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Simultaneous AFM and FLIM imaging with s SiR-DNA reveals structural changes during DNA condensation in live cell nuclei

Poudel C, Curry N, et al. "Simultaneous AFM and FLIM imaging with s SiR-DNA reveals structural changes during DNA condensation in live cell nuclei" Biophysical Journal (2018) 114(3):1, 596A


 

We show that the use of fluorescence lifetime imaging (FLIM) on the cell-permeable DNA reporter dye SiR-DNA reports on the condensation state of DNA in the nucleus of live cells. As opposed to other probes, SiR-DNA offers the advantages of minimal toxicity, photostability, and the use of gentler far-red light illumination compared to alternative, UV sensitive probes. This permits single photon excitation and compatibility with super-resolution techniques and makes for a powerful probe for live-cell imaging. Using a variety of biological and chemical processes known to induce DNA condensation and structural modifications, we demonstrate that SiR-DNA FLIM informs on state changes in live nuclei without the conventional requirement for cell fixation with alternative probes. We verify our FLIM findings with simultaneous atomic force microscopy (AFM) measurements in the same field-of-view and monitor nuclear stiffness in response to treatments that affect DNA condensation. The acquisition of such multi-parametric biophysical data enables us to highlight and study differences between types of nuclear modifications and their secondary effects.