Conducting Ratio Analysis with a Plate Reader

The SC-41 Plate reader facilitates high-throughput measurements on the FS5 Spectrofluorometer using automated measurements of microwell plates. It is possible to measure standard and custom plates with up to 384 wells. One of the unique features of the SC-41 I is its capacity to achieve spectral and lifetime measurements. This article highlights the unique capabilities of the SC-41 Plate Reader when conducting ratio analysis in which the emission spectra of samples containing different ratios of fluorophores can be measured.

Figure 1. SC-41 Plate Reader Sample Module for the FSS Spectrofluorometer. Image Credit: Edinburgh Instruments

Experimental Setup

Two solutions, Fluorophore 1 (F1) and Fluorophore 2 (F2), were prepared with a 50:50 ethanol/water solution at various F1 and F2 ratios, as shown in Table 1. As demonstrated, samples 1 to 6, used for producing a calibration curve, have a known F1:F2 ratio, whereas U1 and U2 have an unknown F1:F2 that is yet to be experimentally established. A total of 300 uL of each sample was transferred into a Greiner 96 well plate using a pipette.

Table 1. Sample Details. Source: Edinburgh Instruments

SC-41 Emission Measurements

The SC-41 Sample Module was automatically recognized by Fluoracle when inserted into the FS5 Spectrofluorometer’s sample compartment. Using the Remove Plate/Load Plate button, the filled microwell plate was loaded into the SC-41.

As shown in Figure 2, the wells display as singular circles, and a right-click of the well moves the plate reader to this position which displays the well’s live emission count rate. The user can verify measurement parameters, including bandwidth, before recording the final measurements. Left-clicking on a well highlights it in blue and prepares it for measurement. It is also possible to save and load well measurement patterns for future use.

Figure 2. Fluoracle software showing auto-recognition of the sample module (outlined in red), and the well plate schematic with plate loading (outlined in purple). Image Credit: Edinburgh Instruments

Wells A1-A8 were selected for measurement, and bandwidths were established by moving the plate reader to the sample with the highest emission. This helped to prevent detector saturation. Emission spectral scans from 480 nm to 750 nm were marked with a 0.5-second integration time and 1 nm step.

Figure 3. SC-41 plate set up in Fluoracle. Image Credit: Edinburgh Instruments

Throughout the measurement process, the live emission scans were displayed alongside a schematic of the microwell plate, which highlighted the well being measured, as demonstrated by the white circle with a red outline (Figure 4). Once the individual well measurement was finalized, the circle changed into a square, and the schematic closed when all measurements were completed.

Figure 4. Live measurement screen for the SC-41 Plate Reader. Image Credit: Edinburgh Instruments

Ratio Trend Analysis

Using Fluoracle^® Trend Analysis, the spectra were analyzed using ratio analysis. The ratio analysis was selected, and the emission peak wavelengths of F1 and F2 were inputted. The intensity ratio was then calculated.

As shown in Figure 5, samples 1 to 6 with a known fluorophore concentration ratio were chosen, with the ratio being inputted as a percentage. Using this data, Fluoracle^® generated a quadratic calibration curve, which determined the fluorophore ratio of the two unknown samples.

Figure 5. Ratio Trend Analysis using Fluoracle. Image Credit: Edinburgh Instruments

Applying the equation produced by Fluoracle^®, it was determined that the percentage ratio of Unknown 1 (U1) was around 74.8% F1 and 25.2 F2. Unknown 2 (U2) comprised a mix of 50.5% F1 and 49.5% F2 (Table 2).

Table 2. Unknown Fluorophore Ratios. Source: Edinburgh Instruments

Conclusion

The SC-41 Plate Reader facilitates automated measurements using the FS5 Spectrofluorometer and microwell plates.

The Fluoracle^® Trend Analysis feature was used to generate and plot a calibration curve that reveals the ratio of F1:F2 in the unknown samples.

This information has been sourced, reviewed and adapted from materials provided by Edinburgh Instruments.

For more information on this source, please visit Edinburgh Instruments.