The glass substrate is cleaned with piranha solution (NH4OH/H2O2/H2O) in 1:1:5 v/v/v/ for 60 minutes at 60℃ and modified the glass substrate with 3-aminopropyltriethoxysilane (APTES). After modification, we assembled multi-channel flow cells and flow in the purified lambda phage DNAs which adsorb and are stretched on the surface. Then YOYO-1 dyes in various concentrations in 10 mM HEPES buffer with 10 mM NaCl are flown in the channel under a fluorescence microscope. The binding of dyes to the DNA is visualized in situ under 473 nm laser excitation with a relatively low power density ~0.74 W/cm 2 to minimize dye photobleaching. Please see the Supporting Information for the detailed sample preparation, data treatment, DNA selecting methods, and data analysis processes. Other Supplementary Material for this manuscript includes the following: