State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China.
Key Laboratory of Image Processing and Intelligent Control of Ministry of Education of China, School of Automation, Huazhong University of Science and Technology, Wuhan, China.
Nat Biotechnol. 2018 Jun;36(5):451-459. doi: 10.1038/nbt.4115. Epub 2018 Apr 11.
To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis.
为了提高超分辨率（SR）显微镜的时间分辨率和最大成像时间，我们开发了一种基于Hessian 矩阵的结构光照明显微镜去卷积算法（Hessian-SIM）。它利用生物结构在多个维度上的连续性作为先验知识来指导图像重建，在使用传统 SIM 光剂量的 10%以下获得最小伪影的 SR 图像，同时在低信号强度下大大优于当前算法。Hessian-SIM 能够在没有运动伪影的情况下快速成像内质网中的移动囊泡或环，具有 88nm 和 188Hz 的时空分辨率。其高灵敏度允许使用亚毫秒级的激发脉冲和暗恢复时间来减少荧光蛋白的光漂白，从而能够在活细胞中进行长达一小时的肌动蛋白丝的长时间延时 SR 成像。最后，我们观察到线粒体嵴的结构动力学以及以前未观察到的结构，例如囊泡胞吐过程中融合孔的扩大。
