A comparative study of high resolution microscopy imaging modalities using a three-dimensional resolution measure.
A comparative study of high resolution microscopy imaging modalities using a three-dimensional resolution measure.
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DOI：
10.1364/oe.17.024377
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发表时间：
2009-12-21
期刊：
影响因子：
3.8
通讯作者：
Ober RJ
中科院分区：
文献类型：
--
作者：
Chao J;Ram S;Ward ES;Ober RJ
从获取的图像，单分子显微镜使得在三维（3D）空间中分离两个紧密间隔的生物分子的距离的确定成为可能。这样的距离信息可以是生物分子相互作用的性质的重要指标。然而，当例如成像点源彼此非常接近或位于成像装置的焦平面附近时，距离确定尤其困难。在这种挑战的背景下，我们比较了几种高分辨率3D成像模式的距离估计精度的限制。比较使用Cramer-Rao下限为基础的3D分辨率的措施，预测最好的可能的准确性，可以估计一个给定的距离。将单个点源的检测分开的模式（例如，使用可光激活的荧光团）被示出为当两个点源彼此非常接近和/或取向为接近平行于光轴时提供最佳的精度极限。与此同时，当给定近焦点源对时，实现多个焦平面点源同时成像的模式表现最好。我们还表明，最大似然估计是能够达到的精度预测为每一种方式的限制。
From an acquired image, single molecule microscopy makes possible the determination of the distance separating two closely spaced biomolecules in three-dimensional (3D) space. Such distance information can be an important indicator of the nature of the biomolecular interaction. Distance determination, however, is especially difficult when, for example, the imaged point sources are very close to each other or are located near the focal plane of the imaging setup. In the context of such challenges, we compare the limits of the distance estimation accuracy for several high resolution 3D imaging modalities. The comparisons are made using a Cramer-Rao lower bound-based 3D resolution measure which predicts the best possible accuracy with which a given distance can be estimated. Modalities which separate the detection of individual point sources (e.g., using photoactivatable fluorophores) are shown to provide the best accuracy limits when the two point sources are very close to each other and/or are oriented near parallel to the optical axis. Meanwhile, modalities which implement the simultaneous imaging of the point sources from multiple focal planes perform best when given a near-focus point source pair. We also demonstrate that the maximum likelihood estimator is capable of attaining the limit of the accuracy predicted for each modality.