Light-Sheet Microscopy with holographic Bessel beams

Beam Profile of a Bessel beam upon propagation through a scattering medium. The main lobe is barely deviated, demonstrating the propagation stability of a Bessel beam.

Penetration depth of a Bessel beam compared to a Gaussian beam inside a shpere cluster.

Separation of ballistic and diffusive fluorescence photons

Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We have developed a theoretical model allowing to extract typical scattering parameters  of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection.

 
The object transfer function H_obj(k_r,y₀) allows to distinguish between ballistic and diffusive photons and their contributions for each image in a depth y₀. It can be approximated as

Sketch of the experimental setup