Phase-Shift Fluorophore Positioning via Modulated Excitation
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Solution Overview
Problem
Current super-resolution optical microscopy techniques face limitations in precision due to the dependence on the number of photons received and anisotropic emission from fluorophores, which restricts field size and introduces uncertainty in fluorophore positioning.
Innovation Solution
A method involving periodic modulation of excitation waves and phase shift analysis using a transducer array to determine physical parameters of a target region, allowing for precise collection of photons and improved imaging beyond the diffraction limit.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a small number of fluorophores are activated to zoom in on a single fluorophore and determine the center of the focal spot, then measurement precision is improved, but the field of observation size is reduced
Solution Approach 1:
The field of observation is divided into multiple regions, each with its own focal spot. Multiple fluorophores can be simultaneously activated and measured in different regions without overlapping, allowing both high precision measurement and a larger total field of observation.
Solution Approach 2:
The patent extends the observation space by using multiple focal spots arranged in a specific geometric configuration. This multi-dimensional arrangement allows simultaneous observation of multiple fluorophores across a larger field while maintaining the precision benefits of focused observation.
2Measurement precision
If the center of the focal spot is measured to determine fluorophore position, then position information is obtained, but precision is limited and dependent on the square root of the number of photons received
Solution Approach 1:
The patent replaces the conventional method of determining position by measuring the center of the focal spot with a phase-based measurement system. By modulating the excitation wave and measuring phase shifts in the emitted light, position information is obtained without relying on photon counting statistics, thereby improving precision especially when photon numbers are limited.
Solution Approach 2:
The patent changes the measurement parameter from intensity-based (photon count) to phase-based measurement. By using phase modulation and detection, the system achieves higher precision in determining fluorophore position and orientation, particularly effective when the number of received photons is small.
3Illumination intensity
If fluorophores are used as emitters, then light emission is obtained, but emission is anisotropic and dependent on polarization of exciting light, creating uncertainty in fluorophore position
Solution Approach 1:
The patent applies periodic modulation to the excitation wave, causing the fluorophores to emit light with a modulated intensity pattern. By measuring the phase shift of this modulated emission relative to the excitation signal, the system can determine fluorophore position and orientation while compensating for the anisotropic emission characteristics.
Solution Approach 2:
The system uses the phase information from the modulated fluorophore emission as feedback to accurately determine position and orientation. The phase shift measurement provides direct information about the fluorophore's spatial coordinates and angular orientation, correcting for polarization-dependent emission variations.
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 precise determination of fluorophore position and orientation with enhanced resolution, capable of observing single fluorophores with limited lifetime and expanding applicability to fields like acoustics and radiofrequency waves.
Implementation Method 1
The fluorophores are excitable nanoscopic light emitters that, when they are excited by incident light, emit a certain number of photons before self-destructing
Implementation Method 2
said return wave is periodically deflected into a deflected wave by means of a controllable deflecting element, so that said deflected wave scans a transducer array in each period of the periodic modulation
Implementation Method 3
a phase image comprising signals generated by each transducer of the transducer array, in response to the deflected wave, is recorded during at least one period of the periodic modulation
Implementation Method 4
a phase shift between a periodic modulation over time of at least one physical characteristic of the return wave and the periodic modulation over time of at least one physical characteristic of the excitation wave is determined from said phase image
Data Source
AI summary
Method and system for measuring a physical parameter of a target region of a medium are provided, wherein: an excitation wave having a periodic modulation over time of a physical characteristic is emitted, a return wave is periodically deflected with a controllable deflecting element so that the deflected wave scans a transducer array, each transducer being associated with a predefined phase range of the periodic modulation, a phase image is recorded during at least one period of the periodic modulation, a phase shift between a periodic modulation of the return wave and the periodic modulation of the excitation wave is determined from the phase image, a physical parameter of the target region is determined from the phase shift.