分类： 生物学 >> 生物物理学 >> 细胞生物学 提交时间： 2016-05-12
摘要： Super-resolution microscopy techniques have overcome the limit of optical diffraction. Recently, the Bayesian analysis of Bleaching and Blinking data (3B) method has emerged as an important tool to obtain super-resolution fluorescence images. 3B uses the change in information caused by adding or removing fluorophores in the cell to fit the data. When adding a new fluorophore, 3B selects a random initial position, optimizes this position and then determines its reliability. However, the fluorophores are not evenly distributed in the entire image region, and the fluorescence intensity at a given position positively correlates with the probability of observing a fluorophore at this position. In this paper, we present a Bayesian analysis of Bleaching and Blinking microscopy method based on fluorescence intensity distribution (FID3B). We utilize the intensity distribution to select more reliable positions as the initial positions of fluorophores. This approach can improve the reconstruction results and significantly reduce the computational time. We validate the performance of our method using both simulated data and experimental data from cellular structures. The results confirm the effectiveness of our method.
分类： 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间： 2016-05-11
摘要： Although the topic of fiducial marker-based alignment in electron tomography (ET) has been widely discussed for decades, alignment without human intervention remains a difficult problem. Specifically, the emergence of subtomogram averaging has increased the demand for batch processing during tomographic reconstruction; fully automatic fiducial marker-based alignment is the main technique in this process. However, the lack of an accurate method for detecting and tracking fiducial markers precludes fully automatic alignment. In this paper, we present a novel, fully automatic alignment scheme for ET. Our scheme has two main contributions: First, we present a series of algorithms to ensure a high recognition rate and precise localization during the detection of fiducial markers. Our proposed solution reduces fiducial marker detection to a sampling and classification problem and further introduces an algorithm to solve the parameter dependence of marker diameter and marker number. Second, we propose a novel algorithm to solve the tracking of fiducial markers by reducing the tracking problem to an incomplete point set registration problem. Because a global optimization of a point set registration occurs, the result of our tracking is independent of the initial image position in the tilt series, allowing for the robust tracking of fiducial markers without pre-alignment. The experimental results indicate that our method can achieve an accurate tracking, almost identical to the current best one in IMOD with half automatic scheme. Furthermore, our scheme is fully automatic, depends on fewer parameters (only requires a gross value of the marker diameter) and does not require any manual interaction, providing the possibility of automatic batch processing of electron tomographic reconstruction. (C) 2015 Elsevier Inc. All rights reserved.
分类： 生物学 >> 生物物理学 >> 细胞生物学 提交时间： 2016-05-05
Shan, Hong
Yin, Chang-Cheng
Sun, Fei
Wang, Zihao
Zhang, Fa
Wang, Zihao
Zhang, Fa
Wang, Zihao
Sun, Fei
Xiong, Yong
Sun, Fei
摘要： Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macromolecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate alignments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orientations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimensional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large (60S/50S) and the small (40S/30S) subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolution single particle analysis of macromolecular complexes with dynamic conformations.
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