Single-Molecule Imaging to Visualize Fusion Protein Condensates Assembly on DNA Curtains

1. Imaging of EWS-FLI1 condensation formation on DNA Curtains

1. Open the imaging software, find and mark the positions of the 3 × 3 zig-zag patterns under the bright field.
2. Turn on the flow at 0.2 mL/min to stain the DNA with the double-stranded DNA dye for 10 min.
3. Dilute mCherry-EWS-FLI1 with the imaging buffer to the concentration of 100 nM in 100 µL in the tube.
4. Load the protein sample through the valve with a 100 µL glass syringe, and change the flow rate to 0.4 mL/min.
5. Turn on the 488 nm laser and pre-scan each region to check the DNA distribution state; select the region where the DNA molecules distribute evenly. Set the laser power to 10% for the 488 nm laser and 20% for the 561 nm laser. Use the power meter to measure the real laser power near the prism: 4.5 mW for the 488 nm laser and 16.0 mW for the 561 nm laser.
6. Image acquisition
   1. Start acquiring images at 2 s intervals with both 488 nm and 561 nm lasers simultaneously.
   2. Change the valve from manual to injection mode to let the imaging buffer flush the protein sample into the flow cell after 60 s.
      NOTE: This process will take ~30 s, and the field of view will be covered with mCherry signals as soon as the EWS-FLI1 proteins reach the flow cell.
   3. To remove free EWS-FLI1, keep washing the flowcell with the imaging buffer for 5 min with only the 561 nm laser switched on. Stop the flow and incubate at 37 °C for 10 min.
   4. Turn on the flow at 0.4 mL/min to let the DNA extend, and acquire images at 2 s intervals between different frames to obtain high-throughput data of EWS-FLI1 condensate formation.

2. Intensity analysis for mCherry-EWS-FLI1

1. Import the data as image sequences into ImageJ software, pick out the puncta at 25× GGAA sites in an 8×8 pixels square, and save the image in .tif format.
2. Calculate the intensity as the summation of the intensity in the 8×8 pixels square, including the whole puncta, and remove the background by subtracting the intensity of the background in an area of the same size near the 8×8 pixels square.