Optimized Super-Resolution Imaging of Nuclear Sites in an Engineered Leukemia Cell Line

Stimulated emission depletion (STED) microscopy and Separation of Photons by Lifetime Tuning (SPLIT) provide optical super-resolution imaging, at the nanometer scale, on intact single cell nuclei. The resolution that can be achieved is limited by factors such as photobleaching and/or reduction of signal-to-noise, which depend on the conditions of image acquisition. Here we use image correlation spectroscopy (ICS) to quantify, in an unbiased way, the resolution and signal-to-noise of STED and SPLIT images and determine how they are affected by multiple parameters such as photobleaching, STED power, and number of averages. Imaging conditions optimization is particularly relevant when it comes to analyzing genomic processes, such as DNA replication, occurring within the crowded nuclear environment. As an application, we optimize the imaging conditions on an engineered cell line, U937-PR9, derived …