In this manuscript, we review the physics and recent developments of the least invasive optical higher harmonic
generation microscopy, with an emphasis on the in vivo molecular imaging applications. Optical higher harmonicgenerations,
including second harmonic generation (SHG) and third harmonic generation (THG), leave no energy
deposition to the interacted matters due to their energy-conservation characteristic, providing the "noninvasiveness"
nature desirable for clinical studies. Combined with their nonlinearity, harmonic generation microscopy provides threedimensional
sectioning capability, offering new insights into live samples. By choosing the lasers working in the high
penetration window, we have recently developed a least-invasive in vivo light microscopy with submicron 3D resolution
and high penetration, utilizing endogenous and resonantly-enhanced multi-harmonic-generation signals in live
specimens, with focused applications on the developmental biology study and clinical virtual biopsy.