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Practical considerations for quantitative light sheet fluorescence microscopy

Abstract

Fluorescence microscopy has evolved from a purely observational tool to a platform for quantitative, hypothesis-driven research. As such, the demand for faster and less phototoxic imaging modalities has spurred a rapid growth in light sheet fluorescence microscopy (LSFM). By restricting the excitation to a thin plane, LSFM reduces the overall light dose to a specimen while simultaneously improving image contrast. However, the defining characteristics of light sheet microscopes subsequently warrant unique considerations in their use for quantitative experiments. In this Perspective, we outline many of the pitfalls in LSFM that can compromise analysis and confound interpretation. Moreover, we offer guidance in addressing these caveats when possible. In doing so, we hope to provide a useful resource for life scientists seeking to adopt LSFM to quantitatively address complex biological hypotheses.

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Fig. 1: Effect of beam waist on image quality.
Fig. 2: Misalignment of light sheet and focal plane compromises feature detection and axial localization.
Fig. 3: Nonuniform illumination intensity in LSFM.
Fig. 4: Degradation of light sheet quality.
Fig. 5: Reducing FOV improves accuracy of stitching.
Fig. 6: Registration failure and motion artifact in multiview fusion.

Data availability

Data used in this article are available at https://doi.org/10.6084/m9.figshare.c.6211429 or from the authors on request.

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Acknowledgements

We thank S. Khuon, L. Eisenman, A. Nain, C. Hernandez-Padillaand and L. Zhang for cell culture and sample preparation; W. Lemon for preparation of D. melanogaster embryos; D. Dalle Nogare and A. Chitnis for preparation of zebrafish samples; R. Christensen for C. elegans sample preparation; Z. Bao for providing the OD58 C. elegans strain; Y. Su, I. Curtin, F. Kyere, J. Stein and M.S. Itano for advice and assistance with brain sample preparation; and M. DeSantis and the Janelia Light Microscopy Facility. This work was supported in part by the intramural research program of the National Institute of Biomedical Imaging and Bioengineering at the National Institutes of Health (M.G., H.D.V., Y.W. and H.S.). The Advanced Imaging Center at Janelia Research Campus was generously supported by the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation (C.M.H. and T.-L.C.).

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C.M.H., M.G., H.D.V. and Y.W. performed the imaging experiments and accompanying analyses. H.S. and T.-L.C. oversaw the project. All authors contributed to writing and editing the manuscript.

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Correspondence to Teng-Leong Chew.

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Extended data

Extended Data Fig. 1 ‘Practical Considerations for Quantitative Light Sheet Fluorescence Microscopy’ Infographic.

Summary of the important considerations for quantitative LSFM described in this Perspective.

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Supplementary Methods and Figs. 1–8.

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Hobson, C.M., Guo, M., Vishwasrao, H.D. et al. Practical considerations for quantitative light sheet fluorescence microscopy. Nat Methods 19, 1538–1549 (2022). https://doi.org/10.1038/s41592-022-01632-x

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