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

Area of Science:

• Cell Biology
• Genomics
• Microscopy

Background:

• Chromatin structure changes are crucial for genomic responses and genome regulation.
• Accurate measurement of chromatin compaction is essential for understanding genome function.
• Existing methods for measuring chromatin compaction can be complex and time-consuming.

Purpose of the Study:

• To develop and validate a streamlined, faster, and more robust method for measuring chromatin compaction states using histone-based fluorescence lifetime imaging microscopy (FLIM).
• To demonstrate the versatility and adaptability of the chromatin FLIM method in various cellular contexts and experimental setups.
• To apply the developed chromatin FLIM method to investigate chromatin organization changes after genotoxic stress and explore the role of ATM signaling.

Main Methods:

• Development and validation of a streamlined histone-based fluorescence lifetime imaging microscopy (FLIM) protocol.
• Utilizing FLIMfit open-source software for data analysis.
• Combining chromatin FLIM with immunofluorescence and applying it to fixed and live cells (2D and 3D), including immortalized and primary cells.

Main Results:

• The developed chromatin FLIM method is simpler, faster, and more robust than previous techniques.
• The method successfully detects chromatin compaction states in various cell types and conditions.
• Application of the method revealed insights into ATM's role in regulating chromatin structure independently of DNA damage following genotoxic stress.

Conclusions:

• The streamlined chromatin FLIM method provides an adaptable and efficient tool for examining chromatin structure in mammalian cells.
• This technique enhances the investigation of genome regulation, function, and response to cellular stress.
• The study establishes the utility of this advanced microscopy method in diverse biological research settings.