Principle
In scanned lightsheet SPIM microscopy, the selective illumination is obtained by rapidly scanning a single focused laser beam on a plane, while the same plane is being imaged by a camera looking from the orthogonal direction.
Using a single scanned beam allows for illuminating the sample both with gaussian or Bessel beams, or allows for using two-photon excitation. Moreover, by modulating the intensity of the illuminating beam during the exposure time, it is possible to create a pattern that can be used for acquiring structured illumination images.
A simple simulation of the imaging process can be seen here.
Setup
We built a scanned light sheet SPIM. Our setup can be quickly configured for using Gaussian or Bessel beam illumination, both using single or two-photon excitation.
By changing the parameters of the illumination beams, such as the numerical aperture, or its nature (gaussin or Bessel profile) it is possible to control the region where the beam stays focused. The experimental results agree with the simulated beam profiles.
Single photon (
=488 nm) gaussian beam, N.A.=0.8
Single photon (=488 nm) Bessel beam, N.A.=0.251/0.238
Single photon (=488 nm) Bessel beam, N.A.=0.251/0.245
Two-photon (=920 nm) gaussian beam, N.A.=0.8
Two-photon (=920 nm) Bessel beam, N.A.=0.251/0.238
Two-photon (=920 nm) Bessel beam, N.A.=0.251/0.245
Experimental results
Imaging of a developing C. elegans embryo, labelled with histone-GFP