Comprehensive database of fluorescence lifetime values for fluorochromes with emission peaks in the visible or near infrared

Multiplexing techniques rely on fluorescent probes to simultaneously detect and visualise multiple mRNA or protein molecules in a single cell. Although more than 1200 fluorochromes are available in the visible and near-infrared spectral range, it is difficult to separate the different fluorochromes spectrally into orthogonal channels as their excitation and emission spectra often overlap. Fluorescence lifetimes can be used as an effective method to segregate fluorochromes for multiplex imaging. However, information on fluorescence lifetimes is not always easy to find as it is often only mentioned in passing on websites or in publications. In an effort to overcome this challenge, we performed a systematic literature review to make it easier to access the information required to attempt unmixing fluorochromes by fluorescence lifetime for multiplexed imaging. We found that at least 88 fluorochromes can be used, in principle, to attempt unmixing fluorochromes by lifetime and thus multiplexing. Our data are summarised in a table as well as in a graph in which we plotted the lifetime (tau) against the emission peak (Em). For all fluorochromes, we found that are available as NHS derivatives for easy coupling to DNA oligonucleotides or antibodies. Some additional, potentially very useful fluorophores, for which no lifetime data are available, are shown within a dashed line in a relatively “sparse” region of the spectrum above 800nm. A key is displayed to describe the colour code used to represent different fluorochrome classes, such as Alexa Fluor.