  Observation times of freely diffusing single molecules in solution are
limited by the photophysics of the attached fluorescence markers and by a small
observation volume in the femtolitre range that is required for a sufficient
signal-to-background ratio. To extend diffusion-limited observation times
through a confocal detection volume, A. E. Cohen and W. E. Moerner have
invented and built the ABELtrap - a microfluidic device to actively counteract
Brownian motion of single nanoparticles with an electrokinetic trap. Here we
present a version of an ABELtrap with a laser focus pattern generated by
electro-optical beam deflectors and controlled by a programmable FPGA chip.
This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds,
increasing the observation time of fluorescent nanoparticles compared to free
diffusion by a factor of 10000. To monitor conformational changes of individual
membrane proteins in real time, we record sequential distance changes between
two specifically attached dyes using F\"orster resonance energy transfer
(smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results
in an improved signal-to-background ratio at lower laser excitation powers.
This increases our measured trap duration of proteoliposomes beyond 2 s.
Additionally, we observe different smFRET levels attributed to varying
distances between the FRET donor (mNeonGreen) and acceptor (Alexa568)
fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase
respectively.
