Performance Benchmarking and Intensity Calibration of a Widefield Fluorescence Microscope Using Fluorescent Glass
Michael W. Halter, Elianna Bier, Paul C. DeRose, Gregory A. Cooksey, Steven J. Choquette, Anne L. Plant, John T. Elliott
Widefield fluorescence microscopy is a highly used tool for visually assessing biological samples and for quantifying cell responses. Despite its widespread use in high content analysis and other screening applications, few published methods exist for evaluating and benchmarking the analytical performance of a microscope. Easy-to use benchmarking methods will facilitate the use of fluorescence imaging as a quantitative analytical tool in research applications, and will aid the determination of instrumental method validation for commercial product development applications. We describe and evaluate a method to characterize a fluorescence imaging systems performance by benchmarking the detection threshold, saturation and linear dynamic range to a physical artifact. The benchmarking procedure is demonstrated using two different materials as the physical artifact, uranyl-ion-doped glass and Schott 475 GG filter glass. Both are suitable candidate reference materials that are homogeneously fluorescent and highly photostable, and the Schott 475 GG filter glass is currently commercially available. In addition to benchmarking analytical performance, we also demonstrate that the reference materials provide for accurate day to day intensity calibration. The method described and the glass reference materials used will help to assure that fluorescence microscopy results are meaningful, traceable, and comparable from day to day and between laboratories.