Confocal detection without striping artifacts exploiting a fast multidirectional DSLM (Conference Presentation)

In the past few years, Light Sheet Fluorescence Microscopy (LSFM) has become a cornerstone imaging technique for neuroscience, improving the quality and capabilities of 3D imaging. Selec-tive illumination of a single plane provides intrinsic optical sectioning and fast image recording, while minimizing out-of-focus fluorescence background, sample photo-damage and photobleach-ing. However, images acquired with LSFM are often affected by light absorption or scattering effects, leading to un-even illumination and striping artifacts. Here we present an optical solution for such problem, via fast multi-directional illumination of the sample, based on an Acousto-Optical Deflector (AOD). Using this device, we were able to pivot the beam respect the propagation axis, with a scanning rate faster than the detector acquisition rate, in order to average the shadows attenuation at different angles over time. We also demonstrate that this scanning AOD system is compatible with Digital Scanned laser Light-sheet fluorescence microscopy (DSLM). Furthermore, we provided a theoretical model of the beam pivoting, looking for the optimal beam parameters to optimize the detection efficiency. It has been done because we wanted to adapt such scanning technique to Confocal Light Sheet Microscopy (CLSM), which intrinsically shows an incompatibility between the beam pivoting and the finite size of a digital slit used to spatially filter out spurious signals. We partially solved the problem expanding the beam and using two cylindrical lenses to create an elliptical-Gaussian beam which enables to cover more the digital slit while pivoting the beam. We test its performance by acquiring several mouse brain areas, observing real-time shadows suppression while preserving confocal detection of the signal emitted by specific fluorophores. A comparison between such scanning beam illumination and a traditional static one has been carried out in term of contrast analysis, striping suppression and Point Spread Function response.