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

Summary

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.

Abstract

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.

Introduction

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 elsewhere^1,2.

STORM can achieve a resolution as high as 10 nm by spatial separation^3,4. For STORM, only one molecule within a diffraction-limited region is activated ("on") and the rest of the molecules are kept inactivated ("off"). By an accumulation of rapid switch-on and -off of single molecules, a "diffraction-unlimited" image can be generated³. Meanwhile, many kinds of organic dyes and fluorescent proteins are applicable in STORM, allowing an easy upgrade from regular fluorescence microscopy to high-resolution microscopy^5,6.

STORM has not been widely applied in photosynthetic cells, such as cyanobacteria, algae, and plant cells with chloroplasts^7,8, which is due to the fact that STORM requires high laser intensity to drive photoswitching. The high-intensity laser unfavorably excites strong autofluorescence background in photosynthetic cells and interferes with the single-molecule localization in STORM imaging. In order to use STORM to investigate the subcellular structures or protein interactions in photosynthetic cells, we developed a photobleaching protocol to quench the background autofluorescence signals⁹. In a routine immunofluorescent staining procedure, specimens are exposed to white light of a high intensity during the blocking step, which lowers the autofluorescence of photosynthetic cells to meet the requirements for STORM. Thus, this protocol makes it feasible to investigate pigmented organisms with STORM.

Here, we describe the protocol to use STORM to image the FtsZ ring organization in the unicellular picocyanobacterium Prochlorococcus. FtsZ is a highly conserved tubulin-like cytoskeletal protein which polymerizes to form a ring structure (the Z ring) around the circumference of a cell¹⁰ and is essential for the cell division¹¹. Preserved Prochlorococcus cells are first photobleached to reduce the autofluorescent background and immunostained with a primary anti-FtsZ antibody, and then a secondary anti-Rabbit IgG (H+L) antibody is conjugated with a fluorophore (e.g., Alexa Fluor 750). Eventually, STORM is used to observe the detailed FtsZ ring organizations in Prochlorococcus during different cell cycle stages.

Protocol

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 appropriate for STORM imaging.
2. Add 100 µL of formaldehyde freshly prepared from 25% paraformaldehyde (PFA) (w/v) and 2 µL of 50% glutaraldehyde into the 1.5-mL tube to fix the culture. Incubate the sample in the dark at room temperature for 20 min.
   NOTE: The sample was kept in the dark for fixation because glutaraldehyde is light sensitive.
3. Spin down the sample at 13,500 x g for 1 min; then, remove the supernatant and resuspend the cells in 100 µL of the Pro99 medium¹². Store the sample at 4 °C until immunostaining.

2. Precoating of the Coverslip with Polystyrene Beads

NOTE: Polystyrene beads are considered as the fiducial marker for drift correction.

1. Vortex the original vial of polystyrene beads and prepare a 1:20,000 dilution with 50% ethanol as working slurry.
2. Turn on the hot plate, set the temperature to 120 °C, and place coverslips onto the hot plate.
3. Load 100 µL of working slurry onto each coverslip. Incubate the coverslip on the hot plate for 10 min and, then, carefully transfer the coverslips to Petri dishes for storage at room temperature.
   NOTE: The side with the beads attached to it should face up, and the cells should be attached on the same side. The beads are immobilized on the coverslips and used for correcting the sample-drifting in real-time during STORM imaging. After immobilizing the beads, the coverslips cannot be flamed.

3. Coating of Poly-L-lysine onto the Bead-coated Coverslip

NOTE: This is done for the immobilization of cyanobacterial cells.

1. Add 100 µL of poly-L-lysine (1 mg/mL) to the center of the coverslip with the bead-coated side facing up. Let it sit for 30 min at room temperature.
2. Use a pipette to carefully aspire the unattached poly-L-lysine and transfer it to a 1.5-mL tube. Save the poly-L-lysine at 4 °C for reuse.
3. Rinse the coverslip with 10 mL of ultra-filtered water in a 60-mm Petri dish and then briefly dry the coverslip on a paper towel.
4. Store the coverslip in a Petri dish (100 mm) for later usage. To prevent the attachment of the coverslip to the dish, line the Petri dish with a layer of parafilm.
   NOTE: The coverslip, precoated with beads and poly-L-lysine, can be stored at room temperature for at least half a year. Therefore, preparing a batch of coverslips in advance is highly recommended.

4. Immobilization of Cells on the Coverslip

1. Transfer a precoated coverslip to a new Petri dish with parafilm at the bottom, which will be referred to as the staining dish henceforth. Load 100 µL of the fixed sample on the coverslip and let it sit for 30 min to allow cell attachment.
2. Transfer the coverslip to a well of a 12-well plate, which will be referred to as the washing dish henceforth. Add 1 mL of phosphate-buffered saline (PBS) (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) to the well to wash the coverslip. Remove the PBS buffer and add a new PBS buffer to wash the coverslip again.

5. Permeabilization of Cyanobacterial Cells

1. Remove the PBS buffer using a pipette. Add 1 mL of freshly prepared permeabilization buffer to the well of the washing dish, which contains 0.05% non-ionic detergent-100 (v/v), 10 mM EDTA, 10 mM Tris (pH 8.0), and 0.2 mg/mL lysozyme.
2. Incubate the washing dish at 37 °C for 20 min. Then, remove the permeabilization buffer.
3. Add 1 mL of PBS buffer and place the washing dish on a shaker which gently shakes the washing dish for 5 min. Remove the PBS buffer. Repeat this step 3x to wash the coverslip.

