Photobleaching Cyanobacterial Pigments for Imaging Growth-Regulating Proteins

Abstract

Source: Zhan, Y., et al. Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus. J. Vis. Exp. (2018).

This video demonstrates the procedure for photobleaching pigments in cyanobacterial cells using xenon light under cold conditions to improve the fluorescence imaging of growth-regulating proteins.

Protocol

1. 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 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 absorbing it with the edge of a paper towel.
   NOTE: The intensity of xenon light is so high that proper sunglasses are needed for eye protection. Meanwhile, wrap the light source and the plate using aluminum foil to avoid light leaking, which may cause eye damage. Check the coverslip every 15 minutes to make sure that it remains moist. If it is dry, add 50 µL of blocking buffer.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polystyrene particles	Spherotech	PP-20-10	2.0-2.4 µm
Coverslip	Marienfeld	111580	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
XD-300 Xenon light source			250 W