Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize...

A fundamental understanding of gene regulation requires a quantitative characterization of the spatial organization and dynamics of chromatin. The advent of fluorescence super-resolution microscopy techniques such as photoactivated localization microscopy (PALM) presented a breakthrough to visualize structural features with a resolution of ~20 nm in fixed cells. However, until recently the long acquisition time of super-resolution images prevented high-resolution measurements in living cells due to spreading of localizations caused by chromatin motion. Here, we present a step-by step protocol for our recently developed approach for correlatively imaging telomeres with conventional fluorescence and PALM, in order to obtain time-averaged super-resolution images and dynamic parameters in living cells. First, individual single molecule localizations are assigned to a locus as it moves, allowing to discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. By subtracting the telomere trajectory from the localization of bound molecules, the motion blurring is then corrected, and high-resolution structural characterizations can be made. These structural parameters can also be related to local chromatin motion or larger scale domain movement. This protocol therefore improves the ability to analyze the mobility and time-averaged nanoscopic structure of locus-specific chromatin with single-molecule sensitivity.