Adaptive wavefront correction in two-photon microscopy using Coherence-Gated Wavefront Sensing
The focus of a two-photon microscope is often degraded by inhomogeneities in the refractive index within biological specimens. In this dissertation it is shown for various specimens, even for living zebrafish, that the resolution and the fluorescence signal of a two-photon microscope can be substantially improved by using adaptive optics, i.e. wavefront correction based on coherence-gated wavefront sensing (CGWS). The advantage of using CGWS relies on the fact that the wavefront distortions are sensed by backscattered instead of fluorescent light. Thus, neither photodamage nor photobleaching occurs and wavefront distortions can be sensed up to several scattering lengths deep within the specimen. Fast wavefront correction can be realized, allowing the measurement of a wavefront in less than 1 microsecond with an accuracy of lambda/50, even in strongly scattering samples. Furthermore, CGWS is thoroughly investigated for all relevant parameters affecting the measurement process, such as coherence length, polarization of the light, density of scatterers, and coherence-gate position. A realistic model of CGWS shows that for all experimentally accessible parameters the speckle contrast is fully developed. Thus, the ensemble-averaged wavefront is the incoherent superposition of spherical wavelets that originate from scatterers located within the coherence volume and then propagate through specimen-induced distortions.
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