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).

1. Sample Preparation and Fixation
<ol>
	<li>Inoculate 1 mL of axenic Prochlorococcus MED4 to 5 mL of the seawater-based Pro99 medium12. Grow Prochlorococcus cultures at 23 &#176;C under the light with an intensity of 35 &#181;mol 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...

<ol>
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J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nature Methods. 3 (10), 793-795 (2006).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Super-Resolution+Imaging+by+Stochastic+Optical+Reconstruction+Microscopy.">Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy.</a> Science. 319, 810-813 (2008).</li><li>Dempsey, G. T., Vaughan, J. C., Chen, K. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QuickPALM:+3D+real-time+photoactivation+nanoscopy+image+processing+in+ImageJ.">QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ.</a> Nature Methods. 7 (5), 339-340 (2010).</li><li>Schmid, B., Schindelin, J., Cardona, A., Longair, M., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-level+3D+visualization+API+for+Java+and+ImageJ.">A high-level 3D visualization API for Java and ImageJ.</a> BMC Bioinformatics. 11, 274 (2010).</li><li>Ting, C. S., Rocap, G., King, J., Chisholm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanobacterial+photosynthesis+in+the+oceans:+The+origins+and+significance+of+divergent+light-harvesting+strategies.">Cyanobacterial photosynthesis in the oceans: The origins and significance of divergent light-harvesting strategies.</a> Trends in Microbiology. 10 (3), 134-142 (2002).</li><li>Biller, S. J., Berube, P. M., Lindell, D., Chisholm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prochlorococcus:+The+structure+and+function+of+collective+diversity.">Prochlorococcus: The structure and function of collective diversity.</a> Nature Reviews Microbiology. 13 (1), 13-27 (2015).</li><li>Morel, A., Ahn, Y. -. H., Partensky, F., Vaulot, D., Claustre, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prochlorococcus+and+Synechococcus+-+a+Comparative-Study+of+Their+Optical-Properties+in+Relation+To+Their+Size+and+Pigmentation.">Prochlorococcus and Synechococcus - a Comparative-Study of Their Optical-Properties in Relation To Their Size and Pigmentation.</a> Journal of Marine Research. 51, 617-649 (1993).</li><li>Holden, S. J., Pengo, T., Meibom, K. L., Fernandez, C., Collier, J., Manley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+3D+super-resolution+microscopy+reveals+Caulobacter+crescentusin+vivo+Z-ring+organization.">High throughput 3D super-resolution microscopy reveals Caulobacter crescentusin vivo Z-ring organization.</a> Proceedings of the National Academy of Sciences. 111 (12), 4566-4571 (2014).</li><li>Zhao, M., Wei, B., Chiu, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+multiple+biomarkers+in+captured+rare+cells+by+sequential+immunostaining+and+photobleaching.">Imaging multiple biomarkers in captured rare cells by sequential immunostaining and photobleaching.</a> Methods. 64 (2), 108-113 (2013).</li><li>Golcuk, K., Mandair, G. S., Callender, A. F., Sahar, N., Kohn, D. H., Morris, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+photobleaching+necessary+for+Raman+imaging+of+bone+tissue+using+a+green+laser?.">Is photobleaching necessary for Raman imaging of bone tissue using a green laser?.</a> Biochimica et Biophysica Acta. 1758 (7), 868-873 (2006).</li><li>Haxo, F. T., Blinks, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosynthetic+Action+Spectra+of+Marine+Algae.">Photosynthetic Action Spectra of Marine Algae.</a> The Journal of General Physiology. 33 (4), 389-422 (1950).</li><li>Bryant, D. 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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...

Super-resolution microscopies can break the diffraction limit of light and provide images within sub-diffraction resolutions (&#60; 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 10 nm by spatial separation3,4. For STORM, only one molecule within a diffraction-limited region is activated (&#34;on&#34;) and the rest of the molecules are kept inactivated (&#34;off&#34;). By an accumulation of rapid switch-on and -off of single molecules, a &#34;diffraction-unlimited&#34; 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 microscopy5,6.
STORM has not been widely applied in photosynthetic cells, such as cyanobacteria, algae, and plant cells with chloroplasts7,8, 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.

