A Quantitative Measure of Field Illumination.
A Quantitative Measure of Field Illumination.
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DOI：
10.7171/jbt.15-2602-001
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发表时间：
2015-07-01
期刊：
影响因子：
--
通讯作者：
Cole, Richard W
中科院分区：
文献类型：
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作者：
Brown, Claire M;Reilly, Andrew;Cole, Richard W
在本文中，我们描述了一种基于统计的算法，以量化的光学光学显微镜成像系统，输出一个单一的质量因子（QF）得分的照明均匀性。在定量光学显微镜中均匀场照明的重要性是众所周知的，经常检查。然而，目前还没有标准的自动定量测量场照明的均匀性。作为显微镜质量评估国际研究的一部分，收集了89种不同的激光扫描共聚焦显微镜（LSCM）的图像，这些图像被用作构建算法的“训练”集。为了验证该算法，并验证其鲁棒性，图像从33个额外的显微镜，包括LSCM和宽场（WF）显微镜，被使用。用于开发质量评分量表的统计范例是监督学习的回归方法。三个强度分布在每个图像-2角到角对角线和中心水平-被用来生成像素强度数据。所有的线条都穿过了图像的中心。然后将强度分布数据转换成0-100范围内的单场照明QF分数，其中0具有极端变化，因此基本上不可用，并且100没有偏差，即，具有恒定均匀强度的直线。根据经验，QF ≥ 83被确定为基于制造商验收测试和合理可实现值的最小可接受值。这个新的QF是一个非常宝贵的指标，可以客观、轻松地确定照明质量的均匀性，为监测随时间变化的场均匀性提供可追溯的参考，并在不同的显微镜之间进行直接比较。QF还可用作系统故障的指示器，以及是否需要对仪器进行校准或维修。
In this paper, we describe a statistically based algorithm to quantify the uniformity of illumination in an optical light microscopy imaging system that outputs a single quality factor (QF) score. The importance of homogeneous field illumination in quantitative light microscopy is well understood and often checked. However, there is currently no standard automatic quantitative measure of the uniformity of the field illumination. Images from 89 different laser-scanning confocal microscopes (LSCMs), which were collected as part of an international study on microscope quality assessment, were used as a "training" set to build the algorithm. To validate the algorithm and verify its robustness, images from 33 additional microscopes, including LSCM and wide-field (WF) microscopes, were used. The statistical paradigm used for developing the quality scoring scale was a regression approach to supervised learning. Three intensity profiles across each image-2 corner-to-corner diagonals and a center horizontal-were used to generate pixel-intensity data. All of the lines passed through the center of the image. The intensity profile data then were converted into a single-field illumination QF score in the range of 0-100, with 0 having extreme variation, and therefore, essentially unusable, and 100 having no deviation, i.e., straight lines with a constant uniform intensity. Empirically, a QF ≥ 83 was determined to be the minimum acceptable value based on manufacturer acceptance tests and reasonably achievable values. This new QF is an invaluable metric to ascertain objectively and easily the uniformity of illumination quality, provide a traceable reference for monitoring field uniformity over time, and make a direct comparison among different microscopes. The QF can also be used as an indicator of system failure and the need for alignment or service of the instrument.