Multiplex-multiphoton microscopy and computational strategy for biomedical imaging.

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.