title: Correcting spherical aberrations in confocal light sheet microscopy: a theoretical study.
abstract: In the last years, fluorescence light sheet microscopy has attracted an increasing interest among the microscopy community. One of the most promising applications of this technique is the reconstruction of macroscopic biological specimens with microscopic resolution, without physical sectioning. To this aim, light sheet microscopy is combined with clearing protocols based on refractive index matching, which render the tissue transparent. However, these protocols lead to a huge drop in the fluorescence signal, limiting their practical applicability. The reduction of signal to background ratio is commonly ascribed to chemical degradation of the fluorophores by the organic solvents used for clearing. This view however completely neglects another important factor of contrast loss, i.e., optical aberrations. In fact, commercially available objectives suitable for light sheet microscopy are not designed for the refractive index of the clearing solutions, and this mismatch introduces severe spherical aberration. Here we simulated the aberrated point spread function (PSF) of a light sheet microscope with confocal slit detection. We investigated the variation of the PSF as a function of objective numerical aperture (NA) and of imaging depth inside the clearing solution. We also explored the possibility of correcting such spherical aberration by introducing extra optical devices in the detection path. By correcting up to the second order spherical aberration, a quasi-diffraction-limited regime can be recovered, and image quality is restored.
authors.affiliation: European Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Via Nello Carrara, 1 - 50019 Sesto Fiorentino (FI), Italy.
authors.forename: L
authors.initials: L
authors.lastname: Silvestri
authors.forename: L
authors.initials: L
authors.lastname: Sacconi
authors.forename: F S
authors.initials: FS
authors.lastname: Pavone
mesh_terms: D007089:Image Enhancement / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods*; D008962:Models, Theoretical; D055620:Optical Phenomena
keywords: brain imaging; fluorescence microscopy; optical clearing