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Parallelized computational 3D video microscopy of freely moving organisms at multiple gigapixels per second

Abstract

Wide-field-of-view microscopy that can resolve three-dimensional (3D) information at high speed and spatial resolution is particularly desirable for studying the behaviour of freely moving organisms. However, it is challenging to design an optical instrument that optimizes all these properties simultaneously. Existing techniques typically require the acquisition of sequential image snapshots to observe large areas or measure 3D information, thus compromising speed and throughput. Here we present 3D-RAPID, a computational microscope based on a synchronized array of 54 cameras that can capture high-speed 3D topographic videos over a 135 cm2 area, achieving up to 230 frames per second at a spatiotemporal throughput exceeding 5 gigapixels per second. 3D-RAPID employs a 3D reconstruction algorithm that, for each synchronized snapshot, fuses all 54 images into a composite that includes a co-registered 3D height map. The self-supervised 3D reconstruction algorithm trains a neural network to map raw photometric images to 3D topography using stereo overlap redundancy and ray-propagation physics as the only supervision mechanism. The reconstruction process is thus robust to generalization errors and scales to arbitrarily long videos from arbitrarily sized camera arrays. We demonstrate the broad applicability of 3D-RAPID with several collections of freely behaving organisms: ants, fruit flies and zebrafish larvae.

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Fig. 1: Overview of 3D-RAPID.
Fig. 2: Computational 3D reconstruction and stitching algorithm for 3D-RAPID.
Fig. 3: Zebrafish larvae (10 dpf) swimming in an open arena with interspersed microcapsule food particles (AP100), acquired at 60 f.p.s. for 10 s (Supplementary Videos 1 and 3).
Fig. 4: Adult fruit flies freely moving across a flat, noise-patterned surface, acquired at 60 f.p.s. for 8 s (Supplementary Videos 8 and 10).
Fig. 5: Harvester ants freely moving across a flat, noise-patterned surface, acquired at 60 f.p.s. for 10 s (Supplementary Videos 11 and 12).

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Data availability

The data that support the findings of this study are available from the Duke Research Data Repository at https://doi.org/10.7924/r4db86b1q. Interactive, full-resolution, reconstructed video frames can be viewed at https://gigazoom.rc.duke.edu/.

Code availability

The Python code used to generate the high-resolution 3D videos featured in this study is available at https://github.com/kevinczhou/3D-RAPID.

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Acknowledgements

We would like to thank K. Branson, S. Turaga, T. Dunn, A. Chakraborty and M. Hoffmann for their helpful feedback on the manuscript. Research reported in this publication was supported by the Office of Research Infrastructure Programs (ORIP), Office of the Director, National Institutes of Health of the National Institutes of Health and the National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health under award number R44OD024879 (M.H., J.P., T.D., P.R., V.S., M.Z., J.P.B., A.B. and G.H.), the National Cancer Institute (NCI) of the National Institutes of Health under award number R44CA250877 (M.H., J.P., T.D., P.R., V.S., M.Z., J.P.B., A.B. and G.H.), the National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health under award number R43EB030979 (M.H., J.P., T.D., P.R., V.S., M.Z., J.P.B., A.B. and G.H.), the National Science Foundation under award number 2036439 (M.H., J.P., T.D., P.R., V.S., M.Z., J.P.B., A.B. and G.H.) and the Duke Coulter Translational Partnership Award (K.C.Z., K.K. and R.H.).

Author information

Authors and Affiliations

Authors

Contributions

K.C.Z. and R.H. conceived the idea and initiated the research. With the help of C.L.C., J.P., P.C.K. and R.H., K.C.Z. developed the algorithms and theory. K.C.Z. wrote the code for and performed 3D video reconstruction and stitching, animal tracking and data analysis. M.H., T.D., P.R., V.S., C.B.C., M.Z. and R.H. developed the MCAM hardware and acquisition software. With the help of J.P.B., J.B., A.B., G.H., K.K. and R.H., K.C.Z. acquired and analysed the biological data. M.M., J.B. and M.B. provided input to and supervision of the biological experiments. T.D., J.J., K.K. and K.C.Z. created the supplementary videos and visualizations. K.C.Z. wrote the manuscript and created the figures, with input from all authors. K.C.Z, L.K. and R.H. revised the manuscript. R.H. supervised the research.

Corresponding authors

Correspondence to Kevin C. Zhou or Roarke Horstmeyer.

Ethics declarations

Competing interests

R.H. and M.H. are cofounders of Ramona Optics, Inc., which is commercializing multi-camera array microscopes. M.H., J.P., T.D., P.R., V.S., C.B.C., M.Z., J.P.B. and G.H. are or were employed by Ramona Optics, Inc. during the course of this research. K.C.Z. is a consultant for Ramona Optics, Inc. The remaining authors declare no competing interests.

