Nonlinear microscopy with adaptive optics

The imaging performance of high-resolution optical microscopes is often compromised by specimen-induced aberrations. This is particularly problematic when imaging thick biological specimens, such as early stage mammalian embryos. To understand the morphogenetic events that lead to the proper positioning of differentiated cells in the early morphogenesis of mammalian embryos, it is important to obtain high-resolution 3D image sequences of this development process. This might be possible using combined third-harmonic generation (THG) and two-photon excited fluorescence (2PEF) microscopy. However, the large size of such embryos are challenging for the imaging system: The large number of pixels requires an optimized signal creation to maintain a high frame rate, which implies almost diffraction limited focusing of the excitation light into the tissue. Unfortunately, the large thickness of the tissue leads to severe degradation of the focal spot due to scattering. The aim of the proposed research is to develop dynamic correction strategies to restore the focus quality by means of adaptive optics, and hence to allow the acquisition of high-resolution 3D image sequences of living mouse embryos. The research will cover the assessment of existing techniques as well as the development of improvements in order to meet the specific demands of examining thick tissues with combined 2PEF/THG microscopy.