Streamlined histone-based fluorescence lifetime imaging microscopy (FLIM) for studying chromatin organisation

Changes in chromatin structure are key determinants of genomic responses. Thus, methods that enable such measurements are instrumental for investigating genome regulation and function. Here, we report further developments and validation of a streamlined method of histone-based fluorescence lifetime imaging microscopy (FLIM) that robustly detects chromatin compaction states in fixed and live cells, in 2D and 3D. We present a quality-controlled and detailed method that is simpler and faster than previous methods, and uses FLIMfit open-source software. We demonstrate the versatility of this chromatin FLIM through its combination with immunofluorescence and its implementation in immortalised and primary cells. We applied this method to investigate the regulation of chromatin organisation after genotoxic-stress and provide new insights into ATM's role in controlling chromatin structure independently of DNA damage. Collectively, we present an adaptable chromatin FLIM method for examining chromatin structure and establish its utility in mammalian cells.

Abstract licence: CC BY

1. 6.

   Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets · 2013

2. 26.

   Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM · 2012

38 more references, whose wording the paper’s licence keeps private.

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.