A perfusion chamber (Grace Bio-labs) was placed on top of the spin-coated plasmid DNA, and 50 μl of imaging buffer consisting of 5 nM YOYO-1 and 50 mM MEA (Sigma-Aldrich) was added to the sample. All samples were imaged using an olympus IX-83 wide field Microscope with a 150× TIRFM objective and an NA of 1.45 (Olympus). The samples were excited by a 200 mW 488 nm and 100 mW 640 nm diode laser (Olympus) using a quad band (405/488/561/635) dichroic filter. Emission was collected via a quad band (25 nm band pass 446/523/600/677) emission filter coupled to a Hamamatsu EM-CCD X2 camera.
Imaging was perfomed as described previously (20 (link),21 (link)). Briefly, for the collection of the plasmid images, the sample was first imaged with 640 nm laser light to detect the Atto647N dye for approximately 1000 frames (exposure time 30 ms). Subsequently, using TIRF microscopy, the YOYO-1 was bleached for 15 s using 488 nm laser light, followed by imaging for ∼2000 frames with 30 ms exposure time. Approximately 10–15 movies were recorded for randomly selected regions for each sample.
Imaging was perfomed as described previously (20 (link),21 (link)). Briefly, for the collection of the plasmid images, the sample was first imaged with 640 nm laser light to detect the Atto647N dye for approximately 1000 frames (exposure time 30 ms). Subsequently, using TIRF microscopy, the YOYO-1 was bleached for 15 s using 488 nm laser light, followed by imaging for ∼2000 frames with 30 ms exposure time. Approximately 10–15 movies were recorded for randomly selected regions for each sample.