1. Start the Nanoimager and let the temperature stabilize to avoid drifts during acquisition. The stable microscope temperature is 6-7 ºC higher than room temperature (RT). To reach this stage quickly enter the desired (RT+ 6 ºC) in the temperature control panel and enable control. It will take roughly 1 to 2h to reach the desired temperature.
2. Channel Mapping: Focus on bead slide and run channel mapping. More details here.
3. Focus on your sample at low laser powers. For particles in solution, you may have to go 2-3 um above the coverslip to avoid the ones attaching to the surface.
4. Set a good Optical control and TIRF angle - changing Illumination modes (TIRF or HILO or epifluorescence). Usually go from 40 degrees (HILO) up to ~53 degrees (TIRF).
5. Z lock - Set focus reference and press Zlock.
6. Use low laser power for the acquisition. Start as low a possible and do small increases until the signal-to-noise ratio is decent without having photobleaching of the fluorophore.
7. Acquisition settings: Enter exposure and number of frames. This will depend on the dynamic process to be followed. For particles moving fast, use 10-20 ms and higher power. For particles moving slowly, something between 30-50 ms and 10000 frames per channel is a good starting point.