Fast, high-contrast imaging of animal development
by scanned light sheet-based structured illumination microscopy
1Cell Biology and Biophysics Unit
European Molecular Biology Laboratory, Germany
2Janelia Farm Research Campus
Howard Hughes Medical Institute, USA
3Institute of Zoology, Department for Developmental Physiology
University of Heidelberg, Germany
4Developmental Biology Program
Sloan-Kettering Institute, USA
5Institute of Toxicology and Genetics
Karlsruhe Institute of Technology, Germany
6Frankfurt Institute for Molecular Life Sciences
Goethe University, Germany
Email: kellerp(at)janelia.hhmi.org, stelzer(at)embl.de
Nature Methods, 4 July 2010 (Advance Online Publication)
1) High-Quality Movies (Movies 1-6)
2) High-Quality Figures (Figures 1-4, S1-S16)
3) Digital Embryo Databases (Data Sets 1 and 2)
Note: If you encounter problems during playback of the DivX movies, you most likely require the latest version of the DivX codec for your media player. The codec is freely available for download on the following websites:
Movie 1: 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.
Download Movie 1 as a QuickTime file (1.4 MB, H.264 codec).
Movie 2: 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 mm.
Download Movie 2 as a DivX AVI file (26.3 MB, DivX 6.8.5 codec).
Download Movie 2 as a QuickTime file (38.9 MB, H.264 codec).
Movie 3: DSLM-SI long-term imaging of a membrane-labeled zebrafish embryo
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 mm. Images were deconvolved with the Lucy-Richardson algorithm (10 iterations). Fluorescence was detected with a Carl Zeiss C-Apochromat 10´/0.45 W objective.
Download Movie 3 as a DivX AVI file (136 MB, DivX 6.8.5 codec).
Download Movie 3 as a QuickTime file (102 MB, H.264 codec).
Movie 4: 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 mm long. In total 137,520 images were recorded for 191 time points (3 min sampling intervals). Temperature: 20 °C during preparation (0-2 h.p.f.), 24 °C during imaging (> 2 h.p.f.). Images were deconvolved with the Lucy-Richardson algorithm (5 iterations). Carl Zeiss Plan-Apochromat 20´/1.0 W.
Download Movie 4 as a DivX AVI file (16.7 MB, DivX 6.8.5 codec).
Download Movie 4 as a QuickTime file (33.7 MB, H.264 codec).
Movie 5: 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.
Download Movie 5 as a DivX AVI file (20.9 MB, DivX 6.8.5 codec).
Download Movie 5 as a QuickTime file (23.4 MB, H.264 codec).
Movie 6: The Drosophila Digital Embryo
Different perspectives of the Drosophila 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 mm min–1, cyan to orange).
Download Movie 6 as a DivX AVI file (22.0 MB, DivX 6.8.5 codec).
Download Movie 6 as a QuickTime file (44.9 MB, H.264 codec).
Figures included in
the PDF documents (Adobe Acrobat
Reader 6.0 or higher required):
Figure 1: Light sheet-based structured illumination with digitally adjustable frequency
Figure 2: Enhancing image contrast with DSLM structured illumination
Figure 3: Multiple-view imaging of Drosophila embryogenesis with DSLM-SI
Figure 4: Spatiotemporal image contrast optimization by DSLM-SI frequency chirping
Figure S1: DSLM-SI implementation
Figure S2: DSLM-SI illumination patterns for the typical range of SI frequencies
Figure S3: DSLM-SI illumination patterns for high SI frequencies
Figure S4: Mirror surface reconstruction with DSLM-SI
Figure S5: Imaging tissue phantoms with DSLM-SI
Figure S6: DSLM-SI performance at low to intermediate scattering coefficients
Figure S7: DSLM-SI performance at high scattering coefficients
Figure S8: Contrast enhancement in Medaka DSLM-SI
Figure S10: DSLM-SI coverage at advanced zebrafish embryonic development
Figure S11: DSLM-SI coverage at late zebrafish embryonic development
Figure S12: Removal of scattered light in zebrafish DSLM-SI recordings
Figure S13: Removal of scattered light in Drosophila DSLM-SI recordings
Figure S14: DSLM-SI penetration depth in early Drosophila embryogenesis
Figure S15: Workflow for processing and visualization of DSLM-SI recordings
Figure S16: Multiple-view fusion of the Drosophila Digital Embryo
Download high-quality Figures S1-S16 (235 MB, ZIP archive).
3) Digital Embryo Databases
Each digital embryo database is provided as a Matlab MAT-file, containing a multi-cell array (with each cell corresponding to one time point) and a two-dimensional parameter table in each cell characterizing the nuclei segmented at the respective time point. Each table has n rows (n being the number of nuclei at the respective time point) and 6 columns listing the following parameters:
Number of time points and temporal
Download the Matlab database for data set 1.
Data set 2: Fused reconstruction of early Drosophila wild-type development
Number of time points and temporal
Download the Matlab database for data set 2.
(c) Philipp J. Keller, Janelia Farm/HHMI; June 2010