Combining Dual-View Plane Illumination Microscopy with Bessel Beams

Schnieder, M.; Voß, F.; Hüve, J.; Klingauf, J.

Abstract

Among the different realisations of light-sheet microscopy, dual-view plane illumination microscopy (diSPIM) has proven to be able to provide an isotropic diffraction-limited resolution. This method’s imaging properties may be further enhanced by using beam shaping, e.g. Bessel-Gaussian beams. For our application to image presynaptic mechanisms in comparatively small regions, we would like to adapt the method to improve its contrast and resolution while keeping the possibility to image 3D dynamics at a sub-second timescale. As a first step, we use beams with emphasized Bessel properties by applying high-NA thin annular slits. In order to drastically reduce the power loss usually connected to this kind of slits, we constructed an optical system containing two lenses and three axicons which shape the incident Gaussian beam to a collimated ring. This configuration that we call SWAN (set ring width and NA) can adjust the output ring's diameter simply by changing the last axicon's position along the optical axis. We plan to characterize the benefit of using Bessel-Gaussian beams in combination with diSPIM and to optimize beam shaping for a good balance of spatial and temporal resolution. Furthermore, we introduce the SWAN system in combination with appropriate annular slits. This enables us to easily adapt the illumination NA and thus parameters determining especially the speed and contrast of image acquisition according to the needs of the experimental setting.