Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea.
Department of Biomedical Engineering, Sungkyunkwan University, Suwon, Korea.
Nat Commun. 2022 May 5;13(1):2475. doi: 10.1038/s41467-022-30168-z.
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.
超多重荧光成像需要使用光谱重叠的荧光染料来标记蛋白质，然后对荧光染料的图像进行解混。然而，由于在这些样本中荧光染料的发射光谱存在高度变化，因此这仍然是一个挑战，特别是在高度异质的样本中，如大脑。在这里，我们提出了 PICASSO，它可以在不使用任何参考发射光谱的情况下，在单个成像轮次中对空间重叠的蛋白质进行超过 15 种颜色的成像。PICASSO 需要相同数量的图像和荧光染料，这使得即使在基于带通滤光片的显微镜下也能实现这种高级多重成像。我们表明，PICASSO 可以用于在各种应用中实现强大的多重化能力。通过将 PICASSO 与循环免疫荧光染色相结合，我们在三个周期内实现了对小鼠大脑的 45 种颜色成像。PICASSO 为广泛的研究人员提供了一种具有高可及性和准确性的多重成像工具。
