Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.
Cytometry A. 2021 Oct;99(10):1022-1032. doi: 10.1002/cyto.a.24291. Epub 2020 Dec 20.
Quantitative phase imaging (QPI) provides an approach for monitoring the dry mass of individual cells by measuring the optical pathlength of visible light as it passes through cells. A distinct advantage of QPI is that the measurements result in optical path length quantities that are, in principle, instrument independent. Reference materials that induce a well-defined optical pathlength shift and are compatible with QPI imaging systems will be valuable in assuring the accuracy of such measurements on different instruments. In this study, we evaluate seven combinations of microspheres embedded in index refraction matching media as candidate reference materials for benchmarking the performance of a QPI system and as calibration standards for the optical pathlength measurement. Poly(methyl metharylate) microspheres and mineral oil were used to evaluate the range of illumination apertures, signal-to-noise ratios, and focus positions that allow an accurate quantitative optical pathlength measurement. The microsphere-based reference material can be used to verify settings on an instrument that are suitable for obtaining an accurate pathlength measurement from biological cells. The microsphere/media reference material is applied to QPI-based dry mass measurements of a population of HEK293 cells to benchmark and provide evidence that the QPI image data are accurate.
定量相位成像 (QPI) 通过测量可见光穿过细胞时的光程长度，提供了一种监测单个细胞干质量的方法。QPI 的一个明显优势是，测量结果产生的光程长度量在原则上与仪器无关。与 QPI 成像系统兼容并能引起明确光程长度变化的参考材料，将有助于确保在不同仪器上进行此类测量的准确性。在这项研究中，我们评估了嵌入折射率匹配介质中的七种微球组合，作为基准 QPI 系统性能的候选参考材料，以及光程长度测量的校准标准。聚甲基丙烯酸甲酯微球和矿物油用于评估允许进行准确定量光程长度测量的照明孔径、信噪比和聚焦位置的范围。基于微球的参考材料可用于验证仪器的设置，这些设置适用于从生物细胞获得准确的路径长度测量。该微球/介质参考材料应用于基于 QPI 的 HEK293 细胞群体的干质量测量，以进行基准测试并提供证据表明 QPI 图像数据是准确的。
