Fluorescence and Phosphorescence Lifetime Imaging Microscopy Reveals a Significant Cell Nuclear Viscosity Changes upon Cell Damage

Session

Using FLIM and FCS to Determine Interactions and Dynamics

Authors

Prof Stanley Botchway (1)

Affiliations

1. UKRI-STFC

Keywords

DNA damage, GFP, mCherry, mNeonGreen, E.coli, viscosity, Refractive Index, FLIM, PLIM, multiphoton, Confocal, imaging

Abstract text

Cytoplasmic viscosity is a crucial parameter in determining diffusion, reaction rates and its changes are associated with several diseases. Nuclear viscosity determines gene integrity, regulation and expression and how drugs including DNA damaging agents affects this 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 µseconds, phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity changes and provides an excellent method to investigate the impact of DNA damage. Together with Fluorescence Lifetime Imaging (FLIM) we show the lifetime of both green and red fluorescent proteins (FP) are also sensitive to cellular viscosity and RI changes. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of µsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP. Lifetimes of live cells with Pt[L]Cl or expressing mCherry showed significant increase from 12.5 µs to 14.1 µs and from 1.54 ns to 1.67 ns respectively upon DNA damage. However, GFP showed a decrease in lifetime from 2.65 ns to 2.29 ns following DNA damage.   These values represent a viscosity change from 8.59 cP to 20.56 cP as well as RI significant changes. 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.   Using mNeonGreen FP over expression, bacterial cells showed a decrease viscosity change from 2.8 ns to 2.4 ns

References

1. Botchway  S,   Charnley   M,   Haycock  J,    Parker  A,   Rochester   D,   Weinstein  J,  and Williams  J.  (2008).   Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes  Proceedings of the National Academy of Sciences   105(42)  pp 16071-16076. Doi: 10.1073/pnas.0804071105 
2. Kuimova  M,   Botchway   S,   Parker  A,    Balaz  M,   Collins   H,   Anderson  H,    Suhling  K,  Ogilby   P.   (2009)  Imaging intracellular viscosity of a single cell during photoinduced cell death  Nature Chemistry  1(1)  69-73. doi: 10.1038/nchem.120.