研究目的
To develop a photobleaching-assisted STORM method for super-resolution imaging of the FtsZ ring in the cyanobacterium Prochlorococcus, enabling the study of protein interactions and subcellular structures with high resolution.
研究成果
The photobleaching-assisted STORM method effectively reduces autofluorescence in Prochlorococcus, enabling super-resolution imaging with a lateral resolution of ~10 nm. This method allows detailed study of the FtsZ ring morphologies during the cell cycle, providing insights into cell division processes. The protocol may be adaptable for other photosynthetic organisms.
研究不足
The method requires careful handling of toxic chemicals and high-intensity light, which can be hazardous. The photobleaching step is time-consuming and may not completely eliminate autofluorescence in all cells. The choice of dyes and fluorescent proteins is limited by the absorption spectra of the photosynthetic pigments.
1:Sample Preparation and Fixation:
Inoculate Prochlorococcus MED4, fix with formaldehyde and glutaraldehyde.
2:Precoating of the Coverslip with Polystyrene Beads:
Use as fiducial markers for drift correction.
3:Coating of Poly-L-lysine onto the Bead-coated Coverslip:
For immobilization of cyanobacterial cells.
4:Immobilization of Cells on the Coverslip:
Attach fixed cells to the coverslip.
5:Permeabilization of Cyanobacterial Cells:
Use lysozyme and detergent for permeabilization.
6:Photobleaching of the Chlorophyll Pigments in a Blocking Step:
Reduce autofluorescence with high-intensity light.
7:Antibody Binding:
Immunostain with anti-FtsZ antibody and secondary antibody conjugated with Alexa Fluor
8:Preparation of the STORM Imaging Buffer:
7 Prepare fresh before imaging.
9:Image Acquisition of STORM Data:
Use STORM software for image acquisition and drift correction.
10:Reconstruction of Super-resolution Images from Raw Data:
Use QuickPALM and 3D Viewer in ImageJ for reconstruction.
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