Restoration of Light Sheet Multi-View Data with the Huygens Fusion and Deconvolution Wizard

Peter J. Verveer , 1 Vincent T.G. Schoonderwoert , 1 * Denis Ressnikoff , 2 Shane D. Elliott , 3 Kiefer

D. van Teutem , 1 Tobias C. Walther , 3 and Hans T.M. van der Voort 1 1 Scientifi c Volume Imaging bv , Laapersveld 63 , 1213VB , Hilversum , T e Netherlands 2 CIQLE , Centre d’imagerie quantitative Lyon-Est , Université Claude Bernard Lyon 1 , Lyon , France 3 Department of Genetics and Complex Diseases , Harvard University , Boston , MA


Abstract: Light sheet fl uorescence microscopy (LSFM) allows for high- resolution three-dimensional imaging with minimal photo-damage. By viewing the sample from different directions, different regions of large specimens can be imaged optimally. Moreover, owing to their good spatial resolution and high signal-to-noise ratio, LSFM data are well suited for image deconvolution. Here we present the Huygens Fusion and Deconvolution Wizard, a unique integrated solution for restoring LSFM images, and show that improvements in signal and resolution of 1.5 times and higher are feasible.

Keywords: Light sheet fl uorescence microscopy (LSFM) , selective plane illumination microscopy (SPIM) , fusion , deconvolution , Huygens


Fluorescence microscopy facilitates selective imaging of cellular components at high resolution in fixed and living samples. In particular, fluorescence imaging of genetically encoded reporters has become an important tool to investigate living specimens in various fields, such as cell biology, neuroscience, and developmental biology. Yet, fluorescence microscopy suffers from a number of fundamental constraints that, depending on the experi- mental conditions, require trade-offs in the acquisition of data. Specifically, the spatial resolution in three dimensions is limited by the laws of physics. Perhaps even more limiting in practice, the ability to observe the sample over long periods with high signal-to-noise ratio is restricted by photo- damage induced by the excitation light. This is exemplified best in developmental biology experiments that require relatively large multi-cellular organisms to be observed at regular intervals over a long period of time. Well-established microscopy methods such as widefield/epi-fluorescence or confocal microscopy fare poorly in such experiments, either because of the lack of three-dimensional (3D) resolution (widefield microscopy) or the extensive photo-damage that they inflict (confocal microscopy). In the last decade, light sheet fl uorescence microscopy (LSFM), also referred to as selective plane illumination microscopy (SPIM), has emerged as a technique that provides a near-optimal compromise, providing good spatial resolution in three dimensions while limiting photo-damage, making long-term observation of large specimens feasible [ 1 , 2 , 3 ]. T e remarkable performance of LSFM is based on a simple idea: using a thin sheet of light, only the plane of interest in the sample is excited (and hence subjected to photo-damage) and imaged. T is is achieved by decoupling the excitation and emission paths of the microscope: the sheet of light is generated by one set of optics, while the image is detected via


a separate optical path that is oriented perpendicular to the optical axis of the excitation light ( Figure 1A ). T is approach to selectively illuminate and image planes in a sample provides for highly effi cient 3D imaging at resolutions close to that of a confocal microscope with a much improved signal-to-noise ratio. T e success of LSFM quickly led to a fl urry of technical developments to address imaging artifacts known to be present in conventional LSFM: photon scattering and sample-dependent photon absorption (leading to stripes and shading) ( Figure 2A ) [ 1 , 2 ]. It was quickly realized that the geometry of the setup allowed the sample to be rotated and viewed from diff erent directions ( Figure 1A ). T e diff erent views could then be fused into a single superior image where all parts of the specimen are imaged optimally [ 4 ]. A second development

Figure 1 : LSFM and PSFs. (A) Typical for light sheet fl uorescence microscopy is the decoupling of excitation and emission paths. One objective lens generates a thin sheet of light that illuminates the focal plane (excitation), and emitted light is detected via a separate optical path oriented perpendicular to the optical axis of the excitation light. Specifi c LSFM setups allow rotation of the sample for acquiring views from multiple directions. (B) The PSF changes spatially as a function of the distance from the focus to the excitation objective. Shown are false color coded XZ views of PSFs for a Gaussian light sheet.

doi: 10.1017/S1551929518000846 • 2018 September

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