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Super-resolution video microscopy of live cells by structured illumination

Abstract

Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.

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Figure 1: Comparison of TIRF-SIM and conventional TIRF microscopy.
Figure 2: Time series live TIRF-SIM of EGFP–α-tubulin in an S2 cell.
Figure 3: Time-series live TIRF-SIM of kinesin-73–EGFP in an S2 cell.

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Acknowledgements

We thank T. Huckaba for generating the kinesin-73-EGFP cell line; L. Shao, S. Haase and the late Melvin Jones for help with software; D. Agard and J. Sedat for their role in the development of structured-illumination microscopy; R. Vale (University of California, San Francisco) for his support of E.R.G, for the GFP-α-tubulin S2 cell line and for useful comments; and R. Oldenbourg for a useful discussion. This work was supported in part by the David and Lucile Packard Foundation, the Sandler Family Supporting Foundation, the Keck Advanced Microscopy Laboratory, the Howard Hughes Medical Institute and by the US National Science Foundation through the Center for Biophotonics, a National Science Foundation Science and Technology Center that is managed by the University of California, Davis, under Cooperative Agreement PHY 0120999.

Author information

Authors and Affiliations

Authors

Contributions

P.K. designed and built the rapid control system and wrote the manuscript draft. B.B.C. acquired and processed the data. L.W., B.B.C. and P.K. built optical hardware. E.R.G. prepared the samples. M.G.L.G. made the conceptual design and wrote the simulation. B.B.C. and M.G.L.G. analyzed data. B.B.C., M.G.L.G. and E.R.G. edited the manuscript.

Corresponding author

Correspondence to Mats G L Gustafsson.

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Competing interests

University of California San Francisco is in the process of commercializing structured illumination technology. M.G.L.G. may receive royalties or consulting income related to such licenses. L.W. has consulted for one licensee, Applied Precision Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Note (PDF 2215 kb)

Supplementary Video 1

Comparison of TIRF-SIM and conventional TIRF videos of EGFP-α-tubulin in an S2 cell. TIRF-SIM (left), conventional TIRF (right). The time series comprises 120 SIM frames acquired at the full 512 × 512 pixel field of view (only a subset is shown here) at 360 ms per SIM frame (40 ms per raw exposure), with consecutive SIM frames deliberately spaced at 1 s intervals. Scale bar, 2 μm. (MOV 4779 kb)

Supplementary Video 2

Frequency space view of the data set in Fig. 1a,b and Supplementary Video 1. The video shows the time evolution of the Fourier transforms of the conventional TIRF (left) and TIRF-SIM (right) images. When the video runs, actual sample information, which (for this type of sample) has a regular appearance and changes only slowly from frame to frame, can be distinguished from noise, which varies rapidly and randomly with both position and time. The sample information is seen to continue out to substantially higher spatial frequencies in the TIRF-SIM data set compared to the conventional data. (MOV 6178 kb)

Supplementary Video 3

Videos of EGFP-α-tubulin dynamics within a tripolar mitotic spindle. TIRF-SIM (left) and conventional TIRF (right). Multiple centrosomes occur commonly in S2 cellsS1. The video contains 120 time points acquired with 512 × 512 camera pixels (a subset is shown) at 270 ms per time point, with consecutive time points deliberately spaced by 1 s. Scale bar, 2 μm. (MOV 6084 kb)

Supplementary Video 4

480-frame time series of EGFP-α-tubulin in an S2 cell. TIRF-SIM (left) and conventional TIRF (right). The video, acquired over 8 minutes, illustrates the ability of TIRF-SIM to follow a sample over many time points. The exposures for one SIM frame required 450 ms; consecutive frames were deliberately spaced by 1 s. In order to visualize directly the moderate degree of photobleaching, this video is shown without intensity normalization between timeframes. Scale bar, 2 μm. (MOV 13392 kb)

Supplementary Video 5

TIRF-SIM reconstruction (left) and conventional TIRF (right) videos of the cell shown in Fig. 2. The data set contains 180 time points acquired at 1 s intervals. Scale bar, 2 μm. (MOV 7446 kb)

Supplementary Video 6

Rotating three-dimensional maximum-intensity kymograph from a TIRF-SIM reconstruction of the cell shown in Fig. 2 and Supplementary Video 5. Time is increasing downward from 0 s to 180 s. Vertical sheets indicate the time evolution of each microtubule. Speckling (random variations in labelling density, seen here as striping of each sheet) allows any movement to be tracked. The near-vertical orientation of most stripes indicates that global movement was slight: as expected, most extension and retraction of microtubules took place through polymerization and depolymerization at their distal ends. Scale bar 2 μm. (MOV 11090 kb)

Supplementary Video 7

Kinesin73-EGFP in an S2 cell. TIRF-SIM reconstruction (left) and conventional TIRF (right). The video contains 120 time points acquired with 256 × 256 camera pixels at 144 ms per SIM frame (16 ms per raw exposure). Scale bar, 2 μm. (MOV 2261 kb)

Supplementary Video 8

Rotating video version of the three-dimensional kymograph shown in Fig. 3d. The field of view is 4.4 × 4.4 μm; time increases upward from 0 to 11.5 s. Movement trajectories of kinesin-cargo complexes can be seen as white paths. (MOV 1515 kb)

Supplementary Video 9

High-speed live SIM. Conventional TIRF (left) and TIRF-SIM (right) videos of kinesin73-EGFP in an S2 cell. The video contains 160 time points from a time series acquired with 128 × 128 camera pixels at 90 ms per SIM frame (i.e. 10 ms per raw exposure, and 11.1 Hz SIM frame rate). The video, shown in green, is overlayed on a maximum-intensity projection of the entire time series, shown in red, to help visualize tracks of kinesin motion. This cell contained a significant density of freely diffusing EGFP-kinesin monomers, which were entering and leaving the TIRF layer rapidly, as can be seen in the conventional video. These single-molecule visits are faster than the SIM frame time, even at this high imaging speed, and result in background noise in the SIM reconstruction. The slower-moving microtubule-bound kinesins are reconstructed clearly, however, and can be seen transporting along microtubules. To reconstruct the freely diffusing molecules well would require even higher acquisition rates. Scale bar, 1 μm. (MOV 6101 kb)

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Kner, P., Chhun, B., Griffis, E. et al. Super-resolution video microscopy of live cells by structured illumination. Nat Methods 6, 339–342 (2009). https://doi.org/10.1038/nmeth.1324

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