Comparison of Monochromator and Filter Based Fluorometers in the Measurement of Narrow Stoke’s Shift Labels; Fluorescent Proteins as Practical Examples
Marika Raitio, Reija-Riitta Harinen, Hanna Grano-Fabritius and Jorma Lampinen, Thermo Fisher Scientific, Sample Preparation & Analysis, Vantaa, Finland
Fluorescent proteins are such a group of fluorometric labels that their Stoke’s shifts are normally quite small. Good examples of the labels with very narrow differences in excitation and emission peaks are Renilla reniformis GFP and EYFP. Renilla GFP has excitation and emission maxima at 498 nm and 507 nm and YFP has 514 and 527 nm. These about 10-15 nanometer Stoke’s shifts are the smallest one can normally find but there are several other labels with 15-20 nm Stoke’s shifts.
This paper describes the general instrumentational difficulties that are present when these small Stoke’s shift labels are measured. Excitation and emission wavelengths can not be selected using label’s maximum efficiencies because placing excitation and emission so close to each others would cause excitation light leakage through the reader’s optical system directly to the detector, resulting in very high background. When the wavelength used are moved outside from the peak wavelengths both excitation and emission efficiencies might decrease drastically, resulting in low signal to background values.
This paper shows the general principles how excitation and emission wavelengths and filters should be optimally selected. The effect of this optimization with narrow Stoke’s shift labels is shown on the assay performance parameters (detection limit, dynamic range, Z-prime values etc.). Optimization procedure is described for the most common fluorescent proteins including Renilla GFP, eGFP, YFP. CFP and mCherry
During the optimization calibration series of the pure labels were measured with different excitation emission wavelength pairs using Thermo Scientific Varioskan Flash microplate spectrofluorometer. Wavelength combinations were selected based on shape of the fluorometric spectra of each label so that as high excitation and emission efficiency as possible was used without any straylight leakage through the optics. Then the detection limits of the labels were determined according to IUPAC principle and other central assay parameters were analyzed. As a comparison, all labels were also measured with Thermo Scientific Fluoroskan Ascent microplate fluorometer using optimal filter sets for each label.
The results will show that the monochromator based spectrofluorometer is clearly more powerful in measuring these small Stoke’s shift labels than traditional filter fluorometer, even with the optimal filters. Monochromator technology makes it possible to use excitation and emission wavelengths closer to each others and therefore both higher excitation and emission efficiency can be used in the assay.
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