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Nature Photonics thanks Chao Zuo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Population-level analysis of the zebrafish larvae featured in Fig. 3 and Supplementary Videos 1,3.

a Fish head height vs. elevation angle for all 40 fish over time. Lines define the approximate physical limits due to geometric fish mobility constraints. b Kernel density estimates of the height distributions of the zebrafish and AP100 food particles. Eye vergence vs. head height (c) and vs. elevation angle (d) plots are color-coded by the maximum height the fish attained in the 10-sec video. Fixed effect components of the linear mixed-effects regression lines are plotted (p = 0.33 and p < 10−5) for c and d, respectively.

Extended Data Fig. 2 Population-level analysis of the adult fruit flies featured in Fig. 4 and Supplementary Videos 8,10.

The six plots show kernel densities of the heights of the head, thorax, and abdomen for various behaviors. Differences of head (p < 10−7), thorax (p < 10−16), and abdomen (p < 10−62) heights across behaviors are statistically significant (n = 43 flies).

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Table 1, Equations 1–13 and Sections 1–7.

Supplementary Video 1

60 f.p.s., 36.6 MP video of freely swimming zebrafish larvae (10 dpf) feeding on mostly floating AP100 food particles. The left panel is the photometric composite and the right panel is the 3D height map. The video zooms into three feeding events (or attempts) by two different fish.

Supplementary Video 2

230 f.p.s., 9.1 MP video of freely swimming zebrafish larvae (10 dpf) feeding on mostly floating AP100 food particles. The left panel is the photometric composite and the right panel is the 3D height map. The video zooms in on three independent feeding events by three different fish. The third fish can be seen swallowing the food particle.

Supplementary Video 3

60 f.p.s., 36.6 MP video of freely swimming zebrafish larvae (10 dpf) feeding on mostly floating AP100 food particles. The left panel shows the full FOV with the trajectories mapped out. The panels on the right each correspond to individual fish, uniquely identified via a two-digit number, whose position and orientation are denoted with red annotations. The border colours of the right-hand panels non-uniquely match those of the tracks in the left-hand panel, to assist the viewer in matching the fish to the trajectories. Right-hand panels appear and disappear when the fish enters or exits the FOV. The first half of the video shows the photometric values, and the second half of the video shows the 3D height maps.

Supplementary Video 4

60 f.p.s., 36.6 MP video of 20 dpf zebrafish larvae feeding on live brine shrimp. The left panel is the photometric composite and the right panel is the 3D height map. The video zooms in on two feeding events from two different fish.

Supplementary Video 5

230 f.p.s., 9.1 MP video of 20 dpf zebrafish larvae feeding on live brine shrimp. The left panel is the photometric composite and the right panel is the 3D height map. The video zooms in on one feeding event.

Supplementary Video 6

60 f.p.s., 36.6 MP video of a large school of 5 dpf zebrafish larvae freely swimming in an open arena at high speed. The left panel is the photometric composite and the right panel is the 3D height map.

Supplementary Video 7

230 f.p.s., 9.1 MP video of a large school of 5 dpf zebrafish larvae freely swimming in an open arena at high speed. The left panel is the photometric composite and the right panel is the 3D height map.

Supplementary Video 8

60 f.p.s., 36.6 MP video of freely moving fruit flies. The left panel is the photometric composite and the right panel is the 3D height map.

Supplementary Video 9

230 f.p.s., 9.1 MP video of freely moving fruit flies. The left panel is the photometric composite and the right panel is the 3D height map.

Supplementary Video 10

60 f.p.s., 36.6 MP video of freely moving fruit flies. The left panel shows the full FOV with the trajectories mapped out. The panels on the right each correspond to individual flies, uniquely identified via a two-digit number, whose position is denoted by a red circle. The border colours of the right-hand panels non-uniquely match those of the tracks in the left-hand panel, to assist the viewer in matching the flies to the trajectories. Right-hand panels appear and disappear when the fish enters or exits the FOV. The first half of the video shows the photometric values, and the second half of the video shows the 3D height maps.

Supplementary Video 11

60 f.p.s., 36.6 MP video of freely moving harvester ants. The left panel is the photometric composite and the right panel is the 3D height map.

Supplementary Video 12

230 f.p.s., 9.1 MP video of freely moving harvester ants. The left panel is the photometric composite and the right panel is the 3D height map.

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Zhou, K.C., Harfouche, M., Cooke, C.L. et al. Parallelized computational 3D video microscopy of freely moving organisms at multiple gigapixels per second. Nat. Photon. 17, 442–450 (2023). https://doi.org/10.1038/s41566-023-01171-7

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