Aberration correction for real-time measurements in adaptive confocal microscopy

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Progress in the in vivo investigation of biological tissue essentially relies on microscopy with ever increasing spatial and temporal resolution. In the present proposal, we address this challenge by aiming to enhance the spatial resolution confocal laser scanning microscopy using novel adaptive optical elements and control techniques. To produce a three dimensional image, confocal microscopy scans the volume with a focused laser beam, typically by moving optical elements and using piezo or galvo mirrors where the speed is limited by mass and inertia. This may further result in motion artefacts and requires a bulky set-up, limiting the potential for miniaturization. The spot size, and hence the resolution, can - in principle - be minimized using microscope objectives with a high numerical aperture. As they are optimized for a single focal plane with a limited field, however, scanning results in significant system-induced aberrations in addition to the sample-induced aberrations and hence limits the achievable resolution. To overcome these problems, one can use adaptive elements both for aberration correction and for motion-free scanning.Our aim is the development of an adaptive confocal microscope that uses ideally only two (one lens and one prism) adaptive optical elements to provide fast three dimensional scanning and real-time aberration correction for diffraction-limited imaging over the whole field of view. For this purpose, we will develop novel adaptive lenses that combine axial scanning and aberration correction, including geometric (defocus, spherical, astigmatism, coma) and chromatic aberrations. Similarly, we will preform the lateral scans with novel bi-axial achromatic adaptive prisms. In contrast to scanning with galvo mirrors, this allows for a more compact collinear geometry. Using these adaptive optical elements, this novel smart microscope has a great potential for miniaturization and for the development of robust handheld systems. We will demonstrate potential applications and the enhanced imaging performance by investigating the effects of goitrogens in zebrafish embryos.