The fly digital embryo

Fast, high-contrast imaging of animal development

by scanned light sheet-based structured illumination microscopy

Philipp J. Keller^1,2*, Annette D. Schmidt³, Anthony Santella⁴, Khaled Khairy¹,
Zhirong Bao⁴, Joachim Wittbrodt^3,5 and Ernst H.K. Stelzer^1,6*

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

^*To whom correspondence should be addressed.

Email: kellerp(at)janelia.hhmi.org, stelzer(at)embl.de

Nature Methods, 4 July 2010 (Advance Online Publication)

Overview of the contents of this data repository

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)

1) Digital Embryo Movies

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:

Apple Mac:	http://www.divx.com/en/software/mac
Microsoft Windows:	http://www.divx.com/en/software/divx-plus

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 DivX AVI file (2.0 MB, DivX 6.8.5 codec).

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).

2) Figures 1-4 and S1-S16 in high quality

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 recordings
Figure S9: DSLM-SI coverage in zebrafish multi-channel recordings

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 1-4 (134 MB, ZIP archive).

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:

Columns 1-3:	x-y-z coordinates of the center-of-mass of the nucleus (length unit = 370 nm)
Column 4:	Diameter of the sphere matching the volume occupied by the nucleus
Column 5:	Average fluorescence intensity of the nucleus (arbitrary units)
Column 6:	Index of the microscopic view the nucleus originates from (only applicable to the raw reconstruction of the multiple-view data set; indices 1-4 correspond to views 0 °/90 °/180 °/270 °)

Data set 1: Raw reconstruction of early Drosophila wild-type development

Specification of data set 1:
Filename: "drosophila_raw.mat"

Number of time points and temporal sampling:
191 time points (120-690 m.p.f.), 180 sec intervals
Total number of nucleus entries: 3,624,857
Database size: 49.5 MB
Related database items: Figures 3a-b, S13-S16; Movie 4, Movie 5

Download the Matlab database for data set 1.

Data set 2: Fused reconstruction of early Drosophila wild-type development

Specification of data set 2:
Filename: "drosophila_fused.mat"

Number of time points and temporal sampling:
191 time points (120-690 m.p.f.), 180 sec intervals
Total number of nucleus entries: 1,486,971
Database size: 20.5 MB
Related database items: Figures 3a-c, S13-S16; Movie 4, Movie 6

Download the Matlab database for data set 2.