Biomedical Engineering Department, Oregon Health & Science University, Portland, Oregon, United States of America.
Thermo Fisher Scientific, Pittsburg, Pennsylvania, United States of America.
PLoS One. 2018 Oct 26;13(10):e0206104. doi: 10.1371/journal.pone.0206104. eCollection 2018.
Multicolor single-molecule localization microscopy (SMLM) expands our understanding of subcellular details and enables the study of biomolecular interactions through precise visualization of multiple molecules in a single sample with resolution of ~10-20 nm. Probe selection is vital to multicolor SMLM, as the fluorophores must not only exhibit minimal spectral crosstalk, but also be compatible with the same photochemical conditions that promote fluorophore photoswitching. While there are numerous commercially available photoswitchable fluorophores that are optimally excited in the standard Cy3 channel, they are restricted to short Stokes shifts (<30 nm), limiting the number of colors that can be resolved in a single sample. Furthermore, while imaging buffers have been thoroughly examined for commonly used fluorophore scaffolds including cyanine, rhodamine, and oxazine, optimal conditions have not been found for the BODIPY scaffold, precluding its routine use for multicolor SMLM. Herein, we screened common imaging buffer conditions including seven redox reagents with five additives, resulting in 35 overall imaging buffer conditions to identify compatible combinations for BODIPY-based fluorophores. We then demonstrated that novel, photoswitchable BODIPY-based fluorophores with varied length Stokes shifts provide additional color options for SMLM using a combination of BODIPY-based and commercially available photoswitchable fluorophores.
多色单分子定位显微镜 (SMLM) 扩展了我们对亚细胞细节的理解，并通过在单个样本中精确可视化多个分子，实现分辨率约为 10-20nm 的生物分子相互作用的研究。探针选择对于多色 SMLM 至关重要，因为荧光团不仅必须表现出最小的光谱串扰，而且还必须与促进荧光团光开关的相同光化学条件兼容。虽然有许多市售的光可切换荧光团在标准 Cy3 通道中最佳激发，但它们仅限于短斯托克斯位移（<30nm），限制了单个样品中可分辨的颜色数量。此外，虽然已经对包括氰基、罗丹明和噁嗪在内的常用荧光团支架的成像缓冲液进行了彻底检查，但尚未找到 BODIPY 支架的最佳条件，从而排除了其在多色 SMLM 中的常规使用。在此，我们筛选了常见的成像缓冲液条件，包括七种氧化还原试剂和五种添加剂，共产生 35 种成像缓冲液条件，以确定适用于 BODIPY 基荧光团的兼容组合。然后，我们证明了具有不同长度斯托克斯位移的新型光可切换 BODIPY 基荧光团为 SMLM 提供了额外的颜色选择，使用了 BODIPY 基和市售光可切换荧光团的组合。
