Scientific Results

Correcting Spherical Aberrations in Confocal Light Sheet Microscopy: A Theoretical Study

Year: 2014

Authors: Silvestri L., Sacconi L., Pavone F.S.

Autors Affiliation: European Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Via Nello Carrara, 1 – 50019 Sesto Fiorentino (FI), Italy;
National Institute of Optics, National Research Council (INO-CNR), Via Nello Carrara, 1 – 50019 Sesto Fiorentino (FI), Italy;
Department of Physics, University of Florence, Via G. Sansone, 1 – 50019 Sesto Fiorentino (FI), Italy;
International Center for Computational Neurophotonics (ICON Foundation), Via Nello Carrara 1 – 50019 Sesto Fiorentino (FI) Italy

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. (C) 2014 Wiley Periodicals, Inc.

Journal/Review: MICROSCOPY RESEARCH AND TECHNIQUE

Volume: 77 (7)      Pages from: 483  to: 491

More Information: Contract grant sponsors: LASERLAB-EUROPE; Contract grant numbers: 228334, 284464; Contract grant sponsors: EC\’s Seventh Framework Programme, European Flasghip Human Brain Project; Contract grant number: 604102; Contract grant sponsors: Regione Toscana; Contract grant number: POR-CreO 2007-2013 (SMAG project); Contract grant sponsors: Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX, Italian Ministry of Health in the framework of the \’Stem Cells Call for proposals\’.
DOI: 10.1002/jemt.22330

Citations: 9
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English