| PhD. Gražvydas Lukinavičius, Department of NanoBiophotinics, Max Plank Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany|
Live-cell compatible fluorescent probes for chromatin nanoscopy
PhD. Gražvydas Lukinavičius
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
One of the most intriguing and challenging structures to image in the cell is chromatin – information carrying biopolymer, composed of DNA, RNA and proteins. Our probes consist of DNA binder – Hoechst conjugated to a series of fluorescent dyes compatible with live-cell fluorescence nanoscopy methods. A minimal change in the attachment point of the dye has dramatic effects on the properties of the final probe. All tested 6′-carboxyl dye-containing probes exhibited dual-mode binding to DNA and formed a dimmer complex at high DNA concentrations. The 5′-carboxyl dye-containing probes exhibited single-mode binding to DNA which translated into increased brightness and lower cytotoxicity. Up to 10-fold brighter nuclear staining by the newly developed probes allowed acquisition of stimulated emission depletion (STED) nanoscopy images of outstanding quality in living and fixed cells. Therefore we were able to estimate a diameter of ∼155 nm of the heterochromatin exclusion zones in the nuclear pore region in living cells. Finally, we have extended this approach to hydroxymethyl silicon-rhodamine and Hoechst conjugates which allowed single molecule localization microscopy imaging of heterochromatin in living cells at sub-30 nm resolution.