Optical tomography complements light sheet microscopy for in toto imaging of zebrafish development.
Bottom Line: Fluorescently labeled structures can be spectrally isolated and imaged at high resolution in living embryos by light sheet microscopy.We found that the bright-field contrast of unstained specimens in a selective plane illumination microscopy (SPIM) setup can be exploited for in vivo tomographic reconstructions of the three-dimensional anatomy of zebrafish, without causing phototoxicity.We report multimodal imaging of entire zebrafish embryos over several hours of development, as well as segmentation, tracking and automatic registration of individual organs.
Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany Politecnico di Milano, Dipartimento di Fisica, Milano 20133, Italy.Show MeSH
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Mentions: To integrate an optical tomography approach in a SPIM microscope we took advantage of three features of our existing SPIM setup: (1) fast image acquisition with high frame rate sCMOS cameras; (2) multiview capability, i.e. the sample can be quickly rotated; and (3) LED for back illumination, which can provide transmission images of the specimen. Owing to the relatively high NA of the detection lens (0.3), the depth of field δz is only ∼15 µm and does not span the depth of a typical sample in SPIM of ∼0.1-1 mm (Fig. 1). Therefore, a stack of ∼20 transmission images was taken by sliding the sample through the detection objective's plane of focus. The in-focus information was extracted by high-pass filtering the images and a weighted average yielded a projection of the sample with enhanced depth of field (Häusler, 1972) (supplementary material Methods). Since a projection represents an approximation of the line integral of light attenuation along a certain direction, by collecting multiple projections from several directions (typically 360) we created a dataset suitable for tomographic reconstruction; optically sectioned volumes of the samples were obtained by a filtered back-projection algorithm (Kikuchi and Sonobe, 1994; Fauver et al., 2005). No modification to the SPIM hardware or calibration was needed for the tomographic reconstruction. This method can therefore be readily adopted by a large number of systems, including commercial light sheet microscopes and simple SPIM implementations such as OpenSPIM (Pitrone et al., 2013; Gualda et al., 2013).Fig. 1.
Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany Politecnico di Milano, Dipartimento di Fisica, Milano 20133, Italy.