Super-resolution particle tracking via moving light minimum
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Solution Overview
Problem
Current methods for tracking the movement of single molecules in samples face challenges such as high photon requirements leading to bleaching and limited suitability of fluorophores, and existing techniques like STED and RESOLFT microscopy require complex optical setups and high intensities, which increase the risk of bleaching and are not suitable for all particles.
Innovation Solution
A method involving a spatially limited light intensity distribution with a minimum point is used to drive particles to emit photons, where the light distribution is moved to minimize photon emission rates, allowing for precise tracking of particles without high photon requirements, reducing bleaching risk, and simplifying the optical setup.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a high number of photons is detected for each position of the molecule to achieve spatial precision beyond the diffraction limit, then the spatial precision is improved, but the molecule is seriously stressed which results in an increased risk of bleaching
Solution Approach 1:
Instead of detecting photons emitted by the molecule at each position (localization method), the patent inverts the approach by using a moving light intensity distribution with a minimum that tracks the molecule. The molecule's position is determined by tracking the minimum of the light distribution rather than by photon detection, thereby achieving high spatial precision without requiring a high number of photons from the molecule.
2Measurement precision
If high absolute intensities of fluorescence inhibiting light are applied in STED microscopy to reduce the spatial region of excitation, then the spatial resolution is improved, but the risk of bleaching the fluorophores is relatively high
Solution Approach 1:
The patent extracts the essential function of STED microscopy (reducing the excitation region to achieve super-resolution) without using the harmful high-intensity fluorescence inhibiting light. By using a moving light intensity distribution with a minimum instead of STED's stationary high-intensity pattern, the patent achieves similar spatial resolution improvement while eliminating the high bleaching risk associated with STED's high absolute intensities.
3Reliability
If RESOLFT fluorescence microscopy is used to reduce the spatial region of excitation with relatively low intensities, then the risk of bleaching is reduced, but only special fluorophores that can be switched into a conformational state are suitable
Solution Approach 1:
The patent creates a universal method that can be applied to any fluorescent particle without requiring special photo-switchable properties. By using a moving light intensity distribution with a minimum that tracks the particle, the method achieves super-resolution tracking with any fluorophore, making it universally applicable rather than limited to special RESOLFT-compatible fluorophores.
4Measurement precision
If different light beams are applied for excitation and fluorescence inhibition in STED/RESOLFT microscopy, then the spatial resolution is improved, but the optical setup becomes complex requiring additional effort for alignment
Solution Approach 1:
The patent merges the excitation and tracking functions into a single moving light intensity distribution with a minimum. Instead of using separate excitation beams and fluorescence inhibiting beams as in STED/RESOLFT, the patent uses one light distribution that both excites the fluorophore and provides super-resolution through its moving minimum, thereby simplifying the optical setup while maintaining spatial resolution improvement.
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 method enables tracking of particles at a precision below the diffraction limit with reduced bleaching risk, allowing for longer tracking periods and distances, and simplifies the optical setup by minimizing photon emission rates and eliminating the need for high-intensity fluorescence inhibition.
Implementation Method 1
selecting a particle from a first group of particles in the sample which are driven to emit photons when subjected to the light of the first wavelength composition
Data Source
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AI summary
A method of measuring a sample (3) comprises the steps of (i) providing light of a first composition; (ii) selecting a particle (2) from a group of particles which are driven to emit photons when subjected to the light (4) of the first composition; (iii) forming the light of the first composition to provide a light intensity distribution comprising a spatially limited minimum (19); (iv) applying the light intensity distribution to the sample such that the particle is located in the spatially limited minimum of the light intensity distribution; (v) detecting the photons emitted by the particle; and tracking the movement of the particle with the minimum of the light intensity distribution by (vi) moving the light intensity distribution with respect to the sample such that a rate of the photons emitted by the particle remains minimal, and (vii) taking an actual position of the minimum of the light intensity distribution in the sample as an actual position of the particle in the sample. For imaging the sample the method comprises the further steps of (viii), for each of a plurality of parts of the sample, determining a dwell time of the particle; and (ix) mapping a distribution of the dwell times over the sample.