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Updated: Jun 24, 2025

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and λ Hyperspectral FRET Imaging and Analysis
Published on: October 27, 2020
Linear spectral unmixing analysis in single-molecule FRET spectroscopy for fluorophores with large spectral overlap
Sohyeon Bae1, Keewon Sung1, Seong Keun Kim1
1Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea. seongkim@snu.ac.kr.
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.
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