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Kilohertz volumetric imaging of in vivo dynamics using squeezed light field microscopy

Nature Methods volume 22, pages 2194–2204 (2025)Cite this article

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Abstract

Volumetric functional imaging of transient cellular signaling and motion dynamics is often limited by hardware bandwidth and the scarcity of photons under short exposures. To overcome these challenges, we introduce squeezed light field microscopy (SLIM), a computational imaging approach that rapidly captures high-resolution three-dimensional light signals using only a single, low-format camera sensor. SLIM records over 1,000 volumes per second across a 550-µm diameter field of view and 300-µm depth, achieving 3.6-µm lateral and 6-µm axial resolution. Here we demonstrate its utility in blood cell velocimetry within the embryonic zebrafish brain and in freely moving tails undergoing high-frequency swings. Millisecond-scale temporal resolution further enables precise voltage imaging of neural membrane potentials in the leech ganglion and hippocampus of behaving mice. Together, these results establish SLIM as a versatile and robust tool for high-speed volumetric microscopy across diverse biological systems.

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Fig. 1: Principle of SLIM.
Fig. 2: 3D imaging of hemodynamics in the embryonic zebrafish brain and tail at 1,000 vps.
Fig. 3: 3D imaging of membrane action potentials and fictive swimming oscillation in medicinal leech ganglia at 800 vps.
Fig. 4: 3D voltage imaging of hippocampus in behaving mice at 800 vps.
Fig. 5: 3D imaging of a beating zebrafish heart with multi-plane scanning light-sheet illumination at 300 vps.

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Data availability

Data underlying the results are publicly available on GitHub at https://github.com/aaronzq/SLIM and via Zenodo at https://doi.org/10.5281/zenodo.15793563 (ref. 70). Source data are provided with this paper.

Code availability

Codes for 3D reconstruction are available on GitHub at https://github.com/aaronzq/SLIM and via Zenodo at https://doi.org/10.5281/zenodo.15793563 (ref. 70) under a BSD-License.

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Acknowledgements

We thank Y. Dong and Y. Zhang at UCLA for their assistance in zebrafish experiments. We acknowledge the David Geffen School of Medicine at UCLA for providing the Zebrafish Core Facilities. This work was supported by the following grants: NIH (grant nos. R01HL165318 (L.G.), RF1NS128488 (L.G.), R35GM128761 (L.G.), R01AI102584 (G.C.L.W.), R01HL129727 (T.K.H.), R01HL159970 (T.K.H.) and T32HL144449 (T.K.H.)). W.C.S. was supported by the National Science Foundation Graduate Research Fellowship Program (grant nos. DGE-1650604 and DGE-2034835) and Ruth L. Kirschstein National Research Service Award ‘Multidisciplinary Training in Microbial Pathogenesis’ (grant no. T32AI007323). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Author information

Author notes

  1. These authors contributed equally: Zhaoqiang Wang, Ruixuan Zhao.

Authors and Affiliations

  1. Department of Bioengineering, Henry Samueli School of Engineering and Applied Science, University of California, Los Angeles, Los Angeles, CA, USA

    Zhaoqiang Wang, Ruixuan Zhao, Ohr Benshlomo, Calvin K. Lee, William C. Schmidt, Aryan Pammar, Gerard C. L. Wong & Liang Gao

  2. Division of Biology and Biological Engineering, Tianqiao and Chrissy Chen Institute for Neuroscience, California Institute of Technology, Pasadena, CA, USA

    Daniel A. Wagenaar

  3. Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Diego Espino, Liron Sheintuch & Peyman Golshani

  4. Wyant College of Optical Science, University of Arizona, Tucson, AZ, USA

    Wenjun Kang & Rongguang Liang

  5. Division of Cardiology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Enbo Zhu, Jing Wang & Tzung K. Hsiai

  6. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Calvin K. Lee, William C. Schmidt & Gerard C. L. Wong

  7. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Calvin K. Lee, William C. Schmidt & Gerard C. L. Wong

  8. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Calvin K. Lee, William C. Schmidt & Gerard C. L. Wong

  9. Greater Los Angeles VA Medical Center, Los Angeles, CA, USA

    Peyman Golshani & Tzung K. Hsiai

  10. Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA

    Peyman Golshani

  11. Integrative Center for Learning and Memory, University of California, Los Angeles, Los Angeles, CA, USA

    Peyman Golshani

  12. Intellectual and Developmental Disability Research Center, University of California, Los Angeles, Los Angeles, CA, USA

