MINFLUX Localization Method Using Pre-localization and Fixed Illumination Patterns
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Solution Overview
Problem
Existing MINFLUX localization methods require complex and expensive equipment for live position estimation and feedback control, limiting their applicability and efficiency, especially in terms of photon usage and positional accuracy.
Innovation Solution
A method that uses a pre-localization step to estimate the position of individual emitters followed by a main localization step with a constant maximum extension of the illumination pattern, allowing for high positional accuracy without the need for fast beam displacement devices like electro-optical deflectors, and utilizing a fiber bundle for illumination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If iterative MINFLUX localization methods with feedback control are used, then positional accuracy is improved, but device complexity and cost increase due to requirements for fast beam displacement devices
Solution Approach 1:
The patent performs a pre-localization step using standard fluorescence microscopy to estimate the emitter position before conducting the main MINFLUX localization. This preliminary action provides an initial position estimate that enables subsequent localization iterations to start from a known reference point, reducing the need for complex real-time feedback control systems while maintaining high positional accuracy.
Solution Approach 2:
The localization process is divided into two distinct segments: pre-localization using wide-field fluorescence microscopy to obtain initial position estimates, and main localization using MINFLUX with a fixed illumination pattern. This segmentation allows each step to use optimized, simpler equipment appropriate to its specific function, avoiding the need for a single complex system to perform all tasks.
2Measurement precision
If iterative MINFLUX localization with adaptive illumination pattern adjustment is used, then positional accuracy is improved, but photon efficiency decreases due to increased light exposure
Solution Approach 1:
The pre-localization step acquires initial position information using minimal photon exposure from standard fluorescence imaging. This preliminary action reduces the photon budget requirement for subsequent MINFLUX iterations, as the main localization can start with a known position estimate and require fewer iterative adjustments, thereby improving overall photon efficiency.
Solution Approach 2:
The patent performs a limited number of MINFLUX localization iterations (typically 1-3) rather than extensive iterative refinement. This partial action approach achieves sufficient positional accuracy for most applications while limiting cumulative light exposure and photon consumption, balancing accuracy requirements with photon efficiency constraints.
3Device complexity
If pre-localization is performed before main localization, then equipment requirements are simplified, but measurement time increases due to additional localization steps
Solution Approach 1:
The patent implements a periodic workflow where pre-localization is performed once to establish initial position estimates for multiple emitters, followed by main MINFLUX localization. This periodic structure allows efficient batch processing where the time cost of pre-localization is amortized across multiple subsequent localizations, reducing the average time per emitter while maintaining simplified equipment requirements.
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 simplifies the equipment requirements and enhances positional accuracy while maintaining photon efficiency, making the method more universally applicable and cost-effective.
Implementation Method 1
The individual light-emitting emitters are, in particular, fluorophores and the illumination light is, in particular, excitation light which excites the fluorophores, whereupon they emit fluorescent light
Implementation Method 2
the position of an individual emitter is determined by detecting light emissions from the emitter, taking advantage of the fact that the smaller the distance between the emitter and the minimum of the light distribution, the less light is emitted from the emitter
Data Source
AI summary
The invention relates to a method for localizing individual emitters in a sample, comprising a pre-localization comprising an illumination of the sample with illumination light, wherein the illumination light induces or modulates light emissions of an individual and stationary emitter in the sample, detecting the light emissions of the emitter and estimating the position of the emitter in the sample from the detected light emissions and a subsequent main localization comprising illuminating the sample with an intensity distribution of the illumination light at illumination positions, the intensity distribution comprising a local minimum, detecting the light emissions of the emitter for the illumination positions, and determining the position of the emitter in the sample from the light emissions detected for the illumination positions, wherein the illumination positions are arranged in a first iteration about the estimated position and wherein the illumination positions in at least one second iteration are arranged around the position of the emitter determined in the preceding iteration, wherein the illumination positions in the first iteration and the at least one second iteration form a respective illumination pattern comprising a maximum extension, wherein the maximum extension of the illumination pattern is kept constant during the main localization. The invention further relates to a light microscope for carrying out the method, a computer program and the use of a fiber bundle for the method.