High-resolution reconstruction of the beating zebrafish heart

Abstract

The heart′s continuous motion makes it difficult to capture high-resolution images of this organ in vivo. We developed tools based on high-speed selective plane illumination microscopy (SPIM), offering pristine views into the beating zebrafish heart. We captured three-dimensional cardiac dynamics with postacquisition synchronization of multiview movie stacks, obtained static high-resolution reconstructions by briefly stopping the heart with optogenetics and resolved nonperiodic phenomena by high-speed volume scanning with a liquid lens.

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Figure 1: Cardiac imaging in SPIM.
Figure 2: Dual-view synchronization improves reconstruction at adverse imaging angles.
Figure 3: Reconstructed hearts at different developmental stages.
Figure 4: Optogenetics yields high-resolution images of the heart, and ETL-SPIM captures intracardiac blood flow.

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Acknowledgements

This work was supported by the Max Planck Society, the Human Frontier Science Program (CDA 00063/2010-C to J.H.) and a fellowship to M.M. from the Boehringer Ingelheim Fonds. We thank H. Otsuna for assistance with FluoRender and A. Reade, T. Op't Hof, R. Coronel, D.Y.R. Stainier and members of the Huisken laboratory for their comments. Initial work on this project was performed by J.H. in the labs of E.H.K. Stelzer, J. Wittbrodt and D.Y.R. Stainier.

Author information

M.M. and M.W. designed and built the SPIM setup. M.M. developed the single-view synchronization routine, performed and analyzed all experiments and visualized the data. B.S. developed the dual-view synchronization algorithm as well as the synthetic-heart-tube model and wrote the software to operate the SPIM setup. M.W. built the hardware for optogenetic manipulation. M.W. and M.M. performed the optogenetic experiments. F.O.F. designed, built, programmed and operated the ETL-SPIM setup. S.H. made the Tg(myl7:Gal4) line, and S.R. made the Tg(myl7:lifeactGFP) line. J.H. designed and supervised the project. M.M. and J.H. wrote the manuscript with contributions by M.W., B.S. and F.O.F.

Correspondence to Jan Huisken.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Fixation introduces artifacts.

(a) Anterio-ventral view of the heart of transgenic zebrafish embryos (Tg(myl7:GFP)) before and (b) after fixation. (c) Merged images after (d) registration. (e) Schematic of merged outlines of the beating heart at various phases in the cardiac cycle and the fixed heart. (f-h) Three individual data sets with outlines of the contraction phases in red lines and cyan area representing the fixed heart. Scale bar 30 µm.

Supplementary Figure 2 SPIM setup.

(a) Configuration of our SPIM setup for cardiac imaging with movie stacks. Illumination optics only partly shown. (b) Additional hardware for alternative methods for cardiac imaging including rotational motor for dual-view recording (i), an orange beam for optogenetical manipulation (ii), an ETL and a scanning mirror for volume scanning (iii), an LED for prospective gating (iv) and an additional illumination arm and camera for dual-plane recording (v). Not to scale.

Supplementary Figure 3 Heartbeat variability.

(a) Irregularities within consecutive heart beats in the same heart (Tg(myl7:GFP)). (b) Section of the heart at which data in (a) was taken. Lines indicate sites for kymographs in (c) through the atrium (A) and the ventricle (V). (d) Overlay of consecutive periods in the same heart with arrowheads indicating different wall motion. Scale bar 30 µm.

Supplementary Figure 4 Quality of synchronization depends on frame rate and movie length.

(a) The quality of synchronization as a function of the camera frame rate. (b) Side view of reconstructed heart walls (Tg(myl7:GFP)) at different frame rates. Arrowheads indicate artifacts and the red dashes indicate the myocardium. Scale bar 10 µm. (c) Image difference as a function of the movie length for high (400 fps) and low frame rate (67 fps) data. Error bars represent standard error of the mean. * p-value < 0.01.

Supplementary Figure 5 Alternative approaches for cardiac imaging.

(a) Principle of prospective gating with an external signal triggering image acquisition in each plane of the heart. (b) Principle of dual plane recording with two parallel light sheets.

Supplementary Figure 6 Phase delay introduces errors in single-view synchronization.

