Fluorescence lifetime imaging separates overlapping proteins in living plant cells

Scientists at the Institute of Transformative Bio-Molecules (ITbM) at Nagoya University have demonstrated that fluorescence lifetime imaging microscopy (FLIM) can distinguish several fluorescent proteins whose emission colors overlap too much to be separated by conventional intensity imaging. The study, published July 30 in Plant Physiology, shows that proteins differing by only a few nanometers in emission wavelength and fractions of a nanosecond in fluorescence lifetime can be resolved in living plant cells.

In fluorescence imaging, researchers typically rely on color differences to track multiple proteins at once. The palette of distinguishable colors is limited, however, making it difficult to observe more than a few proteins simultaneously. FLIM captures the average time a fluorophore remains in its excited state before emitting a photon, providing an independent contrast mechanism.

The team first tested four red fluorescent proteins — mCherry, mRFP, mApple and tdTomato — in purified form. Conventional intensity images showed indistinguishable signals, but FLIM combined with phasor plot analysis separated each protein into distinct populations. Notably, mCherry and mRFP were resolved despite a 3-nanometer emission difference and a lifetime difference of only about 0.2 nanoseconds.

The researchers then moved to living protonemal cells of the moss Physcomitrium patens. They expressed fluorescent proteins targeted to the nucleus, peroxisomes, chloroplasts and plasma membrane. Again, intensity imaging failed to separate the signals, while phasor analysis of FLIM data distinguished both red-emitting pairs (mCherry and tagRFP-T) and green-emitting pairs (GFP and NowGFP).

In a three-color demonstration, proteins localized to peroxisomes, the nucleus and chloroplasts were separated into three distinct phasor clusters. An intermediate lifetime signal appeared at boundaries between adjacent peroxisomes and chloroplasts, suggesting mixed contributions from both fluorophores and hinting at potential for studying protein proximity.

Plant cells present a particular challenge because strong autofluorescence from cell walls and chloroplasts obscures color-based multiplexing. The authors say FLIM-based lifetime contrast bypasses this autofluorescence barrier, expanding the number of proteins that can be tracked simultaneously in live plant imaging.

The work was led by researchers Tsuyoshi Aoyama, Nagisa Sugimoto and Designated Associate Professor Yoshikatsu Sato. The study is available at DOI: 10.1093/plphys/kiag475.