Advances in Multimode Microplate Reading

The hybrid design of the Synergy Neo2 includes a quad monochromator and a filter-based optical system. Using the optical system best suited for a specific workflow provides excellent performance efficiency, and versatility.

Quad monochromator optical system

Use the quad monochromator-based optics (Figure 1) in Synergy Neo2 for a very broad range of fluorescence intensity, glow and flash luminescence and UV-Vis absorbance assays. The quad monochromators enable absorbance measurements from 230 nm to 999 nm in 1 nm increments, accommodating nucleic acid protein quantification in microplates or in microvolume with the Take3 plate. Fluorescence excitation and emission wavelength selection is from 250 nm to 850 nm, with bandwidths user-selectable between 3 nm and 50 nm, in 1 nm increments. Higher bandwidths can increase sensitivity, while lower bandwidths can increase specificity. This capability is important for optimal detection conditions, especially with fluorophores that have a very narrow Stokes shift. The variable bandwidth enables fine-tuning of the channels to prevent crosstalk for multiplex assays like SNPs. Glow and flash luminescence measurements are also available using the monochromator optics for workflows in cell proliferation, gene expression, GPCR, ATP quantification, and more.

Figure 1. The quad monochromator optics in Synergy Neo2.

Filter-based optical system

Along with fluorescence intensity, filtered, glow and flash luminescence measurements, advanced detection modes such as time-resolved fluorescence (TRF), time-resolved fluorescence energy transfer (TR-FRET), fluorescence polarization (FP), BRET, and AlphaScreen are also available using the filter-based optics in Synergy Neo2 (Figure 2). The high-quality filter cubes and dichroic mirrors system enables high transmittance for best performance over a wide range of time-dependent and proximity assays. Top and bottom detection with high transmission and high sensitivity optimizes detection for cell-based assays using fluorescence or luminescence detection. Bottom filter detection can maximize sensitivity and minimize light loss for cell-based assays.

The filter cubes for Synergy Neo2 contain excitation and emission filters, along with dichroic mirrors, and/or polarizing filters in barcoded assemblies, to ensure correct filter cube selection and position. The extensive range of available filter cubes allows the best performance for many advanced fluorescence and luminescence workflows.

Figure 2. Synergy Neo2 uses filter cube assemblies with excitation and emission filters, dichroic mirrors, and polarizing filters (for FP applications).

1. Optimize proximity assays using FRET-based measurements

Proximity assays use energy transfer from a donor to an acceptor moiety. FRET based assays employ two fluorescent molecules with overlapping emission and excitation wavelengths in a manner such that the excitation of the donor will result in emission from the donor molecule and the acceptor. While the FRET signal obtained from the acceptor molecule’s emission is generally considered the more important result, information can also be gleaned from the donor molecule’s emission signal. While the dual-photomultiplier (dual-PMT) configuration allows for the simultaneous capture of both emission signals it is important that both are in the dynamic range of the PMTs. In general, the donor molecule emission will be significantly greater than that observed from the FRET reaction so the gain setting of the two PMTs needs to be set differently to reflect this. While the gain setting for each can be set manually, the AutoScale option in the Gen5 software will automatically integrate the plate to identify the highest well and set the gain appropriately. The Synergy Neo2 also offers an extended dynamic range that extended the signal range 100-fold.

2. Improve the accuracy of FP assays by setting the G factor

Fluorescence polarization (FP) is in essence an assay that measures changes in size. Fluorescence polarization is measured using the ratio of the fluorescence emission returned through two polarizing filters, one parallel (∥) to and one perpendicular (⊥) to the plane of polarized excitatory light. The formula used to calculate fluorescence polarization (P) uses an instrument and assay correction factor, referred to as G. To obtain useful and comparable data it is necessary that this correction factor be adjusted on an instrument-by-instrument basis. Small differences in the angle of the polarizing filters of different instruments can often lead to different results. Likewise, assay buffer that have viscous agents such as glycerol can affect results. It is best to adjust the G-factor against a known standard such as sodium fluorescein in the buffer system employed. This small molecule rotates freely in solution and has a reported mP value of 35. The G-factor can either be set prior to running an experiment by testing a standard independently or by dedicating specific wells on the test plate. The Synergy Neo2 in combination with Gen5 will use these wells to automatically set the PMT gain and G-factor to achieve a requested polarization value.

3. Utilize ratiometric FRET readouts for better data interpretation

The dual signal output of FRET based assays can also be used to correct for well-to-well pipetting variances, as well as increase the assay window. Rather than plotting the change in FRET signal against agonist compound concentration directly, plotting the ratio of the FRET signal to that of the donor molecule fluorescence against drug concentration can often have several advantages. The ratiometic nature of the determination often corrects for pipetting errors in assay reagents. With large FRET signals changes both the Acceptor emission and donor emission will change significantly in opposite directions with drug concentration. This bifold change will dramatically increase the assay window in terms of fold-change.

1. Monitoring of Protein Kinase Activity Using Next Generation CSox-based Substrate Sensors
2. Investigation of Protein: Protein Interactions (PPI) using a Novel Bioluminescence Resonance Energy Transfer (BRET) Proximity based, High Throughput Screening Assay
3. Kinetic Degradation Profiling of IMiD Molecular Glues and Their Target Families
4. Fluorometric Quantitation of dsDNA using PicoGreen