Depth aberrations characterization in linear and nonlinear microscopy schemes using a Shack-Hartmann wavefront sensor

The performance of imaging devices such as linear and nonlinear microscopes (NLM) can be limited by the optical properties of the imaged sample. Such an important aspect has already been described using theoretical models due to the difficulties of implementing a direct wavefront sensing scheme. However, these only stand for simple interfaces and cannot be generalized to biological samples given its structural complexity. This has leaded to the development of sensor-less adaptive optics (AO) implementations. In this approach, aberrations are iteratively corrected trough an image related parameter (aberrations are not measured), being prone of causing sample damage. In this work, we perform a practical implementation of a Shack-Hartman wavefront sensor to compensate for sample induced aberrations, demonstrating its applicability in linear and NLM. We perform an extensive analysis of wavefront distortion effects through different depths employing phantom samples. Aberration effects originated by the refractive index mismatch and depth are quantified using the linear and nonlinear guide-star concept. More over we analyze offaxis aberrations in NLM, an important aspect that is commonly overlooked. In this case spherical aberration behaves similarly to the wavefront error compared with the on-axis case. Finally we give examples of aberration compensation using epi-fluorescence and nonlinear microscopy.