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Cell Res:中科院生物物理所徐平勇组等发表活细胞超分辨率显微技术研究进展

摘要 : 2016年12月31日,国际学术权威刊物自然出版集团旗下子刊《Cell Research》杂志在线发表了中国科学院生物物理研究所徐平勇课题组、中国科学院计算技术研究所张法课题组以及美国科学院院士HHMI研究员Jennifer Lippincott-Schwartz合作题为Live-cell single molecule-guided Bayesian localization super-resolution microscopy 的文章

2016年12月31日,国际学术权威刊物自然出版集团旗下子刊《Cell Research》杂志在线发表了中国科学院生物物理研究所徐平勇课题组、中国科学院计算技术研究所张法课题组以及美国科学院院士HHMI研究员Jennifer Lippincott-Schwartz合作题为Live-cell single molecule-guided Bayesian localization super-resolution microscopy 的文章,论文介绍了一种新型活细胞超分辨率显微技术及其独特优势。张法课题组博士研究生徐帆和徐平勇课题组博士研究生张名姝为论文共同第一作者,徐平勇研究员、张法研究员和Jennifer Lippincott-Schwartz教授为论文通讯作者。

超分辨率荧光显微技术由于打破了传统光学衍射的限制,使得人们能够更深入地理解细胞生物学,获得了2014年诺贝尔化学奖。但是由于设备和时空分辨率的影响,活细胞超分辨率技术仍面临诸多挑战。近年来,贝叶斯定位显微技术(Bayesian analysis of the blinking and bleaching,3B)利用荧光蛋白漂白和闪烁的特性,通过分析整个图像序列的变化得到荧光蛋白的概率分布图,该方法用简单的光学设备就能实现活细胞动态结构的超分辨率成像,成为活细胞超分辨率成像的重要工具之一。作为细胞成像新的重要工具,它仍然有三个关键的问题没有解决:1)在精度方面,存在严重的结构缺失,定位精度不高;2)在速度方面,该方法极其耗时,为了得到1.5μm的超分辨率结构,大约需要6小时,并且随着图像尺寸的增加,计算时间急剧增长;3)在分析尺度方面,由于速度的限制,该方法很难获得全细胞大尺度长时间的动态变化。针对以上问题,实验人员通过将单分子定位和贝叶斯技术相结合,开发了一种新型活细胞超分辨率显微技术(single molecule guided Bayesian localization microscopy,SIMBA),该技术有以下优点:1)适用范围广,不需要任何额外的硬件设备,就能与主流TIRFM、PALM、STROM和light-sheet显微镜相结合,便于推广和使用;2)时空分辨率高,减少了结构伪迹的同时实现了50nm的空间分辨率和0.5-2s的时间分辨率;3)运行速度快,相比3B,加速比超过100倍,并且随着图像尺度的增大,加速效果更加明显;4)分析尺度大,实现了全细胞大尺度长时间动态变化分析。

SIMBA对于固定细胞actin和活细胞CLC重构结果展示

原文链接:

Live cell single molecule-guided Bayesian localization super resolution microscopy

原文摘要:

Many current super resolution (SR) microscopic techniques 1,2,3,4,5,6 have been successfully applied to image cellular dynamics in living cells, but their applications have remained technically challenging. Live cell stimulated emission depletion (STED)/reversible saturable optical linear fluorescence transitions (RESOLFT) microscopy and structured illumination microscopy (SIM)/nonlinear SIM require sophisticated expensive optical setups and specialized expertise for accurate optical alignment. Live cell photo-activated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM) use less complicated setup; however, a scientific complementary metal-oxide-semiconductor (sCMOS) camera, whose pixel-dependent noise must be characterized and calibrated before use 7, is required for extremely high acquisition speed over tens of thousands of frames. Recently, wide field-based SR microscopies have been developed to improve temporal resolution using much fewer time-lapse images (hundreds to thousands) than PALM/STORM 8,9. One of them, Bayesian analysis of the blinking and bleaching (3B) 8, offers enormous potential to resolve ultrastructure and fast cellular dynamics in living cells beyond the diffraction limit. Despite its potential, 3B analysis is impractical when imaging the nanoscale dynamics in large fields of view over long time periods, as the calculation is extremely time-consuming 8 and/or the analysis consumes large amounts of web resources 10. Another major problem of 3B imaging is the artificial thinning and thickening of structures both in simulated image data and in experimental data 8,10.

来源: Cell Research 浏览次数:0

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