Abstract
We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 μm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.
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Acknowledgements
We thank M. Davidson (Florida State University) for the U2OS cells, Z. Bao (Sloan-Kettering Institute) for the BV24 nematode strain, K. Hazelwood for assistance with cell culture, G. Patterson for the use of his cell culture facilities, and H. Eden, P. Winter, Y. Wu and C. Waterman for feedback and suggestions on the manuscript. S.H.P. acknowledges financial support from the American Association for the Advancement of Science. This work was supported by the Intramural Research Program of the NIH National Institute of Biomedical Imaging and Bioengineering, the National Institute of Heart, Lung, and Blood and the National Institute of Child Health and Development.
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A.G.Y. and H.S. conceived the idea. A.G.Y., S.H.P., C.A.C. and H.S. designed and built the experimental setup. A.G.Y. and S.H.P. wrote the analysis code and performed simulations. A.G.Y., S.H.P., D.D.N., R.S.F., C.A.C. and H.S. collected data. A.G.Y., S.H.P., D.D.N., R.S.F., K.T., A.B.C., C.A.C. and H.S. analyzed data. S.H.P., D.D.N., R.S.F. and K.T. prepared samples. M.M., A.B.C. and C.A.C. contributed reagents and materials. A.G.Y., S.H.P., D.D.N., R.S.F. and H.S. wrote the paper with input from all authors. H.S. designed and supervised research.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–10 and Supplementary Notes 1–4 (PDF 1940 kb)
Supplementary Video 1
Multifocal excitation (left), pinholing, scaling (middle) and summing process (right). Data are from the 120-frame acquisition displayed in Figure 1; only the top-right region of the field is displayed. (GIF 10885 kb)
Supplementary Video 2
Dual-color MSIM stack of a fixed cell, to accompany Figure 2. Green, Alexa Fluor 488–immunolabeled microtubules; magenta, Mitotracker Red–labeled mitochondria. (GIF 3332 kb)
Supplementary Video 3
Dual-color wide-field stack of a fixed cell, to accompany Figure 2. Green, Alexa Fluor 488–immunolabeled microtubules; magenta, Mitotracker Red–labeled mitochondria. The illumination dose is the same as in Supplementary Video 2. (GIF 10275 kb)
Supplementary Video 4
A 3D rendering of the zebrafish tissue section shown in Figure 3. The three-dimensional volumetric rendering and video were made from the MSIM stack of two-dimensional images using Imaris software version 7.3 (Bitplane). Display brightness and contrast were adjusted to emphasize the full dynamic range of the intensities in the images. (MOV 9567 kb)
Supplementary Video 5
MSIM z stack of the zebrafish tissue section shown in Figure 3. (MOV 13125 kb)
Supplementary Video 6
Confocal z stack (taken on a Zeiss 510) of zebrafish tissue shown in Supplementary Figure 8. (MOV 8412 kb)
Supplementary Video 7
MSIM maximum intensity projections of the zebrafish dataset described in Figure 4. (MOV 7433 kb)
Supplementary Video 8
MSIM z stack of the first timepoint shown in Figure 4. (MOV 3102 kb)
Supplementary Video 9
A 3D rendering of the inset shown in Figure 4b. The three-dimensional volumetric rendering and video were made from the MSIM stack of two-dimensional images using ImageJ. (MOV 1180 kb)
Supplementary Video 10
MSIM time series of the indicated slice, to accompany data in Supplementary Figure 9. (MOV 9732 kb)
Supplementary Video 11
MSIM maximum intensity projections of the two-color cellular dataset described in Figure 5. Scale bar, 5 μm. (GIF 6302 kb)
Supplementary Software
Processing code for constructing resolution-doubled images from raw MSIM data. (ZIP 17244 kb)
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York, A., Parekh, S., Nogare, D. et al. Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy. Nat Methods 9, 749–754 (2012). https://doi.org/10.1038/nmeth.2025
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DOI: https://doi.org/10.1038/nmeth.2025
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