Advanced biomedical multiphoton fluorescence microscopy with a large band excitation system

摘要

Commercial multiphoton microscopes currently include standard and bulk titanium-sapphire laser sources (Ti: Sa) delivering a narrow spectral bandwidth of 10 nm at the full-width at half maximum. Such a spectral width precludes the instantaneous imaging of more than four fluorophores simultaneously in optimal conditions of target. Femtosecond ultra-wideband laser system (UWLS) appeared since fifteen years with an approach of spectral broadening of Ti: Sa pulses into a photonic crystal fiber associated with a dispersion compensation system. In the current work, we demonstrate the interest of an alternative laser solution, compact, cheap, simple and turn-key based on a UWLS delivering a unique spectral excitation bandwidth named supercontinuum. We have coupled this system to a passive filtering setup. Thanks to this unique spectral bandwidth, eleven fluorophores are imaged with multiphoton processes without any modification on the excitation parameters.