Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet.
Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet.
复制标题
利用微流体和单目标倾斜光片进行全细胞多目标单分子超分辨率 3D 成像。
DOI：
10.1101/2023.09.27.559876
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Gustavsson,Anna-Karin
中科院分区：
文献类型：
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作者：
Saliba,Nahima;Gagliano,Gabriella;Gustavsson,Anna-Karin
多目标单分子超分辨率荧光显微镜提供了一个强大的手段，了解在纳米级的分布和多个亚细胞结构之间的相互作用。然而，整个哺乳动物细胞的单分子超分辨率成像通常受到高荧光背景和缓慢采集速度的阻碍，特别是在3D中成像多个目标时。在这项工作中，我们通过开发一种可操纵的，抖动的，单目标倾斜的光片用于光学切片以减少荧光背景，以及一种用于3D纳米打印微流体系统的管道，用于将光片反射到样品中，来缓解这些问题。这种易于适应的微流体制造流水线允许将反射光学器件并入微流体通道中，而不会破坏高效和自动化的溶液交换。我们将这些创新与点扩散函数工程相结合，用于3D中单个分子的纳米级定位，用于重叠发射器分析的深度学习，用于漂移校正和长期成像的主动3D稳定，以及用于无色偏移的顺序多目标成像的Exchange-PAINT。然后，我们证明了这个平台，称为soTILT 3D，使全细胞多靶点3D单分子超分辨率成像具有更高的精度和成像速度。
Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D. In this work, we have mitigated these issues by developing a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background and a pipeline for 3D nanoprinting microfluidic systems for reflection of the light sheet into the sample. This easily adaptable microfluidic fabrication pipeline allows for the incorporation of reflective optics into microfluidic channels without disrupting efficient and automated solution exchange. We combine these innovations with point spread function engineering for nanoscale localization of individual molecules in 3D, deep learning for analysis of overlapping emitters, active 3D stabilization for drift correction and long-term imaging, and Exchange-PAINT for sequential multi-target imaging without chromatic offsets. We then demonstrate that this platform, termed soTILT3D, enables whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed.