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Assessing crosstalk in simultaneous multicolor single-molecule localization microscopy
- Source :
- Cell Reports Methods; September 2023, Vol. 3 Issue: 9
- Publication Year :
- 2023
-
Abstract
- Single-molecule localization microscopy (SMLM) can reach sub-50 nm resolution using techniques such as stochastic optical reconstruction microscopy (STORM) or DNA-point accumulation for imaging in nanoscale topography (PAINT). Here we implement two approaches for faster multicolor SMLM by splitting the emitted fluorescence toward two cameras: simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) that images spectrally separated red and far-red imager strands on each camera, and spectral demixing dSTORM (SD-dSTORM) where spectrally close far-red fluorophores appear on both cameras before being identified by demixing. Using S2C-DNA-PAINT as a reference for low crosstalk, we evaluate SD-dSTORM crosstalk using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then assess if crosstalk can affect the detection of biologically relevant subdiffraction patterns. Extending these approaches to three-dimensional acquisition and SD-dSTORM to three-color imaging, we show that spectral demixing is an attractive option for robust and versatile multicolor SMLM investigations.
Details
- Language :
- English
- ISSN :
- 26672375
- Volume :
- 3
- Issue :
- 9
- Database :
- Supplemental Index
- Journal :
- Cell Reports Methods
- Publication Type :
- Periodical
- Accession number :
- ejs64026942
- Full Text :
- https://doi.org/10.1016/j.crmeth.2023.100571