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.