Box 2 |.

Photoconversion time course guidelines

CRITICAL These criteria were established after extensive screening of each cell line and culturing condition. Similar screening should be done prior to establishing a photoconversion regimen for other experimental conditions.
Procedure – Timing 1 – 4 h
1. Open the 405 nm shutter and adjust laser power to respective intensity dependent on experimental conditions (see below). Laser intensity may vary by experiment or microscope.
2. Turn down all other laser lines to zero as they will not be in use during photoconversion.
3. Set the number of prebleach, bleach and postbleach intervals in the time course frame. Of note, these settings are dependent on experimental conditions and can be enhanced for optimization.
4. Set ROI and run experiment. Continue as needed until all ROI are photoconverted.
Experimental conditions	Non-adherent	3D spheroid	2D monolayer
405 nm laser intensity for photoconversion	5%	15%	10%
Repetitions	1	1	1
Prebleach interval	1	1	1
Bleach 3 – 5 sec interaction	1	2	3
Postbleach intervals	1	1	1