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. 2018 Oct;96(1):233-243.
doi: 10.1111/tpj.14021. Epub 2018 Jul 30.

Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes

Masakazu Iwai  1   2 , Melissa S Roth  2 , Krishna K Niyogi  1   2
Affiliations

Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes

Masakazu Iwai et al. Plant J. 2018 Oct.

Abstract

The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.

Keywords: Arabidopsis thaliana; Chlamydomonas reinhardtii; live-cell imaging; structured illumination microscopy; technical advance; thylakoid structure.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. The subdiffraction-resolution live-cell imaging analysis of the grana size of A. thaliana chloroplasts.
Chl fluorescence from A. thaliana chloroplasts in the mesophyll tissue observed and analyzed by 3D-SIM. As compared with the reconstructed widefield image (a), reconstructed 3D-SIM image (b) of chloroplasts in the WT showed distinct round shape structures, indicating the individual grana. The reconstructed 3D-SIM images of chloroplasts in (b) the WT control, (c) the stn7 stn8 mutant, (d) the tap38 mutant, and the WT acclimated to (e) far-red (FR) light, and (f) blue light (BL) conditions were analyzed to measure the differences in the grana size. Scale bars, 5 µm. FWHM of grana from (g) the WT control (n = 300 from 8 chloroplasts), (h) the stn7 stn8 mutant (n = 124 from 8 chloroplasts), (i) the tap38 mutant (n = 251 from 8 chloroplasts), and the WT chloroplasts acclimated to (j) FR light (n = 149 from 8 chloroplasts) or (k) BL (n = 172 from 8 chloroplasts). Inset numbers indicate mean FWHM ± SD of all chloroplasts, and data represent means ± SD for each chloroplast.
Figure 2.
Figure 2.. The reconstructed 3D-SIM images of the side-view of A. thaliana chloroplasts showing the thylakoid membrane architecture.
Chl fluorescence from isolated A. thaliana chloroplasts observed by 3D-SIM. Optical sections of chloroplasts isolated from (a) WT, (b) stn7 stn8, and (c) tap38 are shown whole (left column) and enlarged to show detail (right column). Each number corresponds to the selected focal plane of the serial optical sections as shown in Figures S1–3. Scale bars, 1 µm. (d) Representative close-up image of a chloroplast side-view with a line scan for a thickness analysis of thylakoid membranes. (e) The intensity profile of the line indicated in (d) (top panel). The dotted line is the multipeak fit, which is used to extract the Gaussian distribution of each peak (bottom panel). The number indicates FWHM (µm) measured in each Gaussian distribution. (f) Thylakoid membrane length in chloroplasts of WT (n = 173 from 8 chloroplasts), stn7 stn8 (n = 144 from 8 chloroplasts), and tap38 (n = 156 from 8 chloroplasts). Inset numbers indicate mean FWHM ± SD of all chloroplasts, and data represent means ± SD for each chloroplast.
Figure 3.
Figure 3.. The subdiffraction-resolution live-cell imaging analysis of C. reinhardtii chloroplasts.
Chl fluorescence from C. reinhardtii chloroplasts observed by 3D-SIM. As compared to reconstructed widefield images (a,c), reconstructed 3D-SIM images (b,d) of C. reinhardtii chloroplasts revealed the distinct thylakoid membrane structures. Arrows and arrowheads in (d) indicate the basal and lobe regions, respectively. (e) Selected optical sections of close-up 3D-SIM images showing the basal region of the chloroplast as shown in (d). Each number corresponds to the selected focal plane of the serial optical sections as shown in Figure S4. Arrows in (e) indicate thylakoid membranes, which appeared to extend into the pyrenoid. (f) Detail image of the thylakoid tip convergence region. (g) The intensity profile of the line indicated in (f). The dotted line is the multipeak fit, which was used to extract the Gaussian distribution of each peak as shown at the bottom. The number indicates FWHM (nm) measured in each Gaussian distribution. Scale bars, 10 µm (a,b), 2 µm (c–f).
Figure 4.
Figure 4.. Structural comparison of thylakoid structures in the C. reinhardtii cells grown under different conditions.
Chl fluorescence from chloroplasts of C. reinhardtii observed by 3D-SIM. Reconstructed 3D-SIM images of Chl fluorescence showing paired central (top image) and peripheral (bottom image) focal planes of (a) heterotrophically and (b) photoautotrophically grown cells under dim light conditions (~5 µmol photons m−2 s−1). Optical sections from the peripheral to central focal planes of photoautotrophically grown cell under (c) low light (~50 µmol photons m−2 s−1) and (d) high light (~350 µmol photons m−2 s−1) conditions. The numbers indicate selected focal planes of the optical sections. Scale bars, 2 µm.
Figure 5.
Figure 5.. Reconstructed 3D-SIM images of other organelles and photosynthetic organisms.
(a) The mitochondria in heterotrophically grown C. reinhardtii cells were stained by MitoTracker (MT) and observed by 3D-SIM. Top, Chl; middle, MT; bottom, merged. (b) Lipid droplets in C. reinhardtii cells under nitrogen starvation were stained by BODIPY and observed by 3D-SIM. Top, Chl; middle, BODIPY; bottom, merged. (c) MT fluorescence in selected optical sections from the peripheral to central focal planes of the cell as shown in (a). (d) BODIPY fluorescence in the selected optical sections from the peripheral to central focal planes of the cell as shown in (b). Arrows indicate a lipid droplet connecting to another one in the different focal planes. (e) Lipid bodies in heterotrophically grown C. zofingiensis cell were stained by BODIPY and observed by 3D-SIM. Top, Chl; middle, BODIPY; bottom, merged. Mitochondria in (f) the chloronema cell and (g) the caulonema cell of P. patens were stained by MT and observed by 3D-SIM. Left, Chl; middle, MT; right, merged. Numbers indicate the selected focal planes of the serial optical sections. Scale bars, 2 µm (a–e), 5 µm (f, g).
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