Accessing nonlinear phase contrast in biological tissue using femtosecond laser pulse shaping
Nonlinear imaging takes advantage of the localized nature of the interaction to achieve high spatial resolution, optical sectioning, and deeper penetration in tissue. However, nonlinear contrast (other than fluorescence or harmonic generation) is generally difficult to measure because it is overwhelmed by the large background of detected illumination light. Especially challenging to measure is the nonlinear refractive index-accessing this quantity would allow the extension of widely employed phase microscopy methods to the nonlinear regime. We have developed a technique to suppress the background in these types of measurements by using femtosecond pulse shaping to encode nonlinear interactions in background-free regions of the frequency spectrum. Using this individual pulse shaping based technique we have been able to measure self-phase modulation (SPM) in highly scattering environments, such as biological tissue, with very modest power levels. Using our measurement technique we have demonstrated strong intrinsic SPM signatures of glutamate-induced neuronal activity in hippocampal brain slices. We have also extended this measurement method to cross-phase modulation, the two-color analogue to SPM. The two-color approach dramatically improves the measurement sensitivity by reducing undesired background and associated noise. We will describe the nonlinear phase contrast measurement technique and report on its application for imaging neuronal activity. © 2011 SPIE-OSA.