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Int J Mol Sci. 2019 Jul 10;20(14). pii: E3376. doi: 10.3390/ijms20143376.

Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy.

Author information

1
Max Plank Institute of Biochemistry, 82152 Martinsried, 82152 Munich, Germany.
2
Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, 80539 Munich, Germany.
3
Max Plank Institute of Neurobiology, 82152 Martinsried, 82152 Munich, Germany.
4
Max Plank Institute of Neurobiology, 82152 Martinsried, 82152 Munich, Germany. rkasper@neuro.mpg.de.
5
Max Plank Institute of Biochemistry, 82152 Martinsried, 82152 Munich, Germany. jshin@biochem.mpg.de.
6
Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, 80539 Munich, Germany. jshin@biochem.mpg.de.

Abstract

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems.

KEYWORDS:

STED; bacteria; cell division; fluorescent proteins; nanobody; super-resolution microscopy

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