6. Photobleaching of the Chlorophyll Pigments in a Blocking Step

1. Transfer the coverslip to the staining dish. Add 50 µL of blocking buffer, which contains 0.2% (v/v) non-ionic detergent-100 and 3% (v/v) goat serum, on the top of the coverslip.
2. Place the whole staining plate on ice and move it under the xenon light source for photobleaching for at least 60 min, with a light intensity at 1,800 µmol photons/m²·s.
3. After photobleaching and blocking, carefully remove the blocking buffer by adsorbing it with the edge of a paper towel.
   NOTE: The intensity of xenon light is so high that a pair of proper sunglasses is needed for eye protection. Meanwhile, wrap the light source and the plate using aluminum foil to avoid the leaking of light, which may cause eye damage. Check the coverslip every 15 min to make sure that the coverslip remains moist. If the coverslip is dry, add 50 µL of blocking buffer.

7. Antibody Binding

1. Dissolve anti-Anabaena FtsZ antibody in 200 µL of water according to the manufacturer’s instructions.
2. Dilute 1 µL of anti-FtsZ antibody into 99 µL of blocking buffer. Add 50 µL of diluted primary antibody on the coverslip and incubate it at room temperature for 30 min.
3. Transfer the coverslip to the washing dish. Add 1 mL of PBS buffer and place the washing dish on a shake. Gently shake the washing dish for 5 min. Repeat this step 3x to wash the coverslip.
4. Transfer the coverslip to the staining dish. Dilute 1 µL of a secondary antibody into 500 µL of blocking buffer. Add 50 µL of diluted secondary antibody onto the coverslip and incubate it for 30 min in the dark at room temperature.
5. Transfer the coverslip to the washing dish. Wash it with 1 mL of PBS buffer on a shaker. Gently shake the washing dish for 5 min. Wrap the washing dish with aluminum paper to make it light-proof. Repeat this step 3x.
6. Add 500 µL of formaldehyde freshly prepared from 4% paraformaldehyde (PFA) (w/v) to the well. Incubate the coverslip for 15 min in the dark at room temperature.
7. Remove the formaldehyde and repeat the washing step 3x.
8. Store the coverslip in PBS buffer at 4 °C in the dark until imaging.

8. Preparation of the STORM Imaging Buffer

1. Prepare 1 mL of imaging buffer according to Table 1, immediately before the STORM imaging.
   NOTE: As the shelf life of the imaging buffer is about 2 h, prepare it fresh, right before usage. Avoid vortexing after the addition of glucose oxidase and catalase.
   CAUTION: Methyl viologen is poisonous material. Please handle it with particular care. Cyclooctatetraene is classified as a carcinogen Cat. 1 chemical. Please avoid inhalation and skin contact and make the cyclooctatetraene stock solution and imaging buffer in a chemical hood.

9. Image Acquisition of STORM Data

1. Load the coverslip in the loading chamber (Figure 1). Load the freshly prepared imaging buffer gently into the chamber to avoid washing off the cyanobacterial cells. Place a rectangular coverslip on the top of the imaging buffer to prevent its reaction with oxygen in the air. Avoid trapping air bubbles underneath the coverslip.
2. Turn on the camera, the LED light, and the laser. Open STORM software for image acquisition, centroid position determination, and sample drifting correction¹³.
3. Add half a drop of immersion oil on top of the lens. Load the chamber and make sure the lens of the objective makes contact with the coverslip.
4. Examine the signal with a 750-nm laser.
   NOTE: Always include a second antibody-minus-staining negative control to ensure that the autofluorescence is diminished.
5. Identify a sample area that contains both cells and fiducial markers. Start the software for sample drifting correction.
   NOTE: In general, the ideal sample area contains 10 - 30 cells. Meanwhile, the cells should be separated well to avoid any overlapping cells.
6. Acquire one wide-field image as a reference, with the camera electron multiplication (EM) gain at 300 and an exposure time of 30 ms (Figure 2A).
7. Increase the 750-nm excitation laser intensity to a higher power, approximately 4.5 kW/cm². Once the fluorophores have transitioned into a sparse blinking pattern, acquire one super-resolution image by collecting 10,000 frames at 33 Hz (Figure 2B).
   NOTE: If the fluorophores are not isolated, wait for a couple of minutes until more fluorophores are switched to the dark state and the isolated single molecules flicker sequentially. The number of frames described here is suitable for our sample and needs to be optimized for other targeted structures.

10. Reconstruction of Super-resolution Images from Raw Data

1. Use a plugin called QuickPALM (Table of Materials)¹⁴ in ImageJ to reconstruct a 3-D color super-resolution image.
   NOTE: A preliminary chromatic image (Figure 2C) along with a color-coded scale bar (Figure 2C) will appear. The color represents the depth on the z-axis.
2. To demonstrate the 3-D structure in a video, construct a stack of super-resolution images with z-axis sectioned every 10 nm using QuickPALM¹⁴. Adjust the brightness of the stacks and duplicate the stack of one target cell. Use a plugin called 3D Viewer (Table of Materials)¹⁵ in ImageJ to generate the 3-D image of the cell. Record the rotation of the 3-D structure for demonstration purposes.

Results

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...

Discussion

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...

Materials

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