Fluorescence Triple-Correlation Spectroscopy for Ternary Interaction Analysis
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Solution Overview
Problem
Conventional fluorescence correlation spectroscopy systems are unable to perform in-situ analysis of intermolecular interactions between three kinds of molecules, limiting their application in studying complex biological functions within living cells.
Innovation Solution
A fluorescence triple-correlation spectroscopy system is developed, utilizing at least three excitation light sources emitting coaxially, with a configuration that includes a collimating beam expander, dichroic mirrors, a galvanometer scanner, and single photon detectors to collect and analyze fluorescence signals from four channels, enabling real-time calculation of the triple-correlation function and plotting of a fluorescence triple-correlation spectroscopy curve.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional fluorescence correlation spectroscopy (FCS or FCCS) is used, then the system structure is simple and easy to operate, but it cannot analyze interactions between three kinds of molecules
Solution Approach 1:
The system divides the detection process into four independent signal detection channels, each equipped with specific optical filters to detect fluorescence signals of different wavelengths. This segmentation enables simultaneous detection of three different fluorescent molecules while maintaining manageable system complexity through modular channel design.
Solution Approach 2:
The patent extends the traditional two-channel FCCS system by adding a third detection dimension (wavelength channel), transforming it into a four-channel system capable of triple-correlation analysis. This dimensional extension allows analysis of ternary interactions while building upon the existing FCCS framework.
2Adaptability or versatility
If fluorescence autocorrelation spectroscopy is used to detect molecular interactions, then the system structure is simple, but it requires noticeable molecular mass difference for detection
Solution Approach 1:
The system uses fluorescent labels as intermediaries to mark the molecules of interest. By detecting the fluorescence signals of these labels rather than relying on intrinsic molecular properties, the system can detect interactions between molecules of similar mass, overcoming the limitation of FACS while maintaining high detection sensitivity.
3Productivity
If conventional FCCS is used for analyzing intermolecular interactions, then it works well for two molecules, but it fails to analyze ternary interactions
Solution Approach 1:
The system merges three separate autocorrelation measurements and two cross-correlation measurements into a unified triple-correlation analysis framework. By combining the signals from three molecular pairs detected in four channels, the system achieves ternary interaction analysis while optimizing the use of shared optical components.
Solution Approach 2:
The four-channel detection system is designed with universal applicability, where each channel can detect different fluorescent wavelengths and any combination of three channels can perform triple-correlation analysis. This multi-functional design enables the system to analyze various ternary interaction scenarios using the same hardware configuration.
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 system allows for the real-time analysis of intermolecular interactions between three kinds of molecules labeled with different fluorophores, achieving detection volumes less than 1 femtoliter and enabling the analysis of fluorescent molecules with four different emission spectra, thus overcoming the limitations of conventional systems in studying ternary interactions within living cells.
Implementation Method 1
the at least three excitation light sources are configured to coaxially emit a laser beam... so as to excite the sample to generate a fluorescence signal
Implementation Method 2
the fluorescence signal is collected by the objective lens, sequentially passed through the tube lens, the galvanometer scanner and the dichroic mirror and then focused by a focusing lens and then divided into four signal detection channels according to wavelength by three dichroic mirrors
Implementation Method 3
the four signal detection channels are transmitted to a signal acquisition and operation card which is connected to the four single photon detectors for real-time collection and operation between any three signals of the four signal detection channels
Implementation Method 4
the fluctuation of fluorescence signals caused by Brownian motion or chemical reaction of fluorescent molecules in a detection microregion
Data Source
AI summary
A ternary fluorescence correlation spectroscopy system for analyzing an interaction between three kinds of molecules, including at least three excitation light sources with different wavelengths. The excitation light sources are configured to illuminate and excite a sample to generate a fluorescence signal. The fluorescence signal is divided into multiple signals according to wavelength, which are then respectively detected by a single photon detector and transmitted to a signal acquisition and operation card to perform real-time operation of a triple-correlation function, so as to obtain a fluorescence triple-correlation spectroscopy curve.