Feedback holographic control of self-reconstructing laser beams in strongly scattering media.

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Light sheet based microscopy has had a strong impact on the development of novel intelligent illumination methods in modern microscopy, which improved 3D image quality significantly. This led to an advance in the areas of developmental biology and 3-D cell biology, but also made the investigation of laser beam propagation through strongly scattering media come to the fore. Recently it could be shown, that the phase profile of a weekly focused laser beam had a strong influence on light scattering in inhomogeneous, weekly absorbing media. In particular, Bessel beams revealed an amazing capability of beam self-reconstruction and a penetration depth which was increased by about 50% relative to conventional Gaussian beams. However, Bessel beams carry a concentric ring system, which results in a loss of image contrast in light sheet microscopy.In this proposal we want to use linear and nonlinear optical methods to improve the quantity of illumination beams in light sheet microscopy by investigating intensively the dependency of computer- holographically generated phase profiles on the beam propagation properties. In the area of linear optics, we aim to develop different read-out procedures for scattered light in different planes. Based on the extracted data, we then want to test and develop different wavefront correction procedures by feedback and iteration. The contrast diminishing influence of the ring system shall be reduced significantly by using a line-confocal detection system.In addition we aim to improve the quality of single laser beams and thereby of the illuminating light sheet by using nonlinear optical approaches, such that the influence of the Bessel beams ring system is nonlinearly suppressed. On the one hand we will use the principle of two-photon fluorescence excitation, where especially the influence of the holographically shaped phase on the propagation of short laser pulses through the scattering medium is to be investigated. On the other hand, we plan to apply the STED-principle, where we will use a second self-reconstructing Bessel beam of higher order to deplete the fluorescence in the ring system by stimulated emission. Here, we want to maximize the optical resolution by improving the depletion efficiency through time-gating in a first step. In a second step, we want to optimize the phase profiles of the excitation beam and the STED- beam by a feedback holographic control, such that fluorescence generated in the single beams will be improved significantly and thereby the quality of the light sheet.