Simple Method for Proper Analysis of FRET Sensor Titration Data and Intracellular Imaging Experiments Based on Isosbestic Points
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- 作者：Anne M. Hessels ; Maarten Merkx
- 刊名：ACS Sensor
- 出版年：2016
- 出版时间：May 27, 2016
- 年：2016
- 卷：1
- 期：5
- 页码：498-502
- 全文大小：465K
- ISSN：2379-3694
文摘
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 Kd 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.