“Ratio FLIM” of CoroNaGreen. (A) Schematic illustration of the Ratio FLIM approach and the empirical separation of the photon distribution of the FL into two boxes (#1 from 1.12 to 1.37 ns and #2 from 1.37 to 11.52 ns), representing a fast and slow segment, respectively. The normalized photon ratio “R” is then expressed as the quotient of the photon counts of both segments as indicated. (B) Plot of the normalized photon ratio against the [Na⁺] (means ± SD; n = 18). Red line represents a sigmoidal fit (K_d =88.6; R² = 0.97). (C) Ratio FLIM of CoroNaGreen in a HEK cell, revealing changes in [Na⁺] in response to removal of extracellular K⁺ (green bar) for 1 min in the three compartments. Gray triangles show individual data points; the black line represents a smoothened trace (Savitzky-Golay Filter 5); the dotted line indicates the average baseline value. The red arrows point to the first data point (colored in red) that exceeds 2× the SD (as depicted by the gray areas) in each compartment. Note the latency in the response of the nucleus (24 s) and PNR (40 s) as compared with the cytosol.