Adaptive interferometric light-sheets for resolution enhanced imaging with and without labeling

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Understanding light matter interaction is of significant importance in modern imaging systems. This is the more the case, the larger and stronger scattering the object is that need to be investigated. Light propagation through weekly absorbing matter primarily changes the phase of light. Therefore phase correcting, adaptive optics has been used in astronomy for quite a while, but recently also in modern optical microscopy. Hence, also in light sheet based microscopy spatial light modulators are used to modulate the phase and the intensity of illumination beams, enabling in principle many advantages to 3-D image acquisition. In this context, Bessel beams with their conical phase profile reveal an amazing capability of beam self-reconstruction and a penetration depth which is increased by about 50% relative to conventional Gaussian beams. However, around their narrow main intensity peak, Bessel beams carry a concentric ring system, which results in a loss of image contrast in light sheet microscopy, if no special tricks are applied. In this proposal we want to use linear and nonlinear optical methods to improve the quality of illumination beams in light sheet microscopy by investigating the dependency of computer- holographically generated phase profiles on the beam propagation properties. By using nonlinear optical concepts, we aim to improve the quality of single laser beams and thereby of the illuminating light sheet, 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 will 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 minimize the thickness of the light sheet and thereby to maximize the optical resolution by improving the depletion efficiency. 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 the fluorescence generated in the single beams will be improved significantly and thereby the quality of the light sheet.