Light Sheet Deconvolution
Huygens supports a wide range of Light Sheet Fluorescence Microscopy (LSFM) setups and different types of light sheets. Light sheets can vary in their shape and intensity distribution which are important factors that determine image quality. For example, the shape of a singe gaussian beam can be adjusted for a larger field of view (FOV), yet this will be at the expense of a lower axial resolution due to the increased sheet thickness. To maintain a high resolution and thinner sheet across a large FOV, a gaussian sheet can be moved along the propagation direction (2), or alternative light sheets can be used such as the Bessel Beam scanning light-sheet (3).
Huygens versatile Light Sheet Deconvolution option includes a point spread function (PSF) model that offers optimal results, since it takes both the light sheet type and PSF variation within the light sheet into account. The required Light Sheet microscopy-specific parameters needed to build an optimal PSF model are read from the image file, and can also be easily adjusted in the Microscopy parameter Editor. They are explained below. For further stitching and fusing of multiview images, we like to refer you to the Huygens Stitcher and Huygens Fuser.
Image description
De-skewing and deconvolution of lattice light sheet images. A skewed image of a breast carcinoma cell (cell line SUM159), expressing an ER marker linked to OX-GFP and a cytosolic protein that should mainly localize to lipid droplets, was acquired with a dithered, multi-Bessel beam Lattice Light Sheet system. The image was de-skewed in the Huygens Object Stabilizer. MIPs of the raw and deconvolved endoplasmic reticulum signal (A and B, respectively) and their corresponding magnifications (A’ and B’) show a more well-defined reticular ER pattern in the cell periphery and close to the nucleus after deconvolution. The line in panel A’ indicates the location in the raw and deconvolved image at which an intensity profile was produced (C; raw, undashed; deconvolved, dashed line). Panel D shows a MIP of the marker that localizes to rings around lipid droplets. These rings are difficult to discern in single slices of the raw image (D’), whereas deconvolution makes their appearance much clearer (E). The intensity profiles of a line in D’ show an increase of signal (more than 2 times) and resolution (more than 1.5 times) after deconvolution (F; raw, undashed line; deconvolved, dashed line). Scale bars are in micrometers.
De-skewing and deconvolution of lattice light sheet images. A skewed image of a breast carcinoma cell (cell line SUM159), expressing an ER marker linked to OX-GFP and a cytosolic protein that should mainly localize to lipid droplets, was acquired with a dithered, multi-Bessel beam Lattice Light Sheet system. The image was de-skewed in the Huygens Object Stabilizer. MIPs of the raw and deconvolved endoplasmic reticulum signal (A and B, respectively) and their corresponding magnifications (A’ and B’) show a more well-defined reticular ER pattern in the cell periphery and close to the nucleus after deconvolution. The line in panel A’ indicates the location in the raw and deconvolved image at which an intensity profile was produced (C; raw, undashed; deconvolved, dashed line). Panel D shows a MIP of the marker that localizes to rings around lipid droplets. These rings are difficult to discern in single slices of the raw image (D’), whereas deconvolution makes their appearance much clearer (E). The intensity profiles of a line in D’ show an increase of signal (more than 2 times) and resolution (more than 1.5 times) after deconvolution (F; raw, undashed line; deconvolved, dashed line). Scale bars are in micrometers.
Light sheet Microscopic Parameters
To prepare the image for Huygens LSFM deconvolution, it is recommended to verify the microscopic parameters of the image data first. If the image meta-data contains information about these parameters, Huygens will incorporate them into the parameter template.Light Sheet excitation mode
With the Light Sheet excitation mode, you specify the Light Sheet type that was used. Note that only the microscopic parameters that apply to the selected light sheet type are shown.- Gaussian light sheet. Appropriate for LSFM system that use a cylindrical lens or a scanning beam where the fill factor at the entry pupil of the excitation is low. Systems such as LaVision UltraMicroscope and Zeiss Z1 microscopes can use this mode.
- Gaussian MultiView light sheet. As “Gaussian light sheet”, but with simultaneous illumination from opposing sides.
- Gaussian Flat light sheet. Suited for LSFM system that use a cylindrical lens or a scanning beam where the fill factor at the entry pupil of the excitation is low, and which beam waist is moved along the sheet direction for the purpose of improving the axial resolution across the field of view.
- High fill factor, scanning beam. Appropriate for a LSFM system where a scanning beam is used to form the light sheet, and where the entry pupil of the excitation lens is overfilled. Systems such as the Leica Digital Light Sheet can use this mode.
- Scanning Bessel beam. Use this option for a LSFM system where a scanning Bessel beam is used to form the light sheet. If the multi-photon excitation parameter is adjusted, Huygens is able to account for multi-photon illumination as well.
