Multiphoton absorption and ionization in ultrafast pump–probe experiments

Pump–probe spectroscopic and diffraction techniques enable ultrafast measurements of structural dynamics. However, interpretation is complicated by simultaneous multiphoton absorption and associated relaxation pathways. At high excitation intensities, many systems, particularly biomolecules such as proteins, undergo both linear and nonlinear absorption, generating electronic and vibrational relaxation signals that obscure the targeted dynamics. To address this, we introduce a rate-equation framework that quantifies the relative contributions of one and two photon absorption while incorporating the effect of time-dependent detuning of resonances, relaxation lifetimes, and intensity attenuation through the sample. Comparison to published datasets suggests that nonlinear excitation becomes significant for pulses shorter than 200 fs when the modeled multiphoton absorption exceeds ∼20% of the total excitation, which can occur for photon densities near and above one photon per chromophore (PPC) for typical absorption cross sections. To prevent nonlinear effects and preserve biological relevance of the observed dynamics, experiments should use conservative excitation conditions well below the one PPC limit and below the 100 GW/cm2 range to prevent multiphoton absorption and the onset of ionization that is unrelated to the dynamics of interest. This study and the associated open-source graphical user interface for estimating the contribution of multiphoton absorption and ionization provide practical guidance for designing and interpreting ultrafast pump–probe experiments to examine the dynamics of interest within linear response.