Point Source Arrangement Detection Beyond the Diffraction Barrier
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Solution Overview
Problem
Existing methods for determining the arrangement of equal point sources, such as fluorophores, are limited by the diffraction barrier and require switchable or multiple fluorophores, leading to inefficiencies and inaccuracies in resolving structures at very small distances.
Innovation Solution
The method involves scanning point sources with a probe signal having a local intensity minimum, registering measurement signals at different positions of this minimum, and using prior knowledge of the point source number and geometry to determine their arrangement, without the need for switchable sources.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If switchable fluorophores are used to determine positions of multiple point sources simultaneously, then measurement precision is improved, but device complexity and time consumption increase due to transition probabilities and blinking behavior
Solution Approach 1:
Instead of switching fluorophores on and off to resolve them sequentially (conventional approach), the patent inverts the strategy by using a fixed illumination pattern and detecting the combined signal from all fluorophores simultaneously. The position information is extracted from the spatial distribution of the combined fluorescence signal, eliminating the need for switching control and reducing complexity while maintaining precision.
Solution Approach 2:
The patent merges the detection of multiple fluorophores into a single simultaneous measurement by using a fixed illumination pattern that excites all fluorophores at once. The combined fluorescence signal from all point sources is detected and processed to determine their positions, eliminating the sequential switching process and reducing time consumption.
2Measurement precision
If multiple fluorophores with different excitation wavelengths are used to mark structures at small distances, then measurement precision is improved, but loss of time increases due to sequential excitation and registration requirements
Solution Approach 1:
The patent combines the detection of multiple fluorophores into a single simultaneous measurement by using a fixed illumination pattern that excites all fluorophores at once. The combined fluorescence signal from all point sources is detected and processed to determine their positions, eliminating the sequential switching process and reducing time consumption.
3Measurement precision
If conventional fluorescence microscopy is used to image point sources at distances below diffraction limit, then measurement precision is improved, but object-generated harmful factors increase due to cross-talk between fluorophores
Solution Approach 1:
The patent applies local quality by using a fixed illumination pattern with specific spatial characteristics (such as donut-shaped or ring-shaped patterns) that creates localized excitation zones. This allows the system to resolve point sources based on their spatial distribution within these localized patterns, reducing cross-talk between neighboring fluorophores while maintaining high spatial resolution.
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 allows for high-precision determination of point source arrangements at very small distances, overcoming the diffraction barrier and enabling accurate imaging of structures without the limitations of switchable fluorophores, with improved spatial resolution and reduced noise.
Implementation Method 1
Fluorophores can be probed or, more particularly, excited with excitation light, and they can be localized based on fluorescence light emitted in response to their excitation by the excitation light
Data Source
Figure 1~1d
Figure 2~2c
Figure 3~3i
AI summary
In a method of determining an arrangement of point sources in a sample the point sources are scanned (3) with a probe signal comprising a probe signal intensity. A spatial intensity distribution of the probe signal intensity has a local probe signal intensity minimum that is, in at least one spatial direction, delimited on both sides by probe signal intensity maxima. The local probe signal intensity minimum is positioned at different probe signal minimum positions at distances in the at least one spatial direction. A measurement intensity of a measurement signal coming from the point sources is registered (4) for each of the different probe signal minimum positions. The measurement intensity depends on the probe signal intensity at point source positions of the point sources. The point sources are limited (2) both to a known point source number of at least 2, and to such a small spatial area of the sample that a spatial course of the registered measurement intensities over the different probe signal minimum positions has one local measurement signal minimum only. The arrangement of the point sources is determined (5) from the spatial course of the registered measurement intensities over the different probe signal minimum positions utilizing previous knowledge of the arrangement of the point sources, the previous knowledge including the known point source number and, at least with a point source number above 4, a basic geometry of the arrangement of the point sources.