Live-Cell Fluorescence Lifetime Multiplexing Using Organic Fluorophores

We characterized 18 cell-permeable synthetic probes by fluorescence lifetime imaging microscopy (FLIM) and found eight pairwise combinations suitable for fluorescence lifetime multiplexing. When combining multiple pairs in different spectral channels, we could perform spectrally resolved fluorescence lifetime multiplexing of up to four subcellular targets in a straightforward manner. However, synthetic probes are only available for a handful of targets and sometimes show unsuitable lifetime properties.

We, therefore, complemented the use of synthetic organic probes with a second labelling strategy for synthetic fluorophores, namely, self-labeling protein tags. Genetically encodable, self-labeling protein tags can be fused to a protein of interest and therefore allow labeling of almost any desired subcellular target. We engineered variants of the self-labeling protein tag HaloTag7 (i.e., HaloTag9, HaloTag10, and HaloTag11) such that the bound fluorophore shows distinct fluorescence lifetimes.

Combining self-labeling protein tags and synthetic probes enabled us to image up to eight targets in four spectral channels. We hope that the two approaches will promote the use of fluorescence lifetime multiplexing in living cells.