PICASSO allows ultra-multiplexed fluorescence imaging of spatially overlapping proteins without reference spectra measurements

Junyoung Seo, Yeonbo Sim, Jeewon Kim, Hyunwoo Kim, In Cho, Hoyeon Nam, Young-Gyu Yoon () and Jae-Byum Chang ()
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Junyoung Seo: Korea Advanced Institute of Science and Technology
Yeonbo Sim: Sungkyunkwan University
Jeewon Kim: Korea Advanced Institute of Science and Technology
Hyunwoo Kim: Korea Advanced Institute of Science and Technology
In Cho: Korea Advanced Institute of Science and Technology
Hoyeon Nam: Korea Advanced Institute of Science and Technology
Young-Gyu Yoon: Korea Advanced Institute of Science and Technology
Jae-Byum Chang: Korea Advanced Institute of Science and Technology

Nature Communications, 2022, vol. 13, issue 1, 1-17

Abstract: Abstract Ultra-multiplexed fluorescence imaging requires the use of spectrally overlapping fluorophores to label proteins and then to unmix the images of the fluorophores. However, doing this remains a challenge, especially in highly heterogeneous specimens, such as the brain, owing to the high degree of variation in the emission spectra of fluorophores in such specimens. Here, we propose PICASSO, which enables more than 15-color imaging of spatially overlapping proteins in a single imaging round without using any reference emission spectra. PICASSO requires an equal number of images and fluorophores, which enables such advanced multiplexed imaging, even with bandpass filter-based microscopy. We show that PICASSO can be used to achieve strong multiplexing capability in diverse applications. By combining PICASSO with cyclic immunofluorescence staining, we achieve 45-color imaging of the mouse brain in three cycles. PICASSO provides a tool for multiplexed imaging with high accessibility and accuracy for a broad range of researchers.

Date: 2022
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DOI: 10.1038/s41467-022-30168-z

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