FIG. 7.

2chFLIM images of τ₂ mode for bound NAD(P)H and free FAD signals and approximate calibration. (A, D) INS-1E cells cultured in medium with 11 mM glucose (“Glc11”); (B, E) after glucose addition to reach a final concentration of 25 mM (“Glc25”); (C, F) as (B, E) with 10 mM BTC. (G, H) Approximate calibration. Decreases in single-component resolved FAD emission decay (details in H), expressed by the linearized Stern-Volmer relationship (eq. {12}) when 1 mM FAD in glycerol was titrated by using 50 μM NAD⁺ aliquots (gray data and gray fit). A single decay component τ₀ was estimated in the absence of quencher (NAD⁺), whereas component τ corresponded to the studied NAD⁺ additions (H). Data in (G) (except of gray symbols) show the τ0₂^FAD from the FAD channel of 2chFLIM relatively to the decreasing τ₂^FAD. INS-1E cells were permeabilized by using digitonin (0.5–1 μg), whereas we measured and analyzed the FAD emission of either (i) natural cytosolic FAD content (circles, triangles); or (ii) added FAD (10 or 100 μM; squares). Parameter τ0₂^FAD was estimated first, before addition of NAD⁺ aliquots. Blue or red circles and black crosses: experimental data from Figure 8F: Blue circles—maximum acquired shift in τ₂^FAD on GSIS; red circles—an average shift in τ₂^FAD, both from the initial 11 mM glucose; black crosses—an average shift in τ₂^FAD from the initial 3 mM glucose. Shifts in x-axis show 10% and 20% elevations in NAD⁺ concentration. The data approximately fit to the Stern-Volmer relationship for dynamic quenching (eq. {12}), to which also our experimental τ₂^FAD data (lifetimes for free FAD) from Figure 8F fit well under the assumption that mitochondrial matrix NAD⁺ is around 800 μM and was 10% or 20% elevated on GSIS. Note also that if this NAD⁺ was overestimated and may be lower around 500 μM, a slightly different Stern-Volmer relationship (eq. {12}) still would be valid. 2chFLIM, two-channel fluorescence lifetime imaging microscopy. Color images are available online.