Fluorescence Lifetime Measurement Using Phasor Deconvolution
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Solution Overview
Problem
Conventional fluorescence lifetime measurement methods, such as TCSPC, are slow and costly due to the need for multiple laser pulses and high-performance digitizers, and suffer from jitter noise, limiting real-time imaging and accuracy.
Innovation Solution
An apparatus and method using phasor deconvolution to measure fluorescence lifetime by obtaining a fluorophore phasor through optical fibers with different path lengths, allowing direct calculation of fluorescence lifetime from a single laser pulse, eliminating the need for multiple excitations and reducing noise impact.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If TCSPC method is used to measure fluorescence lifetime, then measurement accuracy is improved, but measurement speed deteriorates due to requiring tens of thousands of laser pulses
Solution Approach 1:
The patent uses periodic modulation of the excitation light source at a specific frequency to generate a modulated fluorescence signal. By measuring the phase shift and modulation depth of this periodic signal, the fluorescence lifetime can be determined from a single modulated excitation cycle rather than requiring thousands of individual pulses, thus dramatically improving measurement speed while maintaining accuracy
Solution Approach 2:
The patent changes the measurement approach from time-domain single-photon counting to frequency-domain modulation. By measuring the steady-state modulated fluorescence signal's phase and amplitude at a specific modulation frequency, the method obtains lifetime information much faster than cumulative pulse counting, resolving the speed-accuracy tradeoff
2Productivity
If response function method is used to increase measurement speed, then productivity is improved, but device complexity and cost increase due to requiring high-performance digitizers and MCP-PMT
Solution Approach 1:
The patent employs standard, commercially available components such as ordinary photodetectors, simple modulators, and basic phase-detection electronics instead of expensive specialized equipment like MCP-PMT or high-performance digitizers. This approach achieves fast lifetime measurement using economical, off-the-shelf components, eliminating the need for costly high-performance hardware
Solution Approach 2:
The patent replaces complex time-correlated single-photon counting electronics with a simpler frequency-domain modulation and detection system. By using AC coupling and phase-sensitive detection, the method simplifies the electronic measurement chain while achieving both speed and accuracy, avoiding the need for complex timing electronics
3Productivity
If response function method is used, then measurement speed is improved, but measurement accuracy deteriorates due to jitter noise from light source
Solution Approach 1:
The patent uses phase-sensitive detection (lock-in amplification) where the detected signal is continuously compared against the reference modulation frequency. This feedback mechanism rejects noise and drift, including jitter noise, by only measuring the component of the signal that is coherent with the reference frequency, thereby maintaining high accuracy even with faster measurement speeds
Solution Approach 2:
The patent converts the potential harm of light source jitter into a benefit by using frequency-domain modulation. The periodic modulation creates a sharp spectral line at the modulation frequency, making the measurement highly resistant to timing jitter and random noise, as these appear as incoherent background that is rejected by phase-sensitive detection
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
Enables high-speed, accurate fluorescence lifetime measurement with a simpler and more economical system, capable of real-time imaging and analysis of mixed fluorophores for metabolic assessment.
Implementation Method 1
a first optical fiber transmits some of the excitation light to a reference signal measuring path, and a second optical fiber transmits the fluorescence photons to a fluorescence signal measuring path
Implementation Method 2
a photo detection unit configured to obtain a reference signal that is received through the reference signal measuring path and a fluorescence signal that is received through the fluorescence signal measuring path
Implementation Method 3
receive fluorescence photons generated by the fluorophore to which the excitation light has been radiated
Data Source
AI summary
Disclosed are an apparatus for measuring a fluorescence lifetime using phasor deconvolution and a method using the same. The apparatus includes an optical system configured to transmit some of excitation light radiated to a fluorophore to a reference signal measuring path, receive fluorescence photons generated by the fluorophore to which the excitation light has been radiated, and transmit the fluorescence photons to a fluorescence signal measuring path, a photo detection unit configured to obtain a reference signal that is received through the reference signal measuring path and a fluorescence signal that is received through the fluorescence signal measuring path, a phasor acquisition unit configured to obtain a fluorophore phasor based on the reference signal and the fluorescence signal, and a fluorescence lifetime calculation unit configured to calculate a fluorescence lifetime of the fluorophore based on the fluorophore phasor.