Title
Improved fluorescent phytochromes for in situ imaging
Author
Soshichiro Nagano, Maryam Sadeghi, Jens Balke, Moritz Fleck, Nina Heckmann, Georgios Psakis & Ulrike Alexiev
Year
2022
Journal
Scientific reports
Abstract
Modern biology investigations on phytochromes as near-infrared fluorescent pigments pave the way for the development of new biosensors, as well as for optogenetics and in vivo imaging tools. Recently, near-infrared fluorescent proteins (NIR-FPs) engineered from biliverdin-binding bacteriophytochromes and cyanobacteriochromes, and from phycocyanobilin-binding cyanobacterial phytochromes have become promising probes for fluorescence microscopy and in vivo imaging. However, current NIR-FPs typically suffer from low fluorescence quantum yields and short fluorescence lifetimes. Here, we applied the rational approach of combining mutations known to enhance fluorescence in the cyanobacterial phytochrome Cph1 to derive a series of highly fluorescent variants with fluorescence quantum yield exceeding 15%. These variants were characterised by biochemical and spectroscopic methods, including time-resolved fluorescence spectroscopy. We show that these new NIR-FPs exhibit high fluorescence quantum yields and long fluorescence lifetimes, contributing to their bright fluorescence, and provide fluorescence lifetime imaging measurements in E.coli cells.
Full Article
Instrument
J-715
Keywords
biosensors, quantum yield, cyanobacterial phytochromes