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

被引:15
作者
Cramer, Kimberly [1 ,2 ,3 ]
Bolender, Anna-Lena [4 ]
Stockmar, Iris [1 ,2 ,3 ]
Jungmann, Ralf [1 ,2 ,3 ]
Kasper, Robert [4 ]
Shin, Jae Yen [1 ,2 ,3 ]
机构
[1] Max Plank Inst Biochem, D-82152 Martinsried, Munich, Germany
[2] Ludwig Maximilians Univ Munchen, Fac Phys, D-80539 Munich, Germany
[3] Ludwig Maximilians Univ Munchen, Ctr Nanosci, D-80539 Munich, Germany
[4] Max Plank Inst Neurobiol, D-82152 Martinsried, Munich, Germany
基金
欧洲研究理事会;
关键词
STED; bacteria; super-resolution microscopy; fluorescent proteins; nanobody; cell division; ACTIVATED LOCALIZATION MICROSCOPY; SUPERRESOLUTION MICROSCOPY; RESOLUTION LIMIT; CELL-DIVISION; SINGLE; SUBTILIS; FTSZ;
D O I
10.3390/ijms20143376
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
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页数:11
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