Ff775 di01 dichroic mirror

We measured fluorescence lifetime on a homebuilt beam-scanning two-photon microscope with an 80 MHz, 100 fs tunable pulsed laser (SpectraPhysics Insight DeepSee). Pulse compression in the sample plane was performed through maximizing the fluorescence intensity of a bead-sample, using the motorized prism compressor built into the laser. The citrine measurements were performed at 1040 nm excitation wavelength with a time-averaged excitation power of 30 mW, or 0.4 nJ per pulse, focused down to a ~500 nm spot with a 1.2 NA water immersion objective (Olympus UplanSapo) for a time-averaged intensity of 15 MW cm⁻²; measurements were performed for linear speeds of the scanned spot varying between 0 and 30 cm s⁻¹ without any measurable effect on fluorescence lifetime. Excitation light and fluorescence were separated using a FF775-Di01 dichroic mirror and FF01-790/SP-25 shortpass filter (Both Semrock); fluorescence was detected using a Hamamatsu R943-02 photomultiplier tube in photon counting mode, cooled to −20 °C. THe PMT signal was amplified through an SRS PR325 amplifier and discretized with a Hamamatsu Photon counting unit C9744, before being fed into a Picoharp 300 TCSPC module (Picoquant). The setup was controlled by Labview software written in-house.