Beyond color: Fluorescence lifetime imaging distinguishes multiple proteins in living plant cells
Fluorescent protein imaging is an indispensable tool in life science research, enabling visualization of protein movement and localization, gene expression, signaling pathways, protein-protein interactions and more. For simultaneous analysis of multiple proteins (i.e., multiplex imaging), a variety of fluorescent proteins that emit different colors (blue, green, yellow and red) have been…
Fluorescence lifetime imaging microscopy (FLIM) has been shown to distinguish multiple proteins with overlapping color emissions within living plant cells, overcoming limitations of conventional fluorescence intensity imaging. Researchers from WPI-ITbM at Nagoya University demonstrated that FLIM could distinguish four red fluorescent proteins, despite their similar color emissions, by analyzing their fluorescence lifetimes with phasor plot analysis.
This technique successfully distinguished proteins localized to various cellular regions, including the nucleus, peroxisomes, chloroplasts, and plasma membrane, even when conventional imaging methods failed. The method also distinguished three types of fluorescent proteins localized to peroxisomes, nucleus, and chloroplasts, separated into distinct populations.
Furthermore, an intermediate fluorescence lifetime was observed at the boundary regions between adjacent peroxisomes and chloroplasts, reflecting contributions from both proteins. This method could overcome autofluorescence from the cell wall and chloroplasts, making live imaging in plant cells more flexible and overcoming current limitations on the number of fluorescent proteins that can be observed simultaneously during multiplex imaging.
Written by urgent.news from Phys.org's reporting — not their text. Machine-written — it may contain errors, so check the original before relying on it.
