Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage

• Published in: Scientific reports Vol. 13; no. 1; p. 422
• Main Authors: Clancy, Ellen, Ramadurai, Siva, Needham, Sarah R., Baker, Karen, Eastwood, Tara A., Weinstein, Julia A., Mulvihill, Daniel P., Botchway, Stanley W.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 09.01.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 631/57/2267; 639/638/92; Cytoplasm; Deoxyribonucleic acid; DNA; DNA Damage; Fluorescence; Fluorescent Dyes; Green fluorescent protein; HEK293 Cells; HeLa Cells; Humanities and Social Sciences; Humans; multidisciplinary; Phosphorescence; Refractometry; Science; Science (multidisciplinary); Viscosity
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-022-26880-x

Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5–14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54–1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.