Anti-Stokes Photoluminescence Measurement Error Correction
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Solution Overview
Problem
Anti-Stokes photoluminescence measurements in Foerster-type resonance energy-transfer assays are affected by errors from radiative energy transfer, leading to background signals that reduce the signal-to-background ratio and complicate the measurement of sensitized acceptor emission.
Innovation Solution
A method and device that correct for these errors by exciting anti-Stokes photoluminescent molecules with light greater than the acceptor's emission wavelength, measuring emission signals in multiple time windows, including simultaneously with and after the excitation pulse, to distinguish and subtract radiative energy transfer signals from non-radiative energy transfer signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If radiative energy transfer is present in anti-Stokes photoluminescence measurement, then background signal increases, but signal-to-background ratio deteriorates
Solution Approach 1:
The measurement process is segmented into multiple time windows: a first time window during the excitation pulse to capture both FRET and radiative energy transfer signals, and a second time window after the excitation pulse to capture only radiative energy transfer signals. This temporal segmentation allows separate measurement and subsequent subtraction of the harmful radiative component.
Solution Approach 2:
The harmful radiative energy transfer signal is extracted from the total signal by measuring it separately in the second time window when FRET signal is absent. This extracted radiative component is then subtracted from the first time window signal to obtain the pure FRET signal, effectively removing the harmful background.
2Measurement precision
If measurement is delayed after excitation pulse to avoid background, then total signal per time decreases, but measurement accuracy improves
Solution Approach 1:
The radiative energy transfer signal is preliminarily measured during the excitation pulse itself (first time window) along with the FRET signal. This preliminary capture of the background signal allows for its subsequent removal through subtraction, enabling high-speed measurement without sacrificing accuracy.
Solution Approach 2:
The measurement process maintains continuity by collecting useful signal data during the excitation pulse rather than waiting for it to end. The excitation pulse serves dual purposes: exciting the FRET signal and simultaneously capturing the radiative energy transfer background, maximizing signal utilization.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for improved signal-to-background ratio and accurate measurement of sensitized acceptor emission by simultaneously exciting and measuring the donor, increasing the total signal per time and enhancing the performance of FRET-based assays.
Implementation Method 1
exciting anti-Stokes photoluminescent molecules, ions, phosphors, chelates, or particles
Implementation Method 2
measuring emission light signal at a wavelength, which is said emission wavelength of said acceptor molecules in said Foerster-type resonance energy-transfer assay
Implementation Method 3
measuring emission light signal at a wavelength... in at least two different time windows
Data Source
AI summary
A method to correct measurement error in a resonance energy-transfer assay, including exciting anti-Stokes photoluminescent donors with at least one wavelength of light which is greater than an emission wavelength of acceptor molecules; measuring emission at the acceptor's emission wavelength and which differs from the donor's emission wavelength in at least two different time windows; a first time window within the time window defined by the excitation light pulse and a second non-overlapping time window which follows the first time window; and correcting the emission signal, which includes signals originating from non-radiative and radiative energy transfer, within the first time window by estimating the ratio of the signals from non-radiative and radiative energy transfer or the signal originating from radiative energy transfer using at least one emission signal measured in the second time window.