Simultaneous label-free autofluorescence multi-harmonic microscopy driven by the supercontinuum generated from a bulk nonlinear crystal

Nonlinear microscopy encompasses several imaging techniques that leverage laser technology to probe intrinsic molecules of biological specimens. These native molecules produce optical fingerprints that allow nonlinear microscopes to reveal the chemical composition and structure of cells and tissues in a label-free and non-destructive fashion, information that enables a plethora of applications, e.g., real-time digital histopathology or image-guided surgery. Because state-of-the-art lasers exhibit either a limited bandwidth or reduced wavelength tunability, nonlinear microscopes lack the spectral support to probe different biomolecules simultaneously, thus losing analytical potential. Therefore, a conventional nonlinear microscope requires multiple or tunable lasers to individually excite endogenous molecules, increasing both the cost and complexity of the system. A solution to this problem is supercontinuum generation, a nonlinear optical phenomenon that supplies broadband femtosecond radiation, granting a wide spectrum for concurrent molecular excitation. This study introduces a source for nonlinear multiphoton microscopy based on the supercontinuum generation from a yttrium aluminum garnet (YAG) crystal, an approach that allows simultaneous label-free autofluorescence multi-harmonic imaging of biological samples and offers a practical and compact alternative for the clinical translation of nonlinear microscopy. While this supercontinuum covered the visible spectrum (550-900 nm) and the near-infrared region (950-1200 nm), the pulses within 1030-1150 nm produced label-free volumetric chemical images of ex vivo chinchilla kidney, thus validating the supercontinuum from bulk crystals as a powerful source for multimodal nonlinear microscopy.