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Immunology and Infection

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

Published: November 6th, 2018

DOI:

10.3791/58603

1Department of Ocean Science, Hong Kong University of Science and Technology, 2Division of Life Science, Hong Kong University of Science and Technology, 3HKUST Shenzhen Research Institute

Here we describe a photobleaching method to reduce the autofluorescence of cyanobacteria. After photobleaching, stochastic optical reconstruction microscopy is used to obtain three-dimensional super-resolution images of the cyanobacterial FtsZ ring.

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, however, the lateral resolution of super-resolution imaging is only ~100 nm. The low resolution is mainly due to the high autofluorescence background of photosynthetic cells caused by high-intensity lasers that are required for super-resolution imaging, such as stochastic optical reconstruction microscopy (STORM). Here, we describe a photobleaching-assisted STORM method which was developed recently for imaging the marine picocyanobacterium Prochlorococcus. After photobleaching, the autofluorescence of Prochlorococcus is effectively reduced so that STORM can be performed with a lateral resolution of ~10 nm. Using this method, we acquire the in vivo three-dimensional (3-D) organization of the FtsZ protein and characterize four different FtsZ ring morphologies during the cell cycle of Prochlorococcus. The method we describe here might be adopted for the super-resolution imaging of other photosynthetic organisms.

Super-resolution microscopies can break the diffraction limit of light and provide images within sub-diffraction resolutions (< 200 nm). They have been widely used in many organisms to study protein localization and subcellular structures. Major super-resolution microscopy methods include structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), STORM, and photoactivated localization microscopy (PALM). The mechanisms and applications of these super-resolution microscopes have been reviewed elsewhere1,2.

STORM can achieve a resolution as high as 1....

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1. Sample Preparation and Fixation

  1. Inoculate 1 mL of axenic Prochlorococcus MED4 to 5 mL of the seawater-based Pro99 medium12. Grow Prochlorococcus cultures at 23 °C under the light with an intensity of 35 µmol photons/m2s. Five days later, collect 1 mL of culture into a 1.5-mL tube.
    NOTE: Five days after the inoculation, Prochlorococcus MED4 will reach the late log phase, with approximately 108 cell/mL, which is appropr.......

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STORM achieves super-resolution imaging by activating individual photoswitchable fluorophores stochastically. The location of every fluorophore is recorded and a super-resolution image is then constructed based on these locations4. Therefore, the precision of the fluorophore location is important for the super-resolution image reconstruction. The absorption spectra of Prochlorococcus peak at 447 nm and 680 nm,and Prochlorococcus has a minimum abso.......

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In this protocol, we described a procedure to significantly reduce the autofluorescence of the cyanobacterium Prochlorococcus (Figure 3C) and, then, immunostain the proteins in the cells, which enabled us to utilize STORM to study the 3-D FtsZ ring morphologies in Prochlorococcus (Figure 4). This protocol might be adopted for super-resolution imaging in other photosynthetic organisms.

Previous studies on photosynthet.......

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The authors thank Daiying Xu for her technical assistance and comments on the manuscript. This study is supported by grants from the National Natural Science Foundation of China (Project number 41476147) and the Research Grants Council of the Hong Kong Special Administrative Region, China (project numbers 689813 and 16103414).

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Name Company Catalog Number Comments
Polystyrene particles Spherotech PP-20-10 2.0-2.4 µm
Coverslip Marienfeld 0111580 18 mm ∅, Thickness No. 1
Ethanol Scharlau ET00021000
Poly-L-lysine hydrobromide Sigma-Aldrich P9155 mol wt 70,000-150,000
Paraformaldehyde Sigma-Aldrich 158127
Glutaraldehyde solution, 50% Sigma-Aldrich 340855
PBS Sigma P3813
Triton X-100 Sigma T8787
EDTA Disodium Salt, 2-hydrate Gold biotechnology E-210-500
Trizma base Sigma T1503
Lysozyme Sigma L6876
Goat serum Sigma G9023
anti-Anabaena FtsZ antibody Agrisera AS07217
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Life Technologies A-21039 conjugated with Alexa Fluor 750
D-Glucose Anhydrous Fisher Scientific D16-1
L-Ascorbic Acid Sigma-Aldrich A5960
Methyl Viologen Sigma-Aldrich 856177
Cyclooctatetraene Sigma-Aldrich 138924
tris(2-carboxyethyl)phosphine (TCEP) Sigma-Aldrich 646547
Glucose Oxidase Sigma-Aldrich G2133
Catalase Sigma-Aldrich C9322
XD-300 Xenon light source 250 W
STORM microscope NBI SRiS microscope
Rohdea NBI SRiS 3.0 software for imaging acquisition
Luna NBI SRiS 3.0 software for drifting correction
QuickPALM https://code.google.com/archive/p/quickpalm/wikis
3D Viewer http://132.187.25.13/ij3d/?page=Home&category=Home

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