Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses

The fundamental advantages to using ultrafast (?100 fs) laser pulses in two-photon microscopy for biomedical imaging are seldom realized due to chromatic dispersion introduced by the required high numerical aperture microscope objective. Dispersion is eliminated here by using the multiphoton intrapulse interference phase scan (MIIPS) method on pulses with a bandwidth greater than 100 nm full width at half maximum. Higher fluorescence intensity, deeper sample penetration, and improved signal-to-noise ratio are demonstrated quantitatively and qualitatively. Due to the higher signal intensity obtained after MIIPS compensation, lower laser power is required, which decreases photobleaching. The observed advantages are not realized if group delay dispersion is compensated for while higher-order dispersion is not. By using a pulse shaper and taking advantage of the broad spectrum of the ultrafast laser, selective excitation of different cell organelles is achieved due to the difference in nonlinear optical susceptibility of different chromophores without requiring an emission filter wheel. Experiments on biological specimens, such as HeLa cells and mouse kidney tissue samples, illustrate the advantages to using sub-10 fs pulses with MIIPS compensation in the field of two-photon microscopy for biomedical imaging.