Fig. 1

Fluorescent timer protein and workflow for Timer fluorescence data preprocessing using the TockyPrep package. A Diagram illustrating that Timer Blue fluorescence from the immature chromophore is excited by a 405 nm laser, and Timer Red fluorescence from the mature chromophore is excited by a 561 nm laser. These fluorescences are detected by different detectors during flow cytometric analysis. B Overview of the TockyPrep workflow, showing data setup using prep_tocky and preprocessing steps executed by timer_transform, which include timer thresholding, Timer fluorescence normalization, and trigonometric transformation