Differentiating 2-photon and 3-photon processes: order-resolved nonlinear optical imaging with intensity modulation and frequency-gated demodulation

Nonlinear label-free autofluorescence and harmonic generation microscopy integrate 2-, 3-photon, and higher-order processes, enabling high-precision imaging of biological tissues without external labels. These techniques use intrinsic light from the nonlinear response of endogenous molecules to ultrashort pulsed lasers, creating high-resolution tissue maps. A significant challenge is distinguishing between autofluorescent molecules with overlapping spectra. Our novel method employs intensity modulation and frequency-gated demodulation to separate 2-photon from 3-photon signals. This approach enhances the specificity of nonlinear microscopy, allowing for precise imaging of complex biological samples, crucial for cellular metabolism analysis and optical redox ratio measurements, using a single detector. We compare this method with Fluorescence lifetime Imaging, where different molecules are distinguished by their fluorescence decay characteristics.