Picosecond pulsed two‐photon imaging with repetition rates of 200 and 400 MHz

We show the viability of high‐resolution two‐photon fluorescence imaging of fixed and live cells by exciting the fluorophores with a train of near‐infrared pulses with duration in the picosecond range. This is exemplified with a compact, diode‐pumped Nd:YVO 4 laser, emitting trains of 7‐ps pulses at a wavelength of 1064 nm, with a repetition rate of 200 MHz at two separate outputs. Incoherent combination of the outputs enabled two‐photon excitation with a repetition rate of 400 MHz. For a numerical aperture of 1.4 (oil), we used an average illumination power of up to 20–40 mW at the sample. The pulses were coupled into a beam scanning microscope, either directly or through a single mode glass fibre. Compared with standard femtosecond titanium–sapphire excitation conditions, our experiments were performed with a 2.5 or 5 times higher repetition rate, 30–70 times longer pulses and 10–35 times lower pulse peak intensity. The experiments indicate the possibility of significantly relaxing the temporal pulse width constraints for a series of applications.