Advanced biomedical multiphoton fluorescence microscopy with a large band excitation system

Abstract

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 ultrawideband 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.