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： Background: Phosphatidylinositides in the plasma membrane (PM) are pivotal for cellular functions. Results: Superresolution imaging reveals homogeneous distribution of PI(4,5)P-2, PI4P, and PI(3,4,5)P-3 in the major area of the PM. Conclusion: Phosphatidylinositides detected by PH domains are uniformly distributed in the major regions of the PM, with limited concentration gradients. Significance: This result may imply a new working model of phosphatidylinositides at nanometer scale. Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P-2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P-2-enriched membrane patches/domains of diverse sizes (383 +/- 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P-2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P-3) showed similar spatial distributions as PI(4,5)P-2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale.
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