- Scanning Bessel lattice. Use this option for a LSFM system where a scanning Bessel lattice is used to form the light sheet. If the multi-photon excitation parameter is adjusted, Huygens is able to account for multi-photon illumination as well.
- High fill factor, cylinder. Appropriate for a LSFM system where a cylindrical lens is used to form the light sheet, using a high fill factor at the entry pupil.
Width of Gaussian Sheet
This parameter appears only in the “Gaussian light sheet” and “Gaussian MultiView light sheet” modes. It specifies the width (thickness) of the sheet in micrometer. The width of the Gaussian profile is defined as the distance between the two points where the value is equal to the following equation of the peak value. To convert the FWHM value to this full width of the beam you can multiply it with 1.7.Light sheet NA
The numerical aperture (NA) of the excitation lens parameter appears in the “High fill factor, scanning beam”, “Scanning Bessel beam”, “Scanning Bessel lattice” and “High fill factor, cylinder” modes. Note that the NA of the detection lens is specified under the (Main) Optical Parameters. If the NA of the excitation lens or its fill factor are unknown, but the effective NA is known, proceed as follows:- Set the light sheet NA to 4 times the effective NA
- Set the fill factor to 0.25
Light sheet fill factor
This parameter is only applicable for the “High fill factor, scanning beam” and “High fill factor, cylinder” modes. The value indicates the Fill Factor of the excitation (LSFM) lens. The Fill Factor is the ratio between the beam width and the diameter of the objective pupil. Huygens default value is “0.5”, meaning that the illumination beam is half as wide as the objective pupil of the LSFM lens.Light sheet focus offset
This value defines how far the light sheet is located below (negative value) or above (positive value) the focal point of the detection lens. This parameter cannot be read from the image file. Default value is “0”, which is the optimal value in a well-aligned system.Sheet lateral offset
Specifies the distance between the focal point of the excitation lens and the optical axis of the detection lens. A negative value indicates that the “middle” of the light sheet is shifted towards the excitation lens. A positive value indicates a shift away from the excitation lens. Generally, this value is not read from the image file. The value is equal to zero in a well-aligned system. However, this may be different in cases where the image is cropped, or where the optical setup allows you to change this, or where it is simple not well aligned.Light sheet direction
The parameter defines where the excitation objective is positioned with respect to the detection lens.- From right
- From top
- From left
- From bottom
Scatter Parameters
Huygens is able to address scattering in your sample by adjusting the PSF. The scattering correction can be optimized via the Scatter parameter launch button within the microscopic parameter window. Here, you can adjust the scatter model (uniform exponential, uniform Gaussian, and 1D X-direction exponential), and the length of the free path (in micron) of the emission light, and the percentage of the scattered light with respect to the non-scattered direct light. The effectiveness of the scattering correction needs to be determined empirically since it is nearly impossible to have a priori knowledge of scattering properties of the sample.Deconvolution optimization
As with any type of image acquisition, Nyquist sampling is one of the most important factors that influences image quality and the effectiveness of deconvolution. Please prioritize this acquisition setting over signal intensity, and consult our online SVI Nyquist Calculator before you start imaging. Also, prevent any clipping and avoid zero or maximum values in your image. Set the microscopic image parameters correctly (see above), and when applying deconvolution, use the CMLE algorithm, disable bleaching correction, and optimize the deconvolution by adjusting first the Acuity setting. If this does not give you satisfactory results, you can adjust the SNR value.References
1. Hildebrand, S., Schueth, A., Wangenheim, K.v. et al. (2020). hFRUIT: An optimized agent for optical clearing of DiI-stained adult human brain tissue.https://doi.org/10.1038/s41598-020-66999-3
Huygens PSF distiller, Object Stabilizer for deskewing, and deconvolution was used for restoring Light Sheet data.
2. Düring D, Diales Rocha M, Dittrich F, Gahr M, Hahnloser R (2019). Expansion Light Sheet Microscopy Resolves Subcellular Structures in Large Portions of the Songbird Brain. https://doi.org/10.3389/fnana.2019.00002
Huygens deconvolution was used in combination with expansion microscopy and light sheet imaging
3. Stelzer, E.H.K., Strobl, F., Chang, BJ. et al. Light sheet fluorescence microscopy. Nat Rev Methods Primers 1, 73 (2021). https://doi.org/10.1038/s43586-021-00069-4
4. Liang Gao, "Extend the field of view of selective plan illumination microscopy by tiling the excitation light sheet," Opt. Express 23, 6102-6111 (2015)
5. Fahrbach, F., Simon, P. & Rohrbach, A. Microscopy with self-reconstructing beams. Nature Photon 4, 780–785 (2010). https://doi.org/10.1038/nphoton.2010.204