自闪烁染料解锁高阶多平面超分辨光学起伏成像
Self-blinking Dyes unlock High-order and Multi-plane Super-resolution Optical Fluctuation Imaging
摘要：
衍射无限超分辨率成像关键取决于荧光团在至少两种状态之间的切换，通常使用强激光和特殊缓冲液诱导。适当的闪烁动力学所需的高照明功率或UV光目前阻碍了活细胞实验。最近，引入了所谓的自闪烁染料，其在开放的荧光“开”状态和封闭的无色“关”状态之间自发地切换。在这里，我们利用超分辨率光学波动成像（SOFI）和自发切换荧光团的2D功能和体积成像之间的协同作用。SOFI通过分析几百到几千帧随机闪烁荧光团的高阶统计量，容忍高标记密度、准时率和低信噪比。我们展示了固定细胞的2D成像，具有高达50-60 nm的统一分辨率，在6阶SOFI和表征不断变化的实验条件。我们扩展多平面互相关分析到第四阶使用双平面和8平面体积成像实现多达29个（虚拟）平面。自闪烁SOFI所需的低激光激发强度是活细胞成像的理想选择。我们通过观察细胞中缓慢的膜运动来证明主要的时间分辨成像。自闪烁SOFI为易于使用的2D和3D高分辨率功能成像提供了一条途径，该成像对伪影具有鲁棒性，适合活细胞成像。
英文摘要：
Diffraction unlimited super-resolution imaging critically depends on the switching of fluorophores between at least two states, often induced using intense laser light and special buffers. The high illumination power or UV light required for appropriate blinking kinetics is currently hindering live-cell experiments. Recently, so-called self-blinking dyes that switch spontaneously between an open, fluorescent "on"-state and a closed colorless "off"-state were introduced. Here we exploit the synergy between super-resolution optical fluctuation imaging (SOFI) and spontaneously switching fluorophores for 2D functional and for volumetric imaging. SOFI tolerates high labeling densities, on-time ratios, and low signal-to-noise by analyzing higher-order statistics of a few hundred to thousand frames of stochastically blinking fluorophores. We demonstrate 2D imaging of fixed cells with a uniform resolution up to 50-60 nm in 6th order SOFI and characterize changing experimental conditions. We extend multiplane cross-correlation analysis to 4th order using biplane and 8-plane volumetric imaging achieving up to 29 (virtual) planes. The low laser excitation intensities needed for self-blinking SOFI are ideal for live-cell imaging. We show proof-of-principal time-resolved imaging by observing slow membrane movements in cells. Self-blinking SOFI provides a route for easy-to-use 2D and 3D high-resolution functional imaging that is robust against artefacts and suitable for live-cell imaging.
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