The photoreceptor bacteriorhodopsin (HsBR) from Halobacterium salinarum is a model system for studying ultrafast photoinduced reactions in proteins. Recent time-resolved serial femtosecond crystallography (TR-SFX) experiments require high pump energies, raising concerns about nonlinear excitation and multi-photon effects. Here, we systematically investigate the influence of excitation energy, pulse duration and the sign of the chirp on the initial HsBR photo-reaction using femtosecond Vis-pump IR-probe spectroscopy in the retinal C=C stretching region. An acousto-optic programmable dispersive filter enabled independent control of pulse energy and chirp. Within the tested range, the retinal dynamics were independent of pulse duration and chirp, indicating that fluence alone does not fully describe excitation conditions. Increasing excitation energy leads to nonlinear saturation of the retinal signals and the appearance of an additional band near 1550 cm−1. However, this band rises linearly with the excitation energy. Hence, the additional band is not directly caused by non-resonant multi-photon absorption. Spectral decomposition reveals two components: a low-energy contribution consistent with the known retinal isomerization dynamics and a high-energy contribution attributed to a small population of photo-damaged HsBR likely formed via a resonant two-photon process. These findings clarify the role of excitation conditions in ultrafast HsBR spectroscopy and suggest that spectral changes at high pump energies mainly arise from damaged species upon resonant two-photon excitation.
