Dmlp505

Femtosecond laser pulses (80 MHz repetition rate, 100 fs, up to 25 nJ per pulse) from a Ti:Sapphire oscillator (Tsunami, Spectra-Physics, Andover, MA, USA) were applied at an 8 MHz repetition rate using an acousto-optic modulator (Pulse select, APE, Berlin, Germany) and a frequency doubled in the BBO SHG crystal. Then, femtosecond pulses with a central wavelength of 490 nm were coupled with an inverted optical microscope (IX71, Olympus, Tokyo, Japan) using a dichroic mirror (DMLP505, Thorlabs, Newton, MA, USA) and successfully focused on a sample using an objective lens (40 × 0.75NA UPlanFLN, Olympus, Tokyo, Japan). The samples were prepared as droplets of the purified fluorescent proteins dissolved in buffered solution (pH 5.5–10.0), applied to a standard 24 × 24 mm cover glass (Heinz Herenz, Hamburg, Germany). The average laser power was tuned with a polarizing attenuator in the range of 0.1–10 µW. The fluorescence (>505 nm) passed back through the objective lens and laser coupling dichroic mirror was directed towards the input of an Acton SP300i monochromator with a hybrid single-photon detector on its output (HPM-100-6, Becker&Hickl, Berlin, Germany), registering the fluorescence decay kinetics in the 510–520 nm band. The fluorescence decay data were analyzed using SPCImage software (Becker&Hickl, Berlin, Germany) with the measured instrument response function (IRF).