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Superresolution live imaging of plant cells using structured illumination microscopy

Abstract

Although superresolution (SR) approaches have been routinely used for fixed or living material from other organisms, the use of time-lapse structured illumination microscopy (SIM) imaging in plant cells still remains under-developed. Here we describe a validated method for time-lapse SIM that focuses on cortical microtubules of different plant cell types. By using one of the existing commercially available SIM platforms, we provide a user-friendly and easy-to-follow protocol that may be widely applied to the imaging of plant cells. This protocol includes steps describing calibration of the microscope and channel alignment, generation of an experimental point spread function (PSF), preparation of appropriate observation chambers with available plant material, image acquisition, reconstruction and validation. This protocol can be carried out within two to three working days.

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Figure 1: An overview of the principles of SIM and its use in documenting cortical microtubules of A. thaliana hypocotyl epidermal cells stably transformed with the GFP-MBD microtubule marker.
Figure 2: Effect of grating pattern selection on SIM acquisition and final object resolution using mCherry-TUA5-labeled cortical microtubules in hypocotyl epidermal cells of A. thaliana.
Figure 3: Effects of SR Frequency (SRF) weighting on the final output, exemplified in Arabidopsis cortical microtubules labeled with GFP-MBD.
Figure 4: Effect of Wiener filtering on background and resolution of reconstructed SIM image as exemplified in hypocotyl epidermal cell microtubules labeled with the GFP-MBD marker.
Figure 5: Effects of frequency sectioning on reconstructed SIM image resolution, exemplified in mCherry-TUA5–labeled cortical microtubules of A. thaliana hypocotyl epidermal cells.
Figure 6: Subdiffraction imaging of cortical microtubules in different cell types of A. thaliana stably transformed with different microtubule markers as compared with WF epifluorescence.
Figure 7: Addressing the time-lapse option of SIM imaging with subdiffraction settings.

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Acknowledgements

Development of this protocol was financially supported by grant no. P501/11/1764 from the Czech Science Foundation (GACˇR); by grant no. LO1204 from the Czech Ministry of Education, Youth and Sports, National Program of Sustainability I (NPUI) to the Centre of the Region Haná for Biotechnological and Agricultural Research; and by the Czech National program of sustainability, LO1304.

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Authors and Affiliations

Authors

Contributions

G.K., M.M., O.Š. and J.Š. performed the experiments. G.K., O.Š., M.M. and J.Š. established material preparation and microscope settings for SIM. M.M., O.Š., J.B. and J.Š. provided material and equipment. G.K. carried out quantitative analyses and validation procedures inherent to the protocol. M.O. prepared Supplementary Figure 1 with help from O.Š. and G.K. G.K. and J.Š. wrote the manuscript with editorial help from M.O., O.Š., M.M. and J.B. J.Š. and G.K. designed the experiments, and J.Š. supervised the whole project.

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Integrated supplementary information

Supplementary Figure 1 Stepwise assembly of the observation chamber for plants.

(a) Placement and orientation of seedling (arrow throughout a-d) in a drop of liquid ½ MS on top of a glass slide. (b) Placement of coverslip and filling of observation chamber with liquid ½ MS. (c) Sealing of the coverslip rim with silicone paste. (d) The observation chamber with the seedling in proper orientation (arrow) fully sealed.

Supplementary Figure 2 Comparative characterization of lateral resolution of individual and bundled cortical microtubules between SIM and WF acquisitions.

(a) SIM overview of the cortical microtubule array of a hypocotyl cell stably expressing GFP-MBD microtubule marker. (b) The respective WF image corresponding to (a). (c) Transverse 1 μm profile (small diagonal line) across individual microtubule from the left boxed area of (a). (d) Transverse 1 μm profile (small diagonal line) across three closely adjacent microtubules from the right boxed area of (a). (e) Normalized intensity scatterplot corresponding to the individual microtubule profile of (c) by both SIM and WF. The width of the respective curves (green for SIM and red for WF) at 0.5 of normalized intensity corresponds to the FWHM of the individual microtubule. (f) Normalized intensity scatterplots corresponding to the microtubule bundle profile of (d). In both cases SIM (green lines) clearly separates three peaks instead of a single broad one in WF mode (red lines). All scale bars correspond to 5 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 314 kb)

Time-lapse imaging of cortical microtubule dynamics in a hypocotyl epidermal cell of Arabidopsis thaliana stably transformed with the GFP-MBD microtubule marker.

Video corresponds to Fig. 7a,b. Recording time: 239.4 sec, Frames: 90, Time interval: 2.7 sec, Video frame rate: 16 fps (MOV 24992 kb)

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Komis, G., Mistrik, M., Šamajová, O. et al. Superresolution live imaging of plant cells using structured illumination microscopy. Nat Protoc 10, 1248–1263 (2015). https://doi.org/10.1038/nprot.2015.083

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