A Framework of Multi-View Machine Learning for Biological Spectral Unmixing of Fluorophores with Overlapping Excitation and Emission Spectra

Abstract

The accuracy in assigning fluorophore identity and abundance, termed spectral unmixing, in biological fluorescence microscopy images remains challenging due to the unavoidable and significant overlap in emission spectra among fluorophores. In conventional laser scanning confocal spectral microscopy, fluorophore information is acquired by recording emission spectra with a single combination of discrete excitation wavelengths. As a matter of fact, organic fluorophores have not only unique emission spectral signatures but also have unique and characteristic excitation spectra. In this paper, we propose a generalized multi-view machine learning approach, which makes use of both excitation and emission spectra to greatly improve the accuracy in differentiating multiple highly overlapping fluorophores in a single image. By recording emission spectra of the same field with multiple combinations of excitation wavelengths, we obtain data representing these different views of the underlying fluorophore distribution in the sample. We then propose a framework of multi-view machine learning methods, which allows us to flexibly incorporate noise information and abundance constraints, to extract the spectral signatures of fluorophores from their reference images and to efficiently recover their corresponding abundances in unknown mixed images. Numerical experiments on simulated image data demonstrate the method’s efficacy in improving accuracy, allowing for the discrimination of 100 fluorophores with highly overlapping spectra. Furthermore, validation on images of mixtures of fluorescently labeled E. coli demonstrates the power of the proposed multi-view strategy in discriminating fluorophores with spectral overlap in real biological images.