Accurate construction of photoactivated localization microscopy (PALM) images for quantitative measurements.
Accurate construction of photoactivated localization microscopy (PALM) images for quantitative measurements.
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DOI：
10.1371/journal.pone.0051725
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Xiao J
中科院分区：
文献类型：
--
作者：
Coltharp C;Kessler RP;Xiao J
基于局部化的超分辨率显微镜技术，如光激活局部化显微镜（PALM）和随机光学重建显微镜（STORM）已经允许以前所未有的光学分辨率对细胞结构进行研究。然而，解释超分辨率图像的一个主要障碍是由荧光团光闪烁引起的分子数量的过度计算。使用实验和模拟图像，我们确定的影响photoblinking的准确重建的超分辨率图像和定量测量的结构尺寸和分子密度从这些图像。我们发现，结构尺寸和相对密度的测量可以可靠地从图像，包含光致闪烁相关的计数，但准确的绝对密度测量，并因此忠实的分子计数和位置在细胞结构中的表示，需要应用聚类算法组定位，起源于相同的分子。我们分析了如何应用一个简单的算法与不同的聚类阈值（tThresh和dThresh）影响重建图像的准确性，并开发了一种简单的方法来选择最佳阈值。我们还确定了一个经验标准，以评估是否成像条件是适当的准确的超分辨率图像重建与聚类算法。阈值选择方法和成像条件判据在现有PALM聚类算法和实验条件下易于实现。我们的方法的主要优点是，它生成了一个超分辨率图像和分子位置列表，忠实地代表细胞结构内的分子计数和位置，而不仅仅是将结构特性总结为系综参数。这一特点使其特别适用于异质密度和不规则几何形状的细胞结构，并允许各种定量测量，以适应不同生物系统的特定需求。
Localization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (tThresh and dThresh) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems.
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影响因子：
48
作者：
Dempsey, Graham T.;Vaughan, Joshua C.;Chen, Kok Hao;Bates, Mark;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
影响因子：
48
作者：
Biteen, Julie S.;Thompson, Michael A.;Tselentis, Nicole K.;Bowman, Grant R.;Shapiro, Lucy;Moerner, W. E.
通讯作者：
Moerner, W. E.
影响因子：
56.9
作者：
Bates, Mark;Huang, Bo;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
DOI：
10.1073/pnas.0500489102
发表时间：
2005-07-05
影响因子：
11.1
作者：
Habuchi, S;Ando, R;Hofkens, J
通讯作者：
Hofkens, J
影响因子：
1.6
作者：
Geisbrecht, BV;Bouyain, S;Pop, M
通讯作者：
Pop, M