The fluorescence lifetimes were measured with a partially home-built time-correlated single photon counting (TCSPC) setup as previously described (40 (
link)). For excitation, a mode-locked titanium-doped sapphire (Ti:Sa) laser (Tsunami 3941-X3BB, Spectra-Physics, Darmstadt, Germany) was pumped by a 10 W continuous wave diode pumped solid state laser (
Millennia eV, Spectra-Physics, 532 nm). The Ti:Sa laser provided pulses of 775 nm central wavelength with a repetition rate of 80 MHz. With the help of an acousto-optic modulator, the repetition rate was reduced to 8 MHz and the excitation wavelength of 388 nm was obtained by SHG in a BBO crystal (frequency doubler and pulse selector,
Model 3980, Spectra-Physics). Excitation pulses of about 0.1 nJ at 388 nm were applied to the sample. The sample was prepared in a 10 × 4 mm quartz cuvette (29-F/Q/10, Starna) with a fixed temperature of 20°C. Emission filters (GG395,
GG400, Schott AG, Mainz, Germany) suppressed excitation stray light. The instrument response function (IRF, FWHM 200 ps) was obtained without emission filters using a TiO
2 suspension as scattering sample. For single-photon detection, a photomultiplier tube (PMT,
PMA-C 182-M, PicoQuant, Berlin, Germany) and a
TimeHarp 260 PICO Single PCIe card (PicoQuant) were used. Multi-exponential fitting was carried out with
FluoFit Pro 4.6 (PicoQuant) (64 ).
Gustmann H., Segler A.L., Gophane D.B., Reuss A.J., Grünewald C., Braun M., Weigand J.E., Sigurdsson S.T, & Wachtveitl J. (2018). Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer. Nucleic Acids Research, 47(1), 15-28.