Pulse compression in two-photon excitation fluorescence microscopy.

The use of shorter pulses is a practical way to improve the signal in two-photon excitation fluorescence microscopy. We report on the theoretical and experimental results of pulse compression in a two-photon excitation fluorescence microscope by using approximately 100-fs Ti:Sapphire laser and highly nonlinear photonic crystal fiber. Effects of the fiber parameters, transmitted power, and group-delay dispersion provided by the gratings have been investigated to optimize the compressor performance. By using a 20-mm-long photonic crystal fiber with a zero dispersion wavelength of 850 nm, a compressed pulse of 23.6 fs starting from 94 fs at 790 nm is experimentally demonstrated as a verification of our simulations. By integrating the compressor with a two-photon excitation fluorescence microscope, 5.6 times increase in autofluorescence intensity of NAD(P)H in Nasopharyngeal carcinoma cells is demonstrated, showing its potential in enhanced imaging and sensing for disease diagnosis.