Simultaneous metabolic and oxygen imaging is promising to follow up therapy response, disease development and to determine prognostic factors. FLIM of metabolic coenzymes is now widely accepted to be the most reliable method to determine cellular bioenergetics. Also, oxygen consumption has to be taken into account to understand treatment responses. The phosphorescence lifetime of oxygen sensors is able to indicate local oxygen changes. For phosphorescence lifetime imaging (PLIM) dyes based on ruthenium (II) coordination complexes are useful, in detail TLD1433 which possesses a variety of different triplet states, enables complex photochemistry and redox reactions. PLIM is usually reached by two photon excitation of the drug with a femtosecond (fs) pulsed Ti:Sapphire laser working at 80 MHz repetition rate and (time-correlated single photon counting) (TCSPC) detection electronics. The interesting question was whether it is possible to follow up PLIM using faster repetition rates. Faster repetition rates could be advantageous for the induction of specific photochemical reactions because of similar light doses used normally in standard CW light treatments. For this, a default 2p-FLIM–PLIM system was expanded by adding a second fs pulsed laser (“helixx") which provides 50 fs pulses at a repetition rate of 250MHz, more than 2.3W average power and tunable from 720 nm to 920 nm. The laser beam was coupled into the AOM instead of the default 80MHz laser. We demonstrated successful applications of the 250MHz laser for PLIM which correlates well with measurements done by excitation with the conventional 80 MHz laser source.