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Optimization of sample preparation and green color imaging using the mNeonGreen fluorescent protein in bacterial cells for photoactivated localization microscopy.
- Source :
-
Scientific reports [Sci Rep] 2018 Jul 04; Vol. 8 (1), pp. 10137. Date of Electronic Publication: 2018 Jul 04. - Publication Year :
- 2018
-
Abstract
- mNeonGreen fluorescent protein is capable of photo-switching, hence in principle applicable for super-resolution imaging. However, difficult-to-control blinking kinetics that lead to simultaneous emission of multiple nearby mNeonGreen molecules impedes its use for PALM. Here, we determined the on- and off- switching rate and the influence of illumination power on the simultaneous emission. Increasing illumination power reduces the probability of simultaneous emission, but not enough to generate high quality PALM images. Therefore, we introduce a simple data post-processing step that uses temporal and spatial information of molecule localizations to further reduce artifacts arising from simultaneous emission of nearby emitters. We also systematically evaluated various sample preparation steps to establish an optimized protocol to preserve cellular morphology and fluorescence signal. In summary, we propose a workflow for super-resolution imaging with mNeonGreen based on optimization of sample preparation, data acquisition and simple post-acquisition data processing. Application of our protocol enabled us to resolve the expected double band of bacterial cell division protein DivIVA, and to visualize that the chromosome organization protein ParB organized into sub-clusters instead of the typically observed diffraction-limited foci. We expect that our workflow allows a broad use of mNeonGreen for super-resolution microscopy, which is so far difficult to achieve.
- Subjects :
- Bacillus subtilis metabolism
Bacillus subtilis physiology
Bacterial Proteins metabolism
Cell Division
Chromosomes, Bacterial metabolism
Green Fluorescent Proteins genetics
Green Fluorescent Proteins standards
Microscopy, Fluorescence methods
Microscopy, Fluorescence standards
Single-Cell Analysis standards
Bacillus subtilis cytology
Green Fluorescent Proteins metabolism
Single-Cell Analysis methods
Subjects
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 8
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 29973667
- Full Text :
- https://doi.org/10.1038/s41598-018-28472-0