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Light-Sheet Microscopy with holographic Bessel beams

Background: In light-sheet microscopy only the part of
thick object is illuminated from the side that is in the
focal plane of the objective lens (OL).
Problem: Thick, scattering media make the illumination
beam scattering along the propagation direction
z and
thus degrading image quality.
Approach: Self-reconstructing Bessel beams, generated
by a computer
hologram (SLM), are scanned laterally in
the plane of focus. Self-healing photons in the Bessel
beam's ring system
result in an amazingly constructive
interference in the
Bessel beam center leading to 50%
increase of ballistic photons in propagation direction.


MISERB Propagation Stability w Comments

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.

MISERB Penetration Depth

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 Hobj(kr,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









Arabidopsis Root Tip imaged by Light-Sheet_Microscopy. (Left) Illumination with Gaussian Beam,
conventional detection, and no post-processing (Right) Illumination with Bessel Beam, line
confocal detection and separation of diffusive and ballistic photons by post-processing.




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