Robust blind spectral unmixing for fluorescence microscopy using unsupervised learning

Autoři: Tristan D. McRae aff001;  David Oleksyn aff003;  Jim Miller aff003;  Yu-Rong Gao aff001
Působiště autorů: Multiphoton Research Core Facility, Shared Resource Laboratories, University of Rochester Medical Center, Rochester, NY, United States of America aff001;  Department of Neuroscience, University of Rochester Medical Center, Rochester, NY, United States of America aff002;  Center for Vaccine Biology and Immunology and Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, United States of America aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225410


Due to the overlapping emission spectra of fluorophores, fluorescence microscopy images often have bleed-through problems, leading to a false positive detection. This problem is almost unavoidable when the samples are labeled with three or more fluorophores, and the situation is complicated even further when imaged under a multiphoton microscope. Several methods have been developed and commonly used by biologists for fluorescence microscopy spectral unmixing, such as linear unmixing, non-negative matrix factorization, deconvolution, and principal component analysis. However, they either require pre-knowledge of emission spectra or restrict the number of fluorophores to be the same as detection channels, which highly limits the real-world applications of those spectral unmixing methods. In this paper, we developed a robust and flexible spectral unmixing method: Learning Unsupervised Means of Spectra (LUMoS), which uses an unsupervised machine learning clustering method to learn individual fluorophores’ spectral signatures from mixed images, and blindly separate channels without restrictions on the number of fluorophores that can be imaged. This method highly expands the hardware capability of two-photon microscopy to simultaneously image more fluorophores than is possible with instrumentation alone. Experimental and simulated results demonstrated the robustness of LUMoS in multi-channel separations of two-photon microscopy images. We also extended the application of this method to background/autofluorescence removal and colocalization analysis. Lastly, we integrated this tool into ImageJ to offer an easy to use spectral unmixing tool for fluorescence imaging. LUMoS allows us to gain a higher spectral resolution and obtain a cleaner image without the need to upgrade the imaging hardware capabilities.

Klíčová slova:

Antigen-presenting cells – Fluorescence – Fluorescence imaging – Fluorescence microscopy – Imaging techniques – Lasers – Yellow fluorescent protein – Emission spectra


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