Simple Method for Proper Analysis of FRET Sensor Titration Data and Intracellular Imaging Experiments Based on Isosbestic Points

Abstract

Förster resonance energy transfer (FRET) sensors and other ratiometric probes are increasingly used in life sciences to obtain quantitative information in complex environments such as the cell interior or blood plasma. When using FRET sensors, either to determine the affinity of the sensor for its analyte in vitro, or to apply the sensor to measure an unknown concentration in situ, the ratio of donor and acceptor emission is commonly used as a measure of the sensor occupancy. However, it has been recently demonstrated that the underlying assumption of a linear relationship between the emission ratio and the relative sensor occupancy is not correct. Here we present a simple solution to this problem by using the fluorescence intensity at the isosbestic wavelength as an internal standard. The isosbestic wavelength of FRET-sensors based on the widely used CFP/YFP pair was determined to be at 513 nm, independent of the specific sensor architecture and pH. We show that using the ratio of either the donor or the acceptor emission and the emission at the isosbestic wavelength of 513 nm provides a straightforward method to obtain accurate K d values from in vitro titration experiments. In addition, using two recently developed FRET sensors for Zn2+ we show that the same approach can be used to allow more accurate quantification in live cell imaging experiments. We believe that this approach provides a generic solution to retain the advantages of ratiometric measurements, without compromising on analytical accuracy.

Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc

“…15,42 As expected, Zn 2+ binding could also be monitored by measuring FRET between the two fluorescent domains, showing a dynamic range and Zn 2+ affinity that are comparable to those of the parental eZinCh-2 FRET sensor (Figure S2A–D). 15,42 …”

Quantitative Imaging of Labile Zn2+ in the Golgi Apparatus Using a Localizable Small-Molecule Fluorescent Probe

“…Various FP-based Zn 2+ probes have been developed; ZapCY (Qin et al, 2011), eCALWY (Vinkenborg et al, 2009), and eZinCh (Hessels et al, 2015) are ratiometric fluorescent probes based on Fo ¨rster (or fluorescence) resonance energy transfer (FRET) and have been utilized for the quantitative analysis of labile cytosolic and organellar Zn 2+ . The cytosolic [Zn 2+ ] ([Zn 2+ ] Cyto ) has been estimated to be in the range of pM to nM (Chabosseau et al, 2014;Fudge et al, Cell Chemical Biology 27, 1521-1531, December 17, 2020 ª 2020 Elsevier Ltd. 1521 ll 2018; Hessels et al, 2015;Hessels and Merkx, 2016;Qin et al, 2011Qin et al, , 2013Vinkenborg et al, 2009). These values are consistent with the results estimated by other methods (Kre ˛_ zel and Maret, 2006;Simons, 1991;Taki et al, 2004).…”

Conformational plasticity of the intrinsically disordered protein ASR1 modulates its function as a drought stress-responsive gene

“…Emission spectra were collected using a 420 nm excitation with a 4-nm bandwidth. FRET changes were followed by the ratio between the donor maximum emission and the emission at the isosbestic wavelength (I donor /I Isosbestic ) as recently described for this type of molecules [ 19 ].…”