Differentiating dyes based on their emission peak and lifetime characteristics

Multiplexing techniques rely on fluorescent probes to simultaneously detect and visualize multiple RNA molecules in a single cell. However, the challenge is the limited choice of dyes, as many of them have overlapping emission spectra. We hypothesize that by using the concept of lifetime, it may be possible to separate closely overlapping dyes and thus enable lifetime multiplexing. Information on lifetime is not always easy to find. After a systematic literature search, we collected data from various commercial dyes and found that at least 88 dyes could be used for multiplexing and potentially separated; our goal would be to update this information to make it accessible to all. In the graph we present the different dyes with their emission peak (Em) and lifetime (T). Dyes for which lifetime data is not available are represented by a dashed line, and areas with overlapping dyes can be filtered by excitation peaks (gray area). Taking dye lifetime into account expands the ability to detect a larger number of molecules within a single cell. In addition, it is possible to distinguish even more dyes by taking their absorption spectra into account.