Order-of-magnitude multiphoton signal enhancement based on characterization of absorption spectra of immersion oils at the 1700-nm window

To enhance signal levels in multiphoton microscopy (MPM) at the deep-tissue excitation window (1600-1820 nm) with oil immersion, we demonstrate: First, the absorption spectra of several commonly immersion oils are characterized, which were unknown before. Second, new material with lower absorption based on mixing is proposed. Third, optimal selection of excitation wavelength within this window is proposed based on absorption spectra characterization. Second and third harmonic generation imaging of mouse tissue corroborate our selection: 1600-nm excitation leads to notable orders-of-magnitude increase in MPM signal, compared with 1700-nm excitation, enabling 200-μm imaging depth of mouse skin while 1700-nm excitation could resolve virtually no structure.