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Smart lattice light-sheet microscopy for imaging rare and complex cellular events


Light-sheet microscopes enable rapid high-resolution imaging of biological specimens; however, biological processes span spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality. Here, to overcome this limitation, we present smartLLSM, a microscope that incorporates artificial intelligence-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light-sheet microscopy (LLSM). We apply this approach to two unique processes: cell division and immune synapse formation. In each context, smartLLSM provides population-level statistics across thousands of cells and autonomously captures multicolor three-dimensional datasets or four-dimensional time-lapse movies of rare events at rates that dramatically exceed human capabilities. From this, we quantify the effects of Taxol dose on spindle structure and kinetochore dynamics in dividing cells and of antigen strength on cytotoxic T lymphocyte engagement and lytic granule polarization at the immune synapse. Overall, smartLLSM efficiently detects rare events within heterogeneous cell populations and records these processes with high spatiotemporal four-dimensional imaging over statistically significant replicates.

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Fig. 1: SmartLLSM training and performance.
Fig. 2: smartLLSM workflow.
Fig. 3: Population statistics of Taxol-induced mitotic defects and the effect of antigen strength on immune synapses.
Fig. 4: High-throughput imaging of Taxol-induced mitotic defects and the effect of antigen strength on immune synapses.
Fig. 5: High-throughput imaging of kinetochore dynamics during mitosis.
Fig. 6: Longitudinal tracking of kinetochore motion during mitosis.

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Data availability

Due to the inordinate size of the image data (40 TB), it is not currently feasible to deposit this into a central repository; however, all datasets underlying the results in this paper are available from the corresponding author upon request. To the extent possible, the authors will try to meet all requests for data sharing within 2 weeks from the original request. Source data are provided with this paper.

Code availability

The source code, annotation GUI, the library of annotated training data and the trained YOLOv5 network generated in the current study are available at Code is provided under The MIT License for open source software, a permissive license approved by the Open Source Initiative. Specific terms can be found at


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We thank K. Heath, M. Clynes and V. Augoustides for assistance with annotating images. We thank G. Upadhyayula for assistance with the single particle tracking code and T. Kapoor, M. Emanuele and A. Palmer for helpful discussions and feedback on the manuscript. This work was funded in part by grants from the National Institutes of Health (1DP2GM136653) awarded to W.R.L. W.R.L. acknowledges additional support from the Searle Scholars program, the Beckman Young Investigator Program and the Packard Fellowship for Science and Engineering.

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Authors and Affiliations



W.R.L. conceived the project. A.G. and J.S.T. lead the development of the DL-based cell detection and classification pipeline with input and contribution from Y.S. and W.R.L. D.E.M assisted with integrating the YOLO network together with the microscope control software. T.A.D, C.Q.Y. and A.T.R assisted with sample preparation. Y.S. and W.R.L performed the imaging experiments, analyzed data and wrote the paper with feedback from all authors. W.R.L. supervised and directed the project.

Corresponding authors

Correspondence to Andrea Giovannucci or Wesley R. Legant.

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Competing interests

W.R.L. and D.E.M. are authors on patents related to lattice light-sheet microscopy and its applications including US Patent numbers: US 11,221,476 B2 and US 10,795,144 B2 issued to W.R.L., D.E.M. and co-authors, and assigned to Howard Hughes Medical Institute. The other authors declare no competing interests.

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Nature Methods thanks Mathieu Ducros, Ilaria Testa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team. Peer reviewer reports are available.

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Shi, Y., Tabet, J.S., Milkie, D.E. et al. Smart lattice light-sheet microscopy for imaging rare and complex cellular events. Nat Methods 21, 301–310 (2024).

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