3D MINFLUX Localization for Unknown Axial Fluorophore Positions
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Solution Overview
Problem
Existing MINFLUX nanoscopy methods struggle with accurately localizing isolated fluorophores in three dimensions, especially when the axial position is not well known, and inefficiently utilize fluorescence emission, leading to systematic shifts and reduced accuracy due to assumptions about background signals and quadratic approximations of excitation donut shapes.
Innovation Solution
The method divides localization into axial and lateral steps using a 3D excitation donut, with sequential axial localization followed by lateral localization, and includes an axial scanning range greater than 500 nm, allowing for improved localization of fluorophores with unknown axial positions and enhanced utilization of fluorescence emission.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If MINFLUX nanoscopy uses a 3D excitation donut with sequential axial and lateral localization steps, then localization accuracy in three dimensions is improved, but the complexity of the localization process increases
Solution Approach 1:
The localization process is segmented into two distinct steps: axial localization and lateral localization. The axial localization step determines the z-position using a 3D excitation donut with sequential axial scanning, while the lateral localization step determines x-y position using a 2D excitation donut. This segmentation allows each step to focus on specific spatial dimensions, improving overall localization accuracy while maintaining a structured approach to the complexity.
Solution Approach 2:
The axial localization step is performed as a preliminary action before lateral localization. By first determining the axial position and using this information to define an axial scanning range greater than 500 nm, the system prepares the foundation for subsequent lateral localization. This preliminary action reduces uncertainties in the overall localization process.
2Adaptability or versatility
If the axial scanning range is increased to greater than 500 nm, then the ability to localize fluorophores with unknown axial positions is improved, but the measurement time and data processing requirements increase
Solution Approach 1:
The axial scanning range is dynamically adjusted based on the localization needs. By setting the axial scanning range to greater than 500 nm, the system adapts to fluorophores with unknown axial positions while maintaining efficient measurement through optimized scanning protocols that balance coverage with time constraints.
Solution Approach 2:
The axial scanning range parameter is changed to greater than 500 nm to improve adaptability for unknown axial positions. This parameter change enables the system to handle a broader range of axial positions while the sequential localization steps and ratiometric evaluation methods optimize the measurement process to minimize time loss.
3Measurement precision
If ratiometric evaluation of intensity ratios is used for localization, then localization precision is improved, but systematic shifts due to background signal assumptions and quadratic approximations occur
Solution Approach 1:
The ratiometric evaluation method provides feedback-based localization by comparing intensity ratios at different positions. The system uses the ratio of fluorescence intensities measured at different axial or lateral positions to calculate the fluorophore position. This feedback mechanism improves precision by utilizing relative intensity information, though systematic shifts from background assumptions and quadratic approximations remain as limitations.
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 achieves high-resolution localization of fluorophores in three dimensions with reduced systematic shifts and improved accuracy, even with uncertain axial positions, by optimizing the use of fluorescence emission and minimizing technical and economic costs.
Implementation Method 1
Fluorophores are localized by means of structured excitation light distributions. A fundamental feature of MINFLUX nanoscopy is that the fluorophores are excited in such a way that a fluorophore to be localized is always placed close to or in a minimum of the excitation light distribution
Data Source
AI summary
The present disclosure relates to MINFLUX nanoscopy. The present disclosure improves three-dimensional localization of isolated emitters, particularly of isolated fluorescent emitters. The utilization of the emitted photons of isolated excitable emitters, in particular of isolated excitable fluorescent emitters, for a three-dimensional localization is improved by separating the localization according to the MINFLUX principle using a 3D excitation donut into a sequence of separate steps, wherein in one step either an axial location or a lateral location of the fluorophore is determined. Thereby, the more precise knowledge of the axial position increases the quality of a following lateral localization and vice versa. The three-dimensional localization is further improved by a method for axial localization by scanning an axial scanning area with a 3D excitation donut and a preferably following evaluation of the measured intensities or photon numbers by forming a vector sum.