## 2 Materials and Methods
### Preparation and Characterization of Sequential Monolayers  
Small (1 cm \(\times\) 1 cm square) PVDF membrane pieces were activated in 70% ethanol and blocked in 1% BSA solution in 100 mM HEPES, pH 7.4. Fluorescent protein layers were immobilized on the membrane by crosslinking with EDC + NHS (0.2 mg EDC + 0.25 mg NHS per 100 mL of protein solution). The efficiency of immobilization was largely improved by prior blocking of a membrane in BSA, probably due to a higher number of functional groups (-NH2 and -COOH) in comparison to a mere PVDF surface. The concentration of proteins in the crosslinking solution was 20 \(\upmu\)M GFP, 200 \(\upmu\)M mBanana, and 40 \(\upmu\)M mCherry (in 100 mM HEPES, pH 7.4). These were chosen experimentally to provide appropriately high intensity of fluorescence.  
Additionally, two types of spacers were used to introduce separation distance between two protein layers: BSA (40 A \(\times\) 40 A \(\times\) 140 A) and 2,2'-(ethylenedioxy)bis(ethylamine) (Jeffamine, approx. 10 A spacer arm) in 200 \(\upmu\)M and 100 mM concentrations, respectively. Subsequent layers were immobilized on the membrane in the following order: protein I--spacer (optionally)--protein II. Conjugation of the second protein layer was less efficient (as determined using fluorescence intensity).  
Unequal immobilization efficiencies resulted in different amounts of proteins in two layers and \"saturation\" of protein molecules as FRET acceptors or donors in the second layer. Measurement of fluorescence lifetime, therefore, was performed for the second layer in every sample. Emission spectra of three chosen proteins overlap--the spectrum tail of the GFP extends to intensity maxima of the mBanana (551 nm) and the mCherry (612 nm), and the mBanana tail overlaps the mCherry maximum (612 nm). This resulted in contamination of fluorescence decays, with remnant components of shorter wavelength-emitting proteins. They were subtracted from decay curves according to the ratio of fluorescence intensities of both given proteins in the sample, which were determined using steady-state fluorescence spectra.  
Fluorescence characteristics were obtained using spectrofluorometer FS5 (Edinburg instruments), equipped with a xenon lamp (for steady state emission) and 405 nm and 470 nm laser diodes (for fluorescence lifetime measurement). The samples were mostly measured on a special sample holder, allowing analysis of surfaces. For measurements in solution, a separate holder for cuvettes was used. Decays were fitted using Fluoracle software version 2.4.1 (Edinburg instruments). Average fluorescence lifetime was calculated as amplitude-weighted mean.  
For confocal fluorescence microscopy and fluorescence lifetime imaging (FLIM), sequential monolayers were mounted on cover microscopic glass slides. Imaging was performed using Stellaris confocal system (Leica, WGermany), with a tunable pulsed white laser. HC PL Apo CS2 100\(\times\)/1.4 oil objective was used. Emission ranges were set using a monochromator. Image collection and FLIM processing were performed using LasX software version 3.7 (Leica, Germany). As the setup does not allow for clear analysis ofemission spectra, and, therefore, the correction of acceptor decays for the remnant components of shorter wavelength-emitting donor species, only donor emission decays were analyzed.