Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.
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We thank members of the mechanical workshop of the European Molecular Biology Laboratory for custom hardware; A. Riedinger and G. Ritter for custom electronics; F. Härle and A. Riedinger for custom microscope software; J. Topczewski (Northwestern University) for the ras-eGFP zebrafish strain; M. Ludwig and K. White (University of Chicago) for the histone-eGFP Drosophila strain; and A. Diaspro, F. Cella and P. Theer for helpful discussions. Financial support was provided to A.D.S. by Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology.
E.H.K.S. filed a patent application for light sheet–based structured illumination fluorescence microscopy: US patent application 11/592, 331, 2006.
Supplementary Figures 1– 16 and Supplementary Table 1 (PDF 10119 kb)
DSLM structured illumination. A schematic illustration of the DSLM with an intensity-modulated laser illumination pattern. The sinusoidal intensity profile was generated by scanning the beam through the specimen at a constant speed while synchronously modulating the laser intensity with an acousto-optical tunable filter (AOTF). Inset, close-up of the illuminated specimen inside the specimen chamber. (MOV 1492 kb)
Fast multichannel imaging of early zebrafish embryogenesis with DSLM-SI. Maximum-intensity projections of a DSLM time-lapse recording of a membrane- and nuclei-labeled zebrafish embryo. Membranes were imaged using structured illumination (SI-25, top row), nuclei using standard light sheet illumination (bottom left). The top-most cell layer (the enveloping layer, EVL) was removed computationally in the ras-eGFP channel and is shown on the right, separate from the deeper cell layers (left). The egg diameter is approximately 720 μm. (MOV 13593 kb)
DSLM-SI long-term imaging of a membrane-labeled zebrafish embryo. Maximum-intensity projections of a DSLM-SI time-lapse recording of a zebrafish embryo (ras-eGFP transgenic line), during the period 9–67 h.p.f. To provide an unobstructed view of the embryo, the top-most cell layer (the enveloping layer, EVL) was removed computationally. The egg diameter is approximately 720 μm. Images were deconvolved with the Lucy-Richardson algorithm (10 iterations). Fluorescence was detected with a Carl Zeiss C-Apochromat 10 × 0.45 W objective. (MOV 39342 kb)
Multiple-view imaging of Drosophila embryogenesis with DSLM-SI. Maximum-intensity projections of a DSLM-SI multiple-view time-lapse recording of a nuclei-labeled Drosophila embryo. The embryo is approximately 520 μm long. (MOV 10659 kb)
Reconstructing Drosophila embryogenesis from DSLM-SI data. Computational alignment of the four point clouds representing the nuclei detected in the four DSLM-SI views of the developing Drosophila embryo. Nuclei shown in different colors originate from different microscopic views. (MOV 13702 kb)
The Drosophila digital embryo. Different perspectives of the fly digital embryo, obtained by multiple-view fusion of the four nuclear point clouds and color-coding of directed regional nuclei movement speeds over 10-min intervals (0–0.8 μm min−1, cyan to orange). (MOV 12929 kb)
Raw reconstruction of early Drosophila wild-type development. (ZIP 50725 kb)
Fused reconstruction of early Drosophila wild-type development. (ZIP 21054 kb)
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Keller, P., Schmidt, A., Santella, A. et al. Fast, high-contrast imaging of animal development with scanned light sheet–based structured-illumination microscopy. Nat Methods 7, 637–642 (2010). https://doi.org/10.1038/nmeth.1476
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