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Instant super-resolution imaging in live cells and embryos via analog image processing

Abstract

Existing super-resolution fluorescence microscopes compromise acquisition speed to provide subdiffractive sample information. We report an analog implementation of structured illumination microscopy that enables three-dimensional (3D) super-resolution imaging with a lateral resolution of 145 nm and an axial resolution of 350 nm at acquisition speeds up to 100 Hz. By using optical instead of digital image-processing operations, we removed the need to capture, store and combine multiple camera exposures, increasing data acquisition rates 10- to 100-fold over other super-resolution microscopes and acquiring and displaying super-resolution images in real time. Low excitation intensities allow imaging over hundreds of 2D sections, and combined physical and computational sectioning allow similar depth penetration to spinning-disk confocal microscopy. We demonstrate the capability of our system by imaging fine, rapidly moving structures including motor-driven organelles in human lung fibroblasts and the cytoskeleton of flowing blood cells within developing zebrafish embryos.

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Figure 1: Key steps in implementing instant structured illumination.
Figure 2: Instant SIM enables high-speed two-color super-resolution imaging.
Figure 3: Instant SIM reveals ER dynamics at 100 Hz.
Figure 4: Imaging vascular flow with subcellular resolution at 37 Hz.

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Acknowledgements

We thank G. Patterson for encouragement and the use of his cell culture facilities, L. Maldonado-Baez (US National Heart, Lung, and Blood Institute) for the mCherry-tagged Rab8A plasmid, C. Combs for lending us his objective lenses, S. Parekh for useful discussions and for help in sample preparation, E. Tyler and A. Hoofring for help with illustrations and Y. Wu for help with Huygens deconvolution software. This work was supported by the Intramural Research Programs of the US National Institute of Biomedical Imaging and Bioengineering (to A.G.Y., P.W. and H.S.); the National Institute of Diabetes and Digestive and Kidney Diseases (to P.C.); the National Heart, Lung, and Blood Institute (to R.S.F.) and the National Institute of Child Health and Human Development (to D.D.N., J.H. and A.C.).

Author information

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Authors

Contributions

A.G.Y. and H.S. conceived idea and designed optical system. A.G.Y. built the optical system, designed and implemented data acquisition software and performed simulations. A.G.Y., P.C., D.D.N., J.H., R.S.F. and H.S. acquired data. P.C., D.D.N., R.S.F. and A.C. provided guidance on biological experiments. P.C., D.D.N., J.H., P.W. and R.S.F. prepared samples. P.C., D.D.N., J.H., R.S.F. and A.C. provided biological reagents. All authors analyzed data. A.G.Y., P.C. and H.S. wrote the paper with input from all authors. H.S. supervised research.

Corresponding author

Correspondence to Andrew G York.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Table 1 and Supplementary Note (PDF 2021 kb)

Dual-color instant SIM imaging of mitochondria with Tom20-mCherry (red) and TFAM-GFP (green).

Dual-color volumes (12 imaging planes/volume) were acquired in 1.2 s and imaged every 2.5 s. XY maximum intensity projections are shown. See also Fig. 2. (AVI 1358 kb)

Dual-color spinning disk confocal imaging of Tom20-mCherry (red) and TFAM-GFP (green).

Dual-color volumes (12 imaging planes/volume) were acquired in 12 s and imaged every 12 s. XY maximum intensity projections are shown. Data are deconvolved. See also Fig. 2. (AVI 792 kb)

Dual-color line-scanning confocal imaging of Tom20-mCherry (red) and TFAM-GFP (green).

Dual-color volumes (12 imaging planes/volume) were acquired in 1.2 s and imaged every 5 s. XY maximum intensity projections are shown. Data are deconvolved. See also Fig. 2. (AVI 538 kb)

Instant SIM imaging of GFP-HRAS over 100 volumes.

Two imaging planes at indicated axial distance from coverslip are shown. Volumes (12 imaging planes) were acquired in 0.6 s and imaged every 2 s. (AVI 16979 kb)

Dual-color instant SIM imaging of GFP-HRAS (green) and mCherry-Rab (red) over 60 volumes.

Two imaging planes at indicated axial distance from coverslip are shown. Volumes (12 imaging planes) were acquired in 0.7 s and imaged every 5 s. (AVI 31816 kb)

Dual-color instant SIM imaging of mitochondria with Tom20-mCherry (red) and peroxisomes with PEX-GFP (green) over 60 volumes.

Dual-color volumes (12 imaging planes/volume) were acquired in 1.2 s and imaged every 7.5 s. xy maximum intensity projections are shown. (AVI 5221 kb)

Higher magnification view of Tom20-mCherry and PEX-GFP.

Interactions between mitochondrial entities could be either in a "kiss and run" or prolonged mode (small Tom20-Cherry and PEX-GFP positive densities). (AVI 529 kb)

Dual-color instant SIM imaging of GFP-SEC16B (green) and PEX-mCherry (red).

Dual-color volumes (12 imaging planes/volume) were acquired in 1.2 s and imaged every 5.5 s. xy maximum-intensity projections are shown. (AVI 4020 kb)

Higher magnification view of GFP-SEC16B (green) and PEX-mCherry (red) over 60 volumes.

Note interactions amongst peroxisomes with fusion events, but also between peroxisomes and lamellar ER extensions, likely depicting transfer of material from the endoplasmic reticulum. (AVI 146 kb)

Instant SIM imaging of the endoplasmic reticulum at 100 Hz over 200 time points.

The ER is labeled with GFP-Sec61. See also Fig. 3b. (AVI 1882 kb)

Instant SIM imaging of microtubules inside flowing blood cells in 3 day old zebrafish embryo, over 100 time points.

Imaging was performed at 37 Hz. See also Fig. 4. (AVI 14005 kb)

Supplementary Software

Hardware control and deconvolution scripts for instant SIMFor documentation, and the most recent versions, see http://code.google.com/p/msim (ZIP 27 kb)

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York, A., Chandris, P., Nogare, D. et al. Instant super-resolution imaging in live cells and embryos via analog image processing. Nat Methods 10, 1122–1126 (2013). https://doi.org/10.1038/nmeth.2687

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