Modulated Fluorescence Detection for Selective Species Analysis
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Solution Overview
Problem
Conventional fluorescence detection techniques face limitations in selectively detecting multiple reversibly photoswitchable fluorescent species in a sample due to overlapping emission spectra and complex implementation requirements, particularly in in vivo imaging and remote sensing applications.
Innovation Solution
A method involving periodically modulated illuminating light and detection of the fluorescence emission's amplitude in phase quadrature, optimized by choosing the average intensity and modulation frequency to maximize the amplitude of the intensity component, allowing for selective and quantitative detection of reversibly photoswitchable fluorescent species.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence detection techniques are used, then the detection process is simple, but the selectivity to detect multiple fluorescent species is poor due to overlapping emission spectra
Solution Approach 1:
The patent applies periodic modulation of the excitation light source at a specific frequency to induce periodic changes in the fluorescence emission of reversibly photoswitchable species. By detecting the fluorescence signal at the modulation frequency using lock-in amplification, the method achieves high selectivity for multiple fluorescent species even with overlapping spectra, while maintaining a relatively simple detection setup.
2Measurement precision
If dynamic contrast techniques with modulated excitation are used, then selectivity is improved, but the implementation complexity increases due to requiring two-color excitation and empirical optimization
Solution Approach 1:
The patent changes the excitation parameters by using a single modulated excitation wavelength instead of two-color excitation. The modulation frequency and average intensity are optimized to maximize the dynamic contrast signal, eliminating the need for empirical optimization of multiple parameters and simplifying the implementation while maintaining high selectivity.
Solution Approach 2:
The patent extracts the useful fluorescence signal from the background by using lock-in amplification to detect only the component of the fluorescence signal that oscillates at the modulation frequency. This effectively separates the signal of interest from background fluorescence and noise, achieving high selectivity without requiring complex two-color excitation systems.
3Measurement precision
If OLID technique is used, then dynamic contrast is achieved, but quantitative information on fluorophore concentration is not provided and the system requires reference pixels
Solution Approach 1:
The patent uses lock-in amplification with a reference signal generated from the known modulation waveform to extract the fluorescence signal at the modulation frequency. This feedback mechanism allows for quantitative measurement of the fluorophore concentration by relating the amplitude of the detected signal to the concentration, while eliminating the need for reference pixels used in OLID techniques.
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 enables selective and quantitative detection of multiple fluorescent species, overcoming the limitations of existing techniques by simplifying the implementation and improving selectivity and accuracy in both microscopic and remote sensing applications.
Implementation Method 1
detecting a fluorescence emission emitted by the duly illuminated sample
Implementation Method 2
said or each said reversibly photoswitchable fluorescent species (P) being able to be switched from said first state to said second state by a first photo-induced reaction
Data Source
AI summary
A method for detection of at least one reversibly photo-convertible fluorescent species, comprises the following steps: a) illumination of a sample comprising said or at least one of the reversibly photo-convertible fluorescent species by a periodically modulated illuminating light; and b) detection of fluorescent emission emitted by the sample thus illuminated; wherein the method further comprises the following step: c) extraction of the amplitude of the intensity component of the fluorescent emission exhibiting the same periodicity as the periodically modulated illuminating light and a phase quadrature with respect to the same; and wherein the mean intensity of the illuminating light and the modulation frequency of the same are chosen to maximize the amplitude of the intensity component of the fluorescent emission.