Two-photon fluorescence imaigng using tunable spectral window based on fiber supercontinuum

Two-photon excitation fluorescence (TPEF) microscopy has evolved into a versatile tool in biological research. However, the multiplexing capability of TPEF microscopy is limited by the narrow spectral bandwidth of the light source. In this study, we applied photonic crystal fiber in TPEF microscopy to broaden the excitation source bandwidth. We tuned the spectral window using a spatial light modulator as a programmable diffraction grating which was placed behind a prism pair. In addition, we combined a grating pair to compensate for dispersion to improve the two-photon excitation efficiency. The combination of a broad spectrum and a programmable grating enabled fast spectral window tuning rate on a time scale of tens of milliseconds. We demonstrate the performance of our method by imaging live B16 cells labeled with four emission spectrum overlapped fluorescent proteins.