. Then with a baseline understanding of the exploration volume available to nuclear proteins during target search, we review direct applications of FFS to fluorescently labeled transcription factors (TFs). FFS can detect changes in TF mobility due to DNA binding, as well as the formation of TF complexes via changes in brightness due to oligomerization. Collectively, we find that FFS-based methods can uncover how nuclear proteins in general navigate the nuclear landscape.