Issue 23, 2024

Linear spectral unmixing analysis in single-molecule FRET spectroscopy for fluorophores with large spectral overlap

Abstract

Fluorescence resonance energy transfer (FRET) is a highly useful tool to investigate biomolecular interactions and dynamics in single-molecule spectroscopy and nanoscopy. However, the use of spectrally overlapping dye pairs results in various artifact signals that prevent accurate determination of FRET values. In this paper, an algorithmic method of spectral unmixing was devised to extract FRET values of spectrally overlapping dye pairs at the single molecule level. Application of this method allows the determination of both the donor–acceptor composition and the FRET efficiency of the samples labelled with spectrally overlapping dye pairs.

Graphical abstract: Linear spectral unmixing analysis in single-molecule FRET spectroscopy for fluorophores with large spectral overlap

Supplementary files

Article information

Article type
Paper
Submitted
20 Feb 2024
Accepted
18 May 2024
First published
20 May 2024

Phys. Chem. Chem. Phys., 2024,26, 16561-16566

Linear spectral unmixing analysis in single-molecule FRET spectroscopy for fluorophores with large spectral overlap

S. Bae, K. Sung and S. K. Kim, Phys. Chem. Chem. Phys., 2024, 26, 16561 DOI: 10.1039/D4CP00736K

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