A study was designed to assess variability between different fluorescence spectroscopy devices. Measurements were made with all combinations of three devices, four probes, and thee sets of standards trays. Additionally, we made three measurements on the same day over two days for the same combination of device, probe, and standards tray to assess reproducibility over a day and across days.
Material and Methods
The devices consisted of light sources, fiber optics, and cameras. We measured thirteen standards and present the data from: the frosted cuvette, water, and rhodamine standards. A preliminary analysis was performed with the data that was wavelength calibrated and background subtracted however the system has not been corrected for systematic intensity variations caused by the devices. Two analyses were performed on the rhodamine, water, and frosted cuvette standards data. The first one is based on first clustering the measurements and then looking for association between the 5 factors (device, probe, standards tray, day, measurement number) using chi-squared tests on the cross tabulation of cluster and factor level. This showed that only device and probe were significant. We then did an analysis of variance to assess the percent variance explained by each factor that was significant from the chi-squared analysis.
The data were remarkably similar across the different combinations of factors. The analysis based on the clusters showed that sometimes devices alone, probes alone, but most often combinations of device and probe caused significant differences in measurements. The analysis showed that time of day, location of device, and standards trays do not vary significantly; whereas the devices and probes account for differences in measurement. We expected this type of significance using unprocessed data since the processing corrects for differences in devices. However, this analysis on raw data is useful to explore what combination of device and probe measurements should be targeted for further investigation. This experiment affirms that online quality control is necessary to obtain the best excitation-emission matrices from optical spectroscopy devices.
The fact that the device and probe are the primary sources of variability indicates that proper correction for the transfer function of the individual devices should make the measurements essentially equivalent.