Abstract

Two-photon absorption (TPA) and stimulated Raman scattering (SRS) microscopy enables label-free, chemically specific imaging of absorptive molecules [1] and vibrational molecules [2], respectively. In the case of the small intensity changes of excitation pulses induced by TPA and SRS, the loss or gain in TPA and SRS needs to be discriminated from the laser 1/f noise. The 1/f noise can be reduced by modulating the intensity of one of the two-color pulses at a high frequency and measuring the modulation transfer to the other pulse [3]. In the case of samples where absorptive molecules mix with vibrational molecules, however, one signal may be the background of label-free imaging using the other signal. Recently, we demonstrated SRS imaging by modulating the spatial overlap between two-color pulses [4]. In this technique, the intensity changes of the two-color pulses induced by TPA and SRS can be modulated at a double frequency of the spatial overlap modulation (SPOM) frequency. Thus, the intensity changes of the two-color pulses can be simultaneously detected by two detectors. TPA results in the losses of the two-color pulses (ω₁ and ω₂), while SRS leads to the loss of the pump light (ω₁) and the amplification (gain) of Stokes light (ω₂). By adding the intensity changes of the two-color pulse, the TPA signal is doubled, while the SRS signal is removed. In contrast, by subtracting the intensity change of the pump light (ω₁) from that of the Stokes light (ω₂), the SRS signal is doubled, whereas the TPA signal is removed. Therefore, TPA signals can be separated from SRS signals. We simultaneously detected the TPA and SRS signals by the SPOM technique.

© 2013 IEEE