Molecule Positioning via Deactivation Light Saturation
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Solution Overview
Problem
Existing methods for determining the position of molecules in a sample, such as minflux microscopy, face challenges in reducing background flux and maintaining high detection efficiency, especially when combined with STED microscopy.
Innovation Solution
The procedure involves generating light distributions with local intensity minima and adjacent intensity maxima, using a combination of excitation and deactivation light distributions. The deactivation light distribution is designed to maximize saturation intensity in a fishing area, while minimizing its impact on the effective detection point spread function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-generated harmful factors
If STED light distribution is combined with excitation light distribution to reduce background fluorescence, then background flux is reduced, but the effective detection point spread function is distorted and photon efficiency decreases
Solution Approach 1:
The patent separates the STED light distribution and excitation light distribution into independent components with各自优化的参数。通过独立优化两条光束的参数，避免了叠加后的相互干扰，实现了对背景荧光的有效抑制同时保持了对分子位置的精确测量
Solution Approach 2:
Instead of combining STED and excitation light distributions as done in conventional approaches, the patent uses them separately - first acquiring images with excitation light alone, then using STED light to selectively suppress background fluorescence from specific regions, thereby inverting the