Per-pixel unmixing of spectrally overlapping fluorophores using intra-exposure excitation modulation

Hana Valenta; Franziska Bierbuesse; Raffaele Vitale; Cyril Ruckebusch; Wim Vandenberg; Peter Dedecker

doi:10.1016/j.talanta.2023.125397

Per-pixel unmixing of spectrally overlapping fluorophores using intra-exposure excitation modulation

Talanta. 2024 Mar 1:269:125397. doi: 10.1016/j.talanta.2023.125397. Epub 2023 Nov 10.

Authors

Hana Valenta¹, Franziska Bierbuesse¹, Raffaele Vitale², Cyril Ruckebusch², Wim Vandenberg¹, Peter Dedecker³

Affiliations

¹ Department of Chemistry, KU Leuven, Belgium.
² U. Lille, CNRS, LASIRE, France.
³ Department of Chemistry, KU Leuven, Belgium. Electronic address: peter.dedecker@kuleuven.be.

PMID: 38048682
DOI: 10.1016/j.talanta.2023.125397

Abstract

Multilabel fluorescence imaging is essential for the visualization of complex systems, though a major challenge is the limited width of the useable spectral window. Here, we present a new method, exNEEMO, that enables per-pixel quantification of spectrally-overlapping fluorophores based on their light-induced dynamics, in a way that is compatible with a very broad range of timescales over which these dynamics may occur. Our approach makes use of intra-exposure modulation of the excitation light to distinguish the different emitters given their reference responses to this modulation. We use the approach to simultaneously image four green photochromic fluorescent proteins at the full spatial resolution of the imaging.

Keywords: Multiplexed imaging; Per-pixel unmixing; Reversibly photoswitchable fluorescent proteins.