(a) Synthetic 4D heart tube model at 30°. (b) Image difference and shifts in synchronized synthetic movie stacks at various imaging angles. (c) Shifts in single-view synchronization of two synthetic movie stacks, one along and one orthogonal to the contraction propagation axis (imaging angle 0 and 90°, respectively). (d) Shifts in synchronization of two real perpendicular movie stacks.

Supplementary Figure 7 Optogenetics offers improved contrast and dynamic range.

(a) Outlines of the heart during cardiac contraction and when stopped with optogenetics. (b) Shape of the heart in repetitive optogenetic acquisitions. (c) Intensity profile at various exposure times. (d) Images of the heart (Tg(myl7:H2B-GFP)) taken with different exposure times.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 2885 kb)

Shape of the beating heart at various contraction phases.

Image of a 48 h.p.f. Tg(myl7:GFP) embryo with segmented outlines (pink dotted lines) of the heart over a full cardiac cycle. (MOV 2164 kb)

Movie stack synchronization.

Three synchronized planes of a movie stack of a 48 h.p.f. Tg(myl7:GFP) embryo and the 3D reconstruction. Scale bar 30 μm. (MOV 2299 kb)

Single view synchronization yields almost isotropic resolution.

Synchronized movie stack of a 48 h.p.f. Tg(myl7:GFP) embryo in maximum projected front view and a horizontal and vertical cut. Scale bar 30 μm. (MOV 2127 kb)

Dual view sync of synthetic movie stacks.

Reconstructions from movie stacks of a synthetic heart tube model synchronized using single- or dual-view synchronization. Scale bar 30 μm. (MOV 4874 kb)

Dual view sync of real movie stacks.

Reconstructions from movie stacks of a 30 h.p.f. Tg(myl7:GFP) embryo synchronized using single- or dual-view synchronization. Scale bar 30 μm. (MOV 7184 kb)

Reconstruction of endo- and myocardium in a 30 h.p.f. embryo.

Maximum projections showing cardiac contraction in real speed and slow motion in a 30 h.p.f. Tg(myl7:DsRed, kdrl:GFP) embryo. Scale bar 30 μm. (MOV 8910 kb)

Reconstruction of endo- and myocardium in a 48 h.p.f. embryo.

Volume rendering (left) and single slice (right) showing cardiac cycle in real speed and slow motion in a 48 h.p.f. Tg(myl7:DsRed, kdrl:GFP) embryo. Scale bar 30 μm. (MOV 4281 kb)

Reconstruction of endo- and myocardium in a 72 h.p.f. embryo.

Volume rendering of three different views showing cardiac contractions in real speed and slow motion in a 72 h.p.f. Tg(myl7:DsRed, kdrl:GFP) embryo. (MOV 5332 kb)

Reconstruction of endo- and myocardium in a 5 d.p.f. embryo.

Single slices in front and side view showing cardiac contractions in a 5 d.p.f. Tg(myl7:DsRed, kdrl:GFP) embryo. (MOV 9297 kb)

Optogenetically stopped heart.

Volume rendering showing the optogenetically stopped heart of a 5 d.p.f. Tg(myl7:lifeactGFP, myl7:Gal4, UAS:NpHR-mCherry) embryo. (MOV 3364 kb)

4D reconstruction of blood flow with single view synchronization.

Front and side view of synchronized heart of a 2 d.p.f. Tg(gata1a:DsRed, myl7:GFP) embryo. Scale bar 30 μm. (MOV 2824 kb)

4D reconstruction of blood flow with ETL-SPIM.

Front and side view of synchronized heart of a 2 d.p.f. Tg(gata1a:DsRed, myl7:GFP) embryo. Scale bar 30 μm. (MOV 3186 kb)

Arrhythmic heart imaged with ETL-SPIM.

Maximum projection of heart in 55 h.p.f. Tg(myl7:GFP) embryo treated with terfenadine. Scale bar 30 μm. (MOV 2532 kb)

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Mickoleit, M., Schmid, B., Weber, M. et al. High-resolution reconstruction of the beating zebrafish heart. Nat Methods 11, 919–922 (2014). https://doi.org/10.1038/nmeth.3037

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