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Practical application of microsphere samples for benchmarking a quantitative phase imaging system



Edward J. Kwee, Alexander W. Peterson, Michael Halter, John Elliott


Quantitative phase imaging (QPI) provides an approach for monitoring the growth rate of individual cells by measuring the optical pathlength of visible light as it is passes through cells. A distinct advantage of QPI is that the measurements are, in principle, instrument-independent and can be compared between different experiments even when performed in different laboratories. 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. One of the microsphere/media combinations was selected and used to evaluate the range of illumination apertures, signal to noise ratios, and focus positions which allows an accurate quantitative optical pathlength measurement. The microsphere-based reference material can be used to verify settings on an instrument 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 OPI image data is accurate and comparable across laboratories.
Cytometry Part A


Quantitative phase imaging, microscopy, live cell imaging, reference materials


Kwee, E. , Peterson, A. , Halter, M. and Elliott, J. (2020), Practical application of microsphere samples for benchmarking a quantitative phase imaging system, Cytometry Part A, [online],, (Accessed May 21, 2024)


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Created December 19, 2020, Updated June 24, 2021