Fluorophore unmixing based on bleaching and recovery kinetics using MCR-ALS

Fluorescence microscopy is a key technology in the life sciences, though its performance is constrained by the number of labels that can be recorded. We propose to use the kinetics of fluorophore photodestruction and subsequent fluorescence recovery to distinguish multiple spectrally-overlapping emitters in fixed cells, thus enhancing the information that can be obtained from a single measurement. We show that the data can be directly processed using multivariate curve resolution - alternating least squares (MCR-ALS) to deliver distinct images for each fluorophore in their local environment, and apply this methodology to membrane imaging using DiBAC4(3) and concanavalin A - Alexa Fluor 488 as the fluorophores. We find that the DiBAC4(3) displays two distinct degradation/recovery kinetics that correspond to two different label distributions, allowing us to simultaneously distinguish three different fluorescence distributions from two spectrally overlapping fluorophores. We expect that our approach will scale to other dynamically-binding dyes, leading to similarly increased multiplexing capability. [Display omitted] •Three distinct membranes were visualized using only the green colour channel.•(Bio-)chemical knowledge was included in MCR-ALS as constraints during optimization.•Photodestruction and recovery were used as sources of information for multiplexing.•The approach could potentially be applied to a wide array of dynamically binding dyes.