Fan Junchao, Huang Xiaoshuai, Li Liuju, Chen Liangyi, Tan Shan
Key Laboratory of Image Processing and Intelligent Control of Ministry of Education of China, School of Automation, Huazhong University of Science and Technology, Wuhan 430074, China.
J.F., X. H. and L.L. contributed equally to this work.
Biomed Opt Express. 2019 Feb 5;10(3):1097-1110. doi: 10.1364/BOE.10.001097. eCollection 2019 Mar 1.
Total internal reflection fluorescence microscopy (TIRF microscopy) uses a rapid decay of evanescent waves to excite fluorophores within several hundred nanometers () beneath the plasma membrane, which can effectively suppress excitation of fluorescence signals in the deep layers. From image stacks obtained with a plurality of different incident angles, a three-dimensional spatial structure of the observed sample can be reconstructed by a Multi-Angle-TIRF (MA-TIRF) algorithm that provides an axial resolution of ~50 . Taking into account the point spread function (PSF) of the TIRF microscopes, we further increase its lateral resolution by introducing a fast deconvolution algorithm into the reconstruction of MA-TIRF data (DMA-TIRF), which is approached in just one step of minimizing the reconstruction function. We also introduce a TV regularization term in the deconvolution algorithm to suppress artifacts induced by the excessive noise. Therefore, based on the hardware of existing MA-TIRF microscopes, the proposed DMA-TIRF algorithm has achieved lateral and axial resolutions of ~200 and ~50 , respectively.
全内反射荧光显微镜（TIRF显微镜）利用倏逝波的快速衰减来激发质膜下几百纳米（）内的荧光团，这可以有效抑制深层荧光信号的激发。从多个不同入射角获得的图像堆栈中，可以通过多角TIRF（MA-TIRF）算法重建观察到的样品的三维空间结构，该算法提供约50的轴向分辨率。考虑到TIRF显微镜的点扩散函数（PSF），我们通过在MA-TIRF数据重建中引入快速去卷积算法（DMA-TIRF）进一步提高其横向分辨率，这只需在最小化重建函数的一步中即可实现。我们还在去卷积算法中引入了TV正则化项，以抑制由过多噪声引起的伪影。因此，基于现有MA-TIRF显微镜的硬件，所提出的DMA-TIRF算法分别实现了约200和约50的横向和轴向分辨率。
