Light sheet fluorescence microscopy (LSFM) is a fluorescence microscope with an intermediate-to-high optical resolution, but good optical sectioning capabilities and high speed. Like in a conventional fluorescence microscope, the image is collected via an objective and detected by a camera. The main difference lies in the way the sample is illuminated by the excitation light.
Raw Light Sheet
Light Sheet DeconvolutionHuygens specific Light Sheet Fluorescence Microscopy (LSFM) deconvolution option supports a range of light sheets types, such as a Gaussian-profile, scanning beam, and scanning Bessel beam and lattice (see this overview). Huygens Light Sheet deconvolution improves contrast, reduces noise and blurring, takes depth-dependent spherical aberration correction into account, and is integrated with multiview fusion in Huygens Fuser.
Huygens Light Sheet Software
Image of Drosophila brain taken with Zeiss Z1 Light Sheet microscope. This multiview dataset consisting of eight views (one of the raw views is shown) was deconvolved and fused with the Huygens Software. Image kindly provided by Dr. Denis Ressnikoff, University Claude Bernard, Lyon, France.
Light Sheet basicsThe basic set up of simple Light Sheet microscope is shown below. Like in a conventional fluorescence microscope, the image is collected via an objective and detected by a camera. The main difference lies in the way the sample is illuminated by the excitation light. Instead of illuminating most or all of the sample, including regions outside the focus of the objective, in Light Sheet only a thin plane is illuminated. This allows for optical sectioning of the sample, and reduces photobleaching of the sample in regions above and below the plane that is being imaged.
Principle of Light sheet fluorescence microscopy. The excitation light is shaped by a cylindrical lens in order to illuminate a thin plane of the sample. The objective is oriented perpendicular to the illumination plane to capture an image of a thin section of the sample. (Image adapted from the Wikimedia Commons file "The principle setup of a light sheet fluorescence microscope": https://commons.wikimedia.org/wiki/File:Spim_prinziple_en.svg)
There are various types of Light Sheet setups that differ mainly in the way the light-sheet is generated. The Huygens Light Sheet optical option currently supports the following Light Sheet types:
- Lightsheets with a Gaussian profile, generated by illumination from one side, either by a cylindrical lens, or by scanning a beam.
- Lightsheets with a Gaussian profile, generated by simultaneous illumination from two opposing sides.
- Lightsheets with a Gaussian flat profile, generated by for example moving the focus along the sheet direction.
- Lightsheets generated by excitation lenses that are overfilled at their entry pupils (see ExcitationFillFactor for an explanation), either by using a cylindrical lens, or by scanning a beam.
- Lightsheets generated with a scanning Bessel beam or lattice.
The difference between these types of excitation are important for deconvolution, because they strongly influence the shape of the PSF. Lightsheets that are generated by lenses that are overfilled tend to be thinner, but at the cost of a more complex profile along the optical detection axis, and a sheet thickness that is less uniform over the image.
If you have the Huygens Light Sheet deconvolution option in your Huygens license the procedure below is applicable. If you have the Huygens Light Sheet Fusion & Deconvolution Wizard you can proceed to our Fusion & Deconvolution Wizard page to read more.