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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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.
The authors declare no competing financial interests.
Integrated supplementary information
(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.
(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.
(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.
(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.
(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.
(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.
(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 Figures 1–7 (PDF 2885 kb)
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)
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)
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)
Reconstructions from movie stacks of a synthetic heart tube model synchronized using single- or dual-view synchronization. Scale bar 30 μm. (MOV 4874 kb)
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)
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)
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)
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)
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)
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)
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)
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)
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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