<table>
	<tbody>
		<tr>
			<td>Polystyrene particles</td>
			<td>Spherotech</td>
			<td>PP-20-10</td>
			<td>2.0-2.4 &#181;m</td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Marienfeld</td>
			<td>0111580</td>
			<td>18 mm &#8709;, Thickness No. 1</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Scharlau</td>
			<td>ET00021000</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P9155</td>
			<td>mol wt 70,000-150,000</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde solution, 50%</td>
			<td>Sigma-Aldrich</td>
			<td>340855</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Disodium Salt, 2-hydrate</td>
			<td>Gold biotechnology</td>
			<td>E-210-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Anabaena FtsZ antibody</td>
			<td>Agrisera</td>
			<td>AS07217</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody</td>
			<td>Life Technologies</td>
			<td>A-21039</td>
			<td>conjugated with Alexa Fluor 750</td>
		</tr>
		<tr>
			<td>D-Glucose Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Viologen</td>
			<td>Sigma-Aldrich</td>
			<td>856177</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclooctatetraene</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td></td>
		</tr>
		<tr>
			<td>tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>646547</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Oxidase</td>
			<td>Sigma-Aldrich</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma-Aldrich</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>XD-300 Xenon light source</td>
			<td></td>
			<td></td>
			<td>250 W</td>
		</tr>
		<tr>
			<td>STORM microscope</td>
			<td>NBI</td>
			<td>SRiS microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Rohdea</td>
			<td>NBI</td>
			<td>SRiS 3.0</td>
			<td>software for imaging acquisition</td>
		</tr>
		<tr>
			<td>Luna</td>
			<td>NBI</td>
			<td>SRiS 3.0</td>
			<td>software for drifting correction</td>
		</tr>
		<tr>
			<td>QuickPALM</td>
			<td></td>
			<td></td>
			<td>https://code.google.com/archive/p/quickpalm/wikis</td>
		</tr>
		<tr>
			<td>3D Viewer</td>
			<td></td>
			<td></td>
			<td>http://132.187.25.13/ij3d/?page=Home&#38;category=Home</td>
		</tr>
	</tbody>
</table>

photobleaching enables super-resolution imaging of the ftsz ring in the cyanobacterium prochlorococcus

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.

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...

Watch this Scientific Journal Video about Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus at JoVE.com

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

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.

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

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, ...

