Common in Light Sheet Fluorescence Microscopy (LSFM) is the acquistion of multiple (opposing or rotational) views of the object and to fuse these to compensate for light absorption and scattering issues which are so typical in light sheet imaging. Since Huygens 17.04, the Fusion and Deconvolution Wizard has been used by many of you to deconvolve and fuse multiview images, yet direct visual feedback in the alignment procedure was lacking. Also from the response and many images we received from you all (thank you!), which emphasized again the wide range of light sheet microscopy systems and imaging conditions being used, we recognized that additional expert fusion parameters may be useful to further optimize fusion.
Therefore, the new Huygens FUSER includes besides the existing functionalities, and CPU + GPU support of the Fusion and Deconvolution Wizard, an even more extensive set of fusion parameters, interactive scenes for optimal positioning of each view, real-time visual feedback during the alignment process, and a pre-cropping function for faster alignment and more efficient computational usage.
Optimal interactive positioning of each view and direct visual feedback makes fusion easy, efficient, and fast.
Specific deconvolution (see picture) and fusion settings for various light sheet setups with extensive help information allow optimal image restoration.
Huygens unique Light Sheet variable PSF modeling ensures high-quality LSFM deconvolution.
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Stripes and shading problems are well-known imaging issues in light sheet microscopy. To minimize these, the sample is typically rotated and imaged from different directions. These different views then need to be fused into one single superior image to have all parts of the specimen imaged optimally. Huygens FUSER is extremely versitale as it helps you with the alignment and fusion of multiview 2D-5D (incl. multi-channel and time) images, which can be aquired with any type of Light Sheet microscope and from any imaging direction.
Two examples of Zeiss Z1 Light Sheet datasets deconvolved and fused with the Huygens FUSER. The first dataset is from a Drosophila brain acquired at 360 degrees rotation (45 degrees steps), and the second set is from a chicken embryo imaged from two opposing sides. Courtesy of Prof. Christophe Marcelle, Mrs. Marie Julie Dejardin (INMG) & Dr. Denis Ressnikoff (CIQLE), Université Lyon 1, France.
Huygens FUSER main window shows on the left previews of each image as maximum intensity projections along the selected axis of rotation. Selected views for fusion are displayed in different customizable colors, and each view can be activated, manually placed, and aligned on top of the others within the center scene by simply using the mouse cursor. After initial setting of the angles and approximate relative shifts, Huygens FUSER fine-tunes the alignment of each view. The FUSER permits full control over the alignment by providing real-time visual feedback during the fusion process. Expert crop, fusion, and sampling settings with detailed explanations are available to cover a broad range of needs, like getting a fused image fast at rough voxel grid size using limited computational resouces, or obtaining at higher computational costs the finest voxel grid size and highest possible resolution.
The Huygens Fuser Window showing multiple views as maximum intensity projections which can be manually aligned before the actual fusion process fine-tunes the alignment. Courtesy of Dr. Denis Ressnikoff from the University Claude Bernard Lyon 1, France.
Similar to our previous Huygens Fusion & Deconvolution Wizard, the new Huygens FUSER has the option to deconvolve and fuse Light Sheet images within one single workflow. Several different light sheet setups are supported which include gaussian, high fill factor, scanning and lattice-based systems. By selecting parameter templates customized for your specific LSFM data, both the deconvolution with a light sheet variable PSF and the fusion are easily and reproducibly executed. Huygens unique set of additional restoration options even allow you to correct for additional imaging artefacts like hot&cold pixels, crosstalk (bleedthrough), chromatic aberration, and drift.
Maximum Intensity Projection of a raw (left) and deconvolved (right) 3D image from mouse blastocysts acquired with a Leica Digital Light Sheet microscope. Deconvolution was performed with the CMLE algorithm and the new Huygens module for calculating the theoretical Light Sheet point-spread-function. Courtesy of Dr. Marc Duque Ramirez and Dr. Ritsuya Niwayama (Hiiragi group) and Dr. Stefan Terjung (ALMF) from the EMBL Heidelberg, Germany.