Simultaneous selective two-photon microscopy using MHz rate pulse shaping and quadrature detection of the time-multiplexed signal

A broad-bandwidth oscillator and pulse shaper with a compensation phase mask applied produce sub-10 fs laser pulses is used to induce selective two-photon excitation in the 380 to 500 nm range. The output is split into two arms with different second order dispersion (SOD). The recombined beams create a train of pulses with phase-shape switching at a rate of 162 MHz. Each pulse induces selective TPEF on the sample at wavelengths determined by the amount of SOD in the beam, which tunes the selective TPEF wavelength. Fluorescence is detected by a single fast photomultiplier tube (PMT) detector; therefore, signal from the PMT detector contains fluorescence signals from two different selectively-excited fluorophores. The two separate signals are isolated by quadrature detection using a lock-in amplifier. Images are obtained from the two different fluorophores simultaneously at 81MHz. The wide tunability of the two-photon excitation wavelength, fast switching rate between the selective excitation, and low photodamage (due to low power of laser beam) enables potential application of this method for in vivo dynamic imaging in biological samples