Adaptive Hybrid Illumination Microscopy

Wide field microscopy is well-established in biological and medical applications. But its re-duced depth sectioning capability leads to background signals originating outside the depth of interest that degrade the contrast and limit the usability. To solve this limitation, a variety of microscopic techniques offering adequate depth sectioning have been introduced, the most prominent one being confocal microscopy. However, although confocal microscopy is advantageous, it is a pointwise technique and thus requires scanning in three dimensions to obtain 3D information. Hybrid illumination microscopy enables to record optically sectioned wide field images by analyzing the spatial frequency content of the recorded image. As the maximum spatial fre-quency bandwidthis transported through the system for in-focus sample parts, high-spatial frequencies that inherently occur from the specimen, already lead to an optical sectioning. To get access to the low spatial frequency part of the focal region, a speckled illumination can be used, to artificially introduce high spatial frequencies. Thus, the combination of an uniform and a non-uniform illumination bears the potential to record optically sectioned images, with a strongly reduced scanning requirement. Just one axial scan is required. Using adaptive lenses allows to circumvent any mechanical scanning and to implement fast axial scanning without moving parts enabling rapid volumetric recordings. We use this technique to analyze fluorescence of transgene zebrafish larvae.

The method is suitable for all fluorescence measurements and is not limited to wide field microscopy, but can also be e.g. integrated in light sheet microscopes.

K. Philipp, A. Smolarski, N. Koukourakis, A. Fischer, M. Stürmer, U. Wallrabe, J. Czarske “Volumetric HiLo microscopy employing an electrically tunable lens”, Opt. Express 24(13), 15029-15041 (2016).