Super-resolved multimodal multiphoton microscopy with spatial frequency-modulated imaging

Super-resolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all super-resolution imaging techniques reported to date rely on real energy states of probe molecules to circumvent the diffraction limit, preventing super-resolved imaging of contrast mechanisms that occur via virtual energy states such as harmonic generation (HG). Here we report a super-resolution technique based on SPatIal Frequency modulated Imaging (SPIFI) that permits super-resolved nonlinear microscopy with any contrast mechanism, and with single-pixel detection. We show multimodal super-resolved images with two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) from biological and inorganic media. Multiphoton SPIFI (MP-SPIFI) provides spatial resolution up to 2\(\eta\) below the diffraction limit, where \(\eta\) is the highest power of the nonlinear intensity response. MP-SPIFI has the potential to not only provide enhanced resolution in optically thin media, but shows promise for providing super-resolved imaging at depth in scattering media - opening the possibility of \(\textit{in vivo}\) super-resolved imaging.