Determination of the photoconversion rate in cellulo

Before photoconversion, an image of a cell expressing (unconverted) LSSmOrange was acquired under 445-nm illumination (zooming factor of 1×, scanning speed of 10.6 nm/μs, scanning region of 512 × 512 pixels, and 4× line Kalman filter). This image was used to correct the expression level of the protein in different cells. LSSmOrange was converted by scanning the sample with a laser (405, 445, or 488 nm) at the laser power indicated. After every two scans with the photoconversion laser, fluorescence images of the converted LSSmOrange were acquired under 559-nm illumination (2× line Kalman filter). After background subtraction followed by correction of the expression level, time traces of the fluorescence intensity were subjected to a simple linear regression, and the slope of the fitting curve was defined as the photoconversion rate.