This method can help answer key questions on protein localization and the dynamics in photosynthetic organisms. By combining the photobleaching method with super-resolution microscope, we are able to detect the protein dynamics of photosynthetic organisms in a remarkable detail. This method not only provides insight into the protein dynamics of Prochlorococcus.It can also be applied to many other organisms containing radio-re-dop-sing and bacteriochlorophyll. Demonstrating this procedure will be Yanxin Liu, a graduate student from my laboratory. To begin, add five milliliters of seawater-based Pro99 medium to a culture flask and inoculate it with one milliliter of Prochlorococcus MED4.Grow the Prochlorococcus at 23 degrees Celsius under a light with an intensity of 35 micromole photons per square meter. After five days, collect one milliliter of the culture into a 1.5 milliliter tube. Add 100 microliters of freshly prepared formaldehyde to the tube to fix the culture.Incubate in the dark at room temperature for 20 minutes. Then, spin down the sample at 13, 500 times G for one minute. Remove the supernatant and resuspend the cells in 100 microliters of Pro99 medium.Store the sample at four degrees Celsius in the dark until ready to perform immunostaining. First, vortex the vial of polystyrene beads. Using 50%ethanol, prepare a one-to-20, 000 dilution as a working slurry.Turn on the hotplate and set the temperature to 120 degrees Celsius. Next, place the coverslips onto the hot plate. Load 100 microliters of the working slurry onto each coverslip and incubate the coverslips on the hotplate for 10 minutes.Then carefully transfer the coverslips to Petri dishes bead-side up for storage at room temperature. When ready to coat the coverslips, retrieve one, making sure that it is bead-side up. Add 100 microliters of a one milligram per milliliter poly-L-lysine solution to the center of the coverslip and let it sit at room temperature for 30 minutes.After this, use a pipette to carefully aspirate any unattached poly-L-lysine and transfer it to a 1.5 milliliter tube. Store this poly-L-lysine at four degrees Celsius for later use. Rinse the coverslip with 10 milliliters of ultra filtered water in a 60-millimeter Petri dish and then briefly dry it with a paper towel.Transfer the coated coverslip to a new Petri dish with Parafilm at the bottom, which will be used as the staining dish. Load 100 microliters of the fixed sample onto the coverslip and let it sit for 30 minutes to allow cell attachment. Next, use a paper towel to absorb the remaining solution and then transfer the coverslip to a well in a 12-well plate for washing.Add one milliliter of PBS buffer to the well to wash the coverslip. Then remove the PBS and add one milliliter of fresh PBS to wash the coverslip a second time. Use a pipette to remove the PBS and replace it with one milliliter of freshly prepared permeabilization buffer.Incubate the washing dish at 37 degrees Celsius for 20 minutes. Then remove the permeabilization buffer. Begin the washing process by adding one milliliter of fresh PBS buffer.Place the washing dish on a shaker to gently agitate the dish for five minutes. After this, remove the PBS and repeat the washing process three times. Transfer the washed coverslip to the staining dish.Add 50 microliters of blocking buffer on the top of the coverslip. Place the entire staining dish on ice and then move it under the xenon light source for photobleaching for at least 60 minutes. After photobleaching and blocking, use the edge of a paper towel to remove the blocking buffer.First, dilute one microliter of anti-FtsZ antibody into 99 microliters of blocking buffer. Place 50 microliters of the diluted primary antibody onto the coverslip and incubate it at room temperature for 30 minutes. Transfer the coverslip to a well of the washing dish.To begin the wash, add one milliliter of PBS. Then transfer the washing dish to a shaker and gently shake it for five minutes. Remove the PBS and repeat the entire washing process three times.Next, transfer the coverslip to the staining dish. Place 50 microliters of the diluted secondary antibody onto the coverslip and incubate it in the dark and at room temperature for 30 minutes. Transfer the coverslip back to the washing dish.To begin the wash, add one milliliter of PBS. Wrap the washing dish in aluminum foil to protect it from light. Transfer the plate to the shaker and gently shake it for five minutes.Remove the PBS and repeat the entire washing process three times. Immediately before the STORM imaging, prepare one milliliter of imaging buffer as outlined in the text protocol. Load the coverslip into the loading chamber.Add the freshly prepared imaging buffer, being gentle to avoid washing off the cyanobacterial cells. After this, place a rectangular coverslip on top of the imaging buffer to prevent it from reacting with the oxygen in the air. Turn on the camera, the LED light, and the laser, and open the STORM software.Add half a drop of immersion oil on top of the lens. Load the chamber, making sure that the objective lens is making contact with the coverslip. And examine the signal with a 750-nanometer laser.Identify a sample area that contains both cells and fiducial markers. Then start the software for sample drifting correction. Acquire one wide-filed images as a reference, with the camera electron multiplication gain at 300 and an exposure time of 30 milliseconds.Increase the 750-nanometer excitation laser intensity to approximately 4.5 kilowatts per square centimeter. Once the fluorophores have transitioned into a sparse blinking pattern, acquire one super-resolution image by collecting 10, 000 frames at 33 hertz. Then reconstruct the super-resolution images from the raw data as outlined in the text protocol.In this study, STORM is used to achieve super-resolution imaging by activating individual photo-switchable fluorophores stochastically. The location of every fluorophore is recorded, and a super-resolution image is then constructed based on these locations. While the absorption spectra of Prochlorococcus peaks at 447 and 680 nanometers and has minimum absorption above 700 nanometers, Prochlorococcus MED4 cells still emit high autofluorescence when exposed to an extremely high intensity of the 750-nanometer laser, which is required for STORM imaging.After using the photobleaching method described here for 30 minutes, the cells'autofluorescence is seen to decrease, although several cells with autofluorescence are still detected. Elongating the photobleaching to 60 minutes results in the majority of the cells losing their autofluorescence. These results indicate that the developed photobleaching method is capable of greatly reducing the autofluorescence of photosynthetic organisms.After photobleaching, STORM is used to visualize the cell division protein, FtsZ, in the cells. Using STORM, a detailed morphology of the FtsZ ring is revealed. By rotating the 3D STORM images, four different types of morphologies were identified, clusters, an incomplete ring, a complete ring, and double rings.It is important to remember that the light intensity and duration of the photobleach may differ for different organisms. After its development, this method paves the way for researchers who want to study protein organization and potential function in photosynthetic cells in detail.

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JoVE Journal

Immunology and Infection

6.2K Views.  Hong Kong University of Science and Technology. Questo metodo può aiutare a rispondere a domande chiave sulla localizzazione delle proteine e sulle dinamiche negli organismi fotosintetici. Combinando il metodo di fotobleaching con il microscopio a super risoluzione, siamo in grado di rilevare la dinamica proteica degli organismi fotosintetici in un dettaglio notevole. Questo metodo non solo fornisce informazioni sulla dinamica proteica del proclorococco.Può anche essere applicato a molti altri organismi contenenti radio-re-dop-sin...