Internal Calibration of Microplate Reader Detection System to Improve the Sensitivity, Dynamic Range and the Accuracy of the Spectral Scanning in Fluorometry and Luminometry
Reija-Riitta Harinen and Jorma Lampinen, Thermo Fisher Scientific, Sample Preparation & Analysis, Vantaa, Finland
When fluorescence signals are measured the results are normally referred as “Relative Fluorescence Units” (RFUs) to emphasis the fact that resulting numeric values are heavily dependent on the settings of the instrument electronics, mainly PMT gain voltage. Results are also somewhat dependent on the environmental
aspects, like ambient temperature that affects the electronic noise levels. The practical effect of this relative nature of fluorescence measurements is that result values become quite difficult to be compared, especially if the results are from different measurements done with different instrument settings.
Most of the available instruments partially overcome these difficulties by preventing the use of different electronic settings inside one assay, for example PMT gain voltage is fixed within an assay. This method causes new difficulties that are seen for example as a narrow dynamic measurement range caused by fixed gain voltage. Specially, when high intensity Xenon flash lamps are used for the excitation, the available dynamic range with one gain voltage is commonly reduced to about 3 – 4 orders of magnitude. Fluorometric assay chemistries offer chemical concentration ranges around 5 – 6 orders of magnitude, or even more, so dynamic range of fixed gain voltage is not sufficient.
If Xenon flash lamp is used for the excitation it is necessary to be able to use several PMT gain voltages within one assay to get a good assay performance: both high sensitivity and large dynamic range. When an instrument with fixed PMT gain is used to measure such an assay where the concentration range exceeds the available dynamic range, either high concentration samples are saturated or low concentration samples are not separated from the background, depending on the PMT gain value selected. In both cases certain samples will give incorrect result values.
In addition, the detection system affects also on spectral scanning measurements. The detector system, mainly PMT and monochromator grating, efficiency varies according to the wavelength and that will have an effect on the peak and shape of the spectra.
Thermo Scientific Varioskan Flash® multitechnology reader has in-built calibration system for all measurement technologies covering all mentioned problems in the measurement. This paper shows how detection systems for fluorescence intensity, time resolved fluorescence and luminescence are calibrated to ensure the best possible performance in each individual measurement.