## 3 Results and Discussion
In all these cases, the yield indicated about 50% occupation of the second layer, referring to the computational results shown in Figure 2. When particular \(\tau\) values of two-exponential decays are taken into account (Table S2), one may conclude that the average lifetime reduction mostly results from the change in amplitudes and, to a lesser extent, from the shortening of \(\tau\)1 or \(\tau\)2. Interestingly, the addition of a spacer did not reduce the energy transfer yield, and there was even a tendency to strengthen the interaction. The BSA presence almost did not change the transfer yield between the GFP layer and the mBanana layer and the mCanary layer and the mCherry layer; however, it slightly increased the yield of the sequential monolayers of GFP-mCherry (Figure 3A). With Jefamine, the transfer yield was significantly higher for all three pairs. For the GFP-mBanana, the calculated yield of 0.158 was higher than theoretically enabled for those two fluorophores. All this suggests that the Jefamine influence is behind the simple spacer effect and leads to the modification of protein properties. For the change in the acceptor lifetime, we were not formally able to calculate the FRET yield. However, we may simply observe the 8% increase in the mBanana fluorescence lifetime in the presence of a GFP layer (Figure 3B) and the 45% increase in the mCherry fluorescence lifetime in the presence of a GFP layer (Figure 3C). Again, the presence of BSA did not decrease FRET efficiency but slightly increased it, although in the range of standard deviation. Jefamine reduced the mBanana fluorescence lifetime below the control mBanana-Jefamine layer. Unexpected values were measured for the mCherry fluorescence lifetime, with an mBanana as a donor; here, the mCherry fluorescence lifetime was decreased significantly compared to the control mCherry-only (or mCherry-BSA/mCherry-Jefamine) monolayers. These results suggest that an mCherry is somehow quenched by an mBanana. Figure 2: FRET efficiencies calculated for a varied relative concentration of acceptor proteins. (**A**) GFP as the first layer, and mBanana as the second layer. (**B**) GFP as the first layer, and mCherry as the second layer, and (**C**) mBanana as the first layer, and mCherry as the second layer. As sequential monolayers are easily visualized using confocal microscopy, we applied a FLIM setup for the observation of the FRET process (Figure 4). We, indeed, found a shortening of the donor fluorescence lifetime, indicating energy transfer from a GFP to an mBanana and an mCherry and from an mBanana to an mCherry. Again, the average lifetime reduction mostly results from the change in amplitudes and not the \(\tau\)1 or \(\tau\)2 values (Table S3). Interestingly, the energy transfer yield differed from those calculated based on the Figure 2 data and was higher for the GFP-mBanana pair, reaching the maximum level, as predicted by MC computation. Again, the spacers did not clearly reduce the FRET yield. Figure 3: Mean fluorescence lifetime values of fluorescent protein immobilized on PVDF membrane, measured in a spectrofluorometer. Samples were excited by a 471 nm LED laser, and the emission wavelengths were 503 nm for GFP (**A**), 551 nm for mBanana (**B**), and 612 nm for mCherry (**C**). Decay curves were fitted to a two-exponential decay equation. Particular values of decays and amplitudes are presented in Supplementary Table S2. Statistical significance of differences (\(p\)-values) is presented in figures as * (\(p<0.5\)) and ** (\(p<0.01\)).