Supplementary Figure 5: Acceptor fluorescence was not directly influenced by the change in ionic strength, and change in objective focus during solution exchange had a negligibly impact on MCS FRET measurement.

(a) Change of donor intensity (green), acceptor intensity (red) under 488 nm excitation and acceptor intensity (blue) under 561 nm excitation of MCS⁺-containing membrane lawns exposed to solution of increasing ionic strengths. (b) The acceptor/ donor (R/G) ratio under 488 excitation at the corresponding ionic strengths. Images were taken sequentially under 488 nm and 561 nm excitation at each ionic strength. Note that the directly excited acceptor (blue) was unaffected by the ionic strength of the solution and did not mirror the intensity change of donor acceptor under 488 nm excitation. This suggested that the acceptor fluorescence was not directly influence by the ionic strength of the solution. Data are representative of n = 5 biological replicates. (c-d) Solution exchange during ionic strength titration experiments could cause a shift in focus that potentially affected R/G ratio values. Here, the maximal variations in R/G values (Max - Min) were determined by adding 300 μL of 10 mM PBS to 600 μL of 10 mM PBS. 6 reprehensive solution exchanges are shown in c, causing an average variation in R/G ratio of 7.5 ±1.0%. Data are mean and standard error of n = 10 measurements. (d) The impact of the loss of focus on R/G values was also directly measured by manually changing the objective focus up and down, causing a maximal change in R/G ratio of 11.8 ± 3.1%. Note the concurrent change of donor (green) and acceptor (red) intensity due to change in focus.