Xu Xinzhu, Wang Wenyi, Qiao Liang, Fu Yunzhe, Ge Xichuan, Zhao Kun, Zhanghao Karl, Guan Meiling, Chen Xin, Li Meiqi, Jin Dayong, Xi Peng
Department of Biomedical Engineering, College of Future Technology, Peking University, Beijing, 100871, China.
Wallace H. Coulter Dept. of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, 30332, GA, USA.
Light Sci Appl. 2024 May 29;13(1):125. doi: 10.1038/s41377-024-01464-8.
Structured illumination microscopy (SIM) has emerged as a promising super-resolution fluorescence imaging technique, offering diverse configurations and computational strategies to mitigate phototoxicity during real-time imaging of biological specimens. Traditional efforts to enhance system frame rates have concentrated on processing algorithms, like rolling reconstruction or reduced frame reconstruction, or on investments in costly sCMOS cameras with accelerated row readout rates. In this article, we introduce an approach to elevate SIM frame rates and region of interest (ROI) coverage at the hardware level, without necessitating an upsurge in camera expenses or intricate algorithms. Here, parallel acquisition-readout SIM (PAR-SIM) achieves the highest imaging speed for fluorescence imaging at currently available detector sensitivity. By using the full frame-width of the detector through synchronizing the pattern generation and image exposure-readout process, we have achieved a fundamentally stupendous information spatial-temporal flux of 132.9 MPixels · s, 9.6-fold that of the latest techniques, with the lowest SNR of -2.11 dB and 100 nm resolution. PAR-SIM demonstrates its proficiency in successfully reconstructing diverse cellular organelles in dual excitations, even under conditions of low signal due to ultra-short exposure times. Notably, mitochondrial dynamic tubulation and ongoing membrane fusion processes have been captured in live COS-7 cell, recorded with PAR-SIM at an impressive 408 Hz. We posit that this novel parallel exposure-readout mode not only augments SIM pattern modulation for superior frame rates but also holds the potential to benefit other complex imaging systems with a strategic controlling approach.
结构照明显微镜（SIM）已成为一种很有前景的超分辨率荧光成像技术，它提供了多种配置和计算策略，以减轻生物样本实时成像过程中的光毒性。传统上提高系统帧率的努力主要集中在处理算法上，如滚动重建或减少帧数重建，或者投资于具有加速行读出速率的昂贵的sCMOS相机。在本文中，我们介绍了一种在硬件层面提高SIM帧率和感兴趣区域（ROI）覆盖率的方法，而无需增加相机成本或采用复杂的算法。在这里，并行采集-读出SIM（PAR-SIM）在当前可用的探测器灵敏度下实现了荧光成像的最高成像速度。通过同步图案生成和图像曝光-读出过程，利用探测器的全帧宽度，我们实现了高达132.9兆像素·秒的巨大信息时空通量，是最新技术的9.6倍，最低信噪比为-2.11 dB，分辨率为100纳米。PAR-SIM证明了其在双激发下成功重建各种细胞器的能力，即使在由于超短曝光时间导致信号较低的情况下也是如此。值得注意的是，在活的COS-7细胞中捕捉到了线粒体动态微管形成和正在进行的膜融合过程，用PAR-SIM以令人印象深刻的408 Hz记录。我们认为，这种新颖的并行曝光-读出模式不仅增强了SIM图案调制以实现更高的帧率，而且有可能通过一种策略性控制方法使其他复杂成像系统受益。
