Light-sheet microscopy in thick media using scanned Bessel beams and two-photon fluorescence excitation

Florian Fahrbach (1, 2) , Vasily Gurchenkov (3, 4) , Kévin Alessandri (3) , Pierre Nassoy (5, 3) , Alexander Rohrbach (2, 6)

1 MPI-CBG - Max Planck Institute of Molecular Cell Biology and Genetics
2 Laboratory for Bio- and Nano-Photonics, University of Freiburg
3 PCC - Physico-Chimie-Curie
4 CDC - Compartimentation et dynamique cellulaires
5 lp2n-04,lp2n-16
6 Centre for Biological Signalling Studies (bioss)

In this study we show that it is possible to successfully combine the benefits of light-sheet microscopy, self-reconstructing Bessel beams and two-photon fluorescence excitation to improve imaging in large, scattering media such as cancer cell clusters. We achieved a nearly two-fold increase in axial image resolution and 5-10 fold increase in contrast relative to linear excitation with Bessel beams. The light-sheet penetration depth could be increased by a factor of 3-5 relative to linear excitation with Gaussian beams. These findings arise from both experiments and computer simulations. In addition, we provide a theoretical description of how these results are composed. We investigated the change of image quality along the propagation direction of the illumination beams both for clusters of spheres and tumor multicellular spheroids. The results reveal that light-sheets generated by pulsed near-infrared Bessel beams and two photon excitation provide the highest image resolution, contrast and light-sheet penetration at a minimum amount of artifacts.

Keywords: DEEP; FIELD; SELECTIVE PLANE ILLUMINATION; PROPAGATION; DEPTH

Domains: Biological Physics [physics.bio-ph]; Optics [physics.optics]

Florian Fahrbach, Vasily Gurchenkov, Kévin Alessandri, Pierre Nassoy, Alexander Rohrbach. Light-sheet microscopy in thick media using scanned Bessel beams and two-photon fluorescence excitation. Optics Express, 2013, 21 (11), pp.13824-13839. ⟨10.1364/OE.21.013824⟩. ⟨hal-00994649⟩