Fig. 2: Principle of FLIM data processing.

a Workflow to analyze fluorescence lifetime images by multiexponential reconvolution fitting. Live mammalian cells are transfected with the Riboglow reporter, a transfection marker, and loaded with the Riboglow probe. Time-Correlated Single Photon Counting (TCSPC) is used to acquire a FastFlim image using SymPhoTime 64 (Picoquant) for a specific region of interest (ROI) in live mammalian cells, resulting in the unfit raw lifetimes of each individual pixel. This data then undergoes a multiexponential reconvolution fitting, producing representative quantitative data such as lifetime values (τ₁ and τ₂) for further processing. The average fluorescence weighted lifetime value represents an average value for the entire ROI where each ROI is defined as an entire cell. This analysis is used for dot plots throughout this study. Alternatively, the component lifetime image yields the lifetime value on a pixel-by-pixel basis, useful for image representations. b Example of different lifetime representations for two cells with different variants of the Riboglow reporter. Lifetime values can be represented using a false-color scale for the component lifetime or average lifetime representation vs. intensity-based detection. A ROI was defined as the entire cell (estimated by a dotted line). Fluorescence intensity was quantified pixel-by-pixel and the background was subtracted. Lifetime of the representative cell is indicated. Scale bar = 10 µm. c A whole-cell was defined as an ROI and fluorescence intensity or fluorescence lifetime were extracted (6 independent experiments, 94 cells, 1 symbol = 1 cell). One-way ANOVA (95% confidence limit); post hoc test (Tukey HSD), ***p ≤ 0.001, ****p ≤ 0.0001. Error bars indicate mean and standard deviation (+/−SD).