Chemigenetic tools for live-cell fluorescence microscopy
- Abstract number
- 5
- Presentation Form
- Invited
- Corresponding Email
- [email protected]
- Session
- Session 4 - New Technologies: Recent advances from Acquisition to Analysis
- Authors
- Michelle Frei (1)
- Affiliations
-
1. University of California San Diego
- Abstract text
Self-labeling protein tags have become critical tools in fluorescence microscopy. Their use in combination with fluorogenic fluorophores, which only become fluorescent when bound to their protein target, makes them particularly suitable for live-cell applications. The increase in fluorescence observed upon labeling and the photophysical properties of the fluorophore are mainly determined by the protein surface near the fluorophore binding site. However, most effort has been invested in the development of new fluorophores and little attention has been paid to the engineering of the self-labeling protein tag.
Here, we report on the engineering of HaloTag7 to modulate the photophysical properties of the bound rhodamines. Specifically, we developed a series of new HaloTag variants with altered brightness and lifetime properties in comparison to HaloTag7. The generated HaloTag variants are labeled with a single fluorophore but show distinct fluorescence lifetimes. This renders them distinguishable by fluorescence lifetime imaging microscopy (FLIM), enabling live-cell multiplexing of three targets in one spectral channel. Fluorescence lifetime multiplexing previously required the fastidious investigation of the fluorescence lifetime of each fluorophore. The introduced HaloTag series simplifies this task tremendously offering the first generalized strategy to create lifetime contrast in living cells. Additionally, we used the differences in fluorescence lifetime to generate a chemigenetic fluorescence lifetime based biosensor to monitor cell cycle progression.
Overall, our work highlights that the combination of protein engineering and chemical synthesis can generate imaging tools with outstanding properties. We expect the HaloTag series will facilitate the widespread use of FLIM and enable researchers to increase the number of targets measured simultaneously. This will ultimately enhance our understanding of multifactorial biological processes.