Effect of pulse phase and shape on the efficiency of multiphoton processes: Implications for fluorescence microscopy

Summary form only given. To date, the primary laser variables in ultrashort pulse excitation microscopy which are used to optimize efficiency have been wavelength, average power and peak power. In this paper we introduce two more parameters: pulse shape and phase (or chirp). Normally the pulse shape is usually assumed to be a simple analytical form, such as a Gaussian, and the pulse is assumed to be transform limited (no chirp). However, this also means that the peak power, and consequently intensity at focus have also contained certain assumptions. Thus, knowledge of these parameters is not only useful for maximizing the efficiency of the excitation, but it is also necessary for an exact description of the fields generated at focus. The effects of spectral shape on two-photon fluorescence efficiency were investigated using an acousto-optic pulse shaper to modify femtosecond pulses from a Ti:sapphire laser. By using different shapes, we find that the measured two-photon efficiency can vary by a factor of 2 for differently shaped spectra with the same full-width-half-maximum. We find that these effects are well described by a simple model assuming transform-limited pulses. The fact that even small changes in the spectral wings can significantly affect the efficiency of nonlinear processes has implications for biological multiphoton imaging, where it is desirable to minimize sample exposure to radiation and maximize fluorescence efficiency. In the case of single photon excitation of a fluorophore at high energy densities the fluorescence shows a strong chirp or phase dependence. The method is quite robust and applicable to very large molecules in room temperature liquid.