Nanosecond Two-photon excitation fluorescence imaging with a multi color fiber MOPA laser

摘要

A system is presented that uses a fiber based Master Oscillator Power Amplifier (MOPA) with nanosecond-range pulses for two-photon excitation fluorescence (TPEF) imaging. The robust laser in the extended near infrared is based on an actively modulated electro-optical modulator (EOM), enabling free synchronization of the pulses to any other light source or detection unit. Pulses with a freely programmable duration between 0.4 and 10 ns are generated and then amplified to up to kilowatts of peak power with ytterbium doped fiber amplifiers (YDFA). Since we achieve peak power and duty cycles comparable to standard femto- and picosecond setups, the TPEF signal levels are similar, but realized with a robust and inexpensive fiber-based setup. The delivery fiber is further used as an optional, electronically controllable Raman shifter to effectively shift the 1064 nm light to 1122 nm and to 1186 nm. This allows imaging of a manifold of fluorophores, like e.g. TexasRed, mCherry, mRaspberry and many more. We show TPEF imaging of the autofluorescence of plant leaves of moss and algae, acquired in epi-direction. This modular laser unit can be integrated into existing systems as either a fiber-based, alignment free excitation laser or an extension for multi-modal imaging.