    Peyman Golshani

Authors
  1. Zhaoqiang Wang
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Contributions

Z.W. and L.G. conceived of the idea. L.G., D.A.W., R.L., G.C.L.W., T.K.H. and P.G. oversaw the project. Z.W. constructed the microscopes and developed the reconstruction algorithm. R.Z., D.A.W. and Z.W. prepared medicinal leech samples and electrophysiology study. D.E. and L.S. performed mouse surgery. Z.W., R.Z., A.P. and J.W. bred the zebrafish. C.K.L. and W.C.S. prepared bacteria samples. W.K. fabricated the lenslet array. Z.W., R.Z., D.A.W., D.E., L.S., O.B. and E.Z. collected the imaging data. Z.W. and R.Z. processed and analyzed the data. Z.W., R.Z. and L.G. wrote the paper. All authors reviewed, edited and consulted on the text.

Corresponding author

Correspondence to Liang Gao.

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Competing interests

L.G. has a financial interest in Lift Photonics. However, it was not involved in the research presented in this paper. The other authors declare no competing interests.

Peer review

Peer review information

Nature Methods thanks Robert Prevedel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Nina Vogt, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 3D voltage imaging of hippocampus pyramidal neurons in awake mice.

a. MIP of SLIM reconstruction of the 3D located neurons. Neuron indices are partially labeled in y-z view to avoid cluttered markers. Representative result from five FOVs using mice labeled for excitatory pyramidal neurons. Scale bar, 100 µm. b. 3D distribution of neuron center locations and their corresponding index. c. Detrended signal traces and the detected spikes labeled in black dots.

Extended Data Fig. 2 Observation of subthreshold oscillations in hippocampus interneurons.

a. Example MIP of identical dataset used in Fig. 4, with neurons of interest labeled with number. Representative result from nine FOVs conducted using mice labeled for OLM interneurons. Scale bar, 100 μm. b. The power spectral densities of 18 neuron traces throughout the entire recording (around three minutes). Red regions denote the frequency band known for theta oscillations. c. Raw traces of ten seconds with red lines plot the band-pass filtered signal in 4–10 Hz.

Extended Data Fig. 3 Comparison between SLIM reconstruction and ground truth.

High-resolution ground truth images are acquired with a reference camera (sharing same objective with SLIM) under either widefield or light-sheet illumination. Sample is axially scanned with a motorized stage to acquire a z stack. SLIM and ground truth image are acquired sequentially on the same sample. a. Tilted mouse kidney slice (FluoCells™ Prepared Slide from ThermoFisher) b. Live mouse hippocampus (CA1, excitatory pyramidal neurons). Green arrows denote representative neuron candidates. Yellow arrows mark the overlaps between sub-aperture views (fabrication tolerance of prisms and holder). When sample exhibits a strong background, this overlap could induce reconstruction artefacts. Blue arrows indicate the MIP artifacts due to the bright boundary of circular FOV. c. Embryonic zebrafish vasculature (endothelial cells, Tg(flk:mCherry)). Scale bar, 100 μm.

Supplementary information

Supplementary Information

Supplementary Figs. 1–18, Notes 1–4 and Tables 1–4.

Reporting Summary

Peer Review File

Supplementary Video 1

3D tracking of blood flow dynamics in the zebrafish vasculature (dorsal view) using SLIM.

Supplementary Video 2

3D tracking of blood flow dynamics in the zebrafish vasculature (ventral view) using SLIM.

Supplementary Video 3

High-speed 3D imaging of a freely moving zebrafish tail using SLIM.

Supplementary Video 4

3D imaging of neuronal action potentials in leech ganglion with SLIM.

Supplementary Video 5

3D imaging of a beating zebrafish heart with SLIM.

Supplementary Video 6

3D imaging of free-swimming Vibrio cholerae bacteria with SLIM.

Source data

Source Data Fig. 3

Source data of Fig. 3.

Source Data Fig. 4

Source data of Fig. 4.

Source Data Fig. 5

Source data of Fig. 5.

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Wang, Z., Zhao, R., Wagenaar, D.A. et al. Kilohertz volumetric imaging of in vivo dynamics using squeezed light field microscopy. Nat Methods 22, 2194–2204 (2025). https://doi.org/10.1038/s41592-025-02843-8

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