Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

• Published in: Proceedings of the National Academy of Sciences - PNAS 2016-10, Vol.113 (42), p.E6372-E6381
• Main Authors: Almassalha, Luay M., Bauer, Greta M., Chandler, John E., Gladstein, Scott, Cherkezyan, Lusik, Stypula-Cyrus, Yolanda, Weinberg, Samuel, Zhang, Di, Ruhoff, Peder Thusgaard, Roy, Hemant K., Subramanian, Hariharan, Chandel, Navdeep S., Szleifer, Igal, Backman, Vadim
• Format: Article
• Language: English
• Subjects: Animals; Binding sites; Biological Sciences; CHO Cells; Chromatin; Chromatin - chemistry; Cricetulus; DNA damage; HeLa Cells; Human Umbilical Vein Endothelial Cells; Humans; Macromolecular Substances - chemistry; Microscopy; Microscopy - methods; Molecular Imaging - methods; Organelles - chemistry; PNAS Plus; Spectrum analysis
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1608198113

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure–function relationship in live cells.