Fig. 4

Timer fluorescence normalization. This figure illustrates the challenges in Timer fluorescence data analysis: (1) the absence of a calibration standard for Timer Blue, (2) acquisition settings that induce signal bias between Timer Blue and Red fluorescence, and (3) the resultant skew in Timer Angle estimation due to these biases. To address these issues, the TockyPrep package includes a function for Timer fluorescence normalization. This function utilizes autofluorescence levels to estimate inherent signal biases and employs MAD to normalize differences between Blue and Red fluorescence, thereby enhancing the accuracy of Timer Angle calculations