We demonstrate the benefit of a novel laser strategy in multiphoton microscopy (MPM). The cheap, simple, and turn‐key supercontinuum laser system with its spectral shaping module, constitutes an ideal approach for the one‐shot microscopic imaging of many fluorophores without modification of the excitation parameters: central wavelength, spectral bandwidth, and average power. The polyvalence of the resulting multiplex‐multiphoton microscopy (M‐MPM) device is illustrated by images of many biomedical models from several origins (biological, medical, or vegetal), generated while keeping constant the spectral parameters of excitation. The resolution of the M‐MPM device is quantified by a procedure of point‐spread‐function (PSF) assessment led by an original, robust, and reliable computational approach FIGARO. The estimated values for the PSF width for our M‐MPM system are shown to be comparable to standard values found in optical microscopy. The simplification of the excitation system constitutes a significant instrumental progress in biomedical MPM, paving the way to the imaging of many fluorophores with a single shot of excitation without any modification of the lighting device. Research Highlights A new solution of multiplex‐multiphoton microscopy device is shown, resting on a supercontinuum laser. The one‐shot excitation device has imaged biomedical and vegetal models. Our original computational strategy measures usual microscopy resolution.
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. .
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