Orth Antony, Ghosh Richik N, Wilson Emma R, Doughney Timothy, Brown Hannah, Reineck Philipp, Thompson Jeremy G, Gibson Brant C
ARC Centre of Excellence for Nanoscale BioPhotonics, School of Science, RMIT University, Melbourne, VIC 3001, Australia.
Thermo Fisher Scientific, 100 Technology Drive, Pittsburgh, PA 15219, USA.
Biomed Opt Express. 2018 Jun 6;9(7):2943-2954. doi: 10.1364/BOE.9.002943. eCollection 2018 Jul 1.
Fluorescence microscopy is widely used to observe and quantify the inner workings of the cell. Traditionally, multiple types of cellular structures or biomolecules are visualized simultaneously in a sample by using spectrally distinct fluorescent labels. The wide emission spectra of most fluorophores limits spectral multiplexing to four or five labels in a standard fluorescence microscope. Further multiplexing requires another dimension of contrast. Here, we show that photostability differences can be used to distinguish between fluorescent labels. By combining photobleaching characteristics with a novel unmixing algorithm, we resolve up to three fluorescent labels in a single spectral channel and unmix fluorescent labels with nearly identical emission spectra. We apply our technique to organic dyes, autofluorescent biomolecules and fluorescent proteins. Our approach has the potential to triple the multiplexing capabilities of any digital widefield or confocal fluorescence microscope with no additional hardware, making it readily accessible to a wide range of researchers.
荧光显微镜被广泛用于观察和量化细胞的内部运作。传统上，通过使用光谱不同的荧光标记，样品中的多种类型的细胞结构或生物分子可以同时可视化。大多数荧光团的宽发射光谱将光谱复用限制在标准荧光显微镜中的四到五个标记。进一步的复用需要另一个对比度维度。在这里，我们表明光稳定性差异可用于区分荧光标记。通过将光漂白特性与一种新颖的解混算法相结合，我们在单个光谱通道中解析多达三种荧光标记，并解混发射光谱几乎相同的荧光标记。我们将我们的技术应用于有机染料、自发荧光生物分子和荧光蛋白。我们的方法有可能在不增加额外硬件的情况下将任何数字宽场或共聚焦荧光显微镜的复用能力提高两倍，使广大研究人员都能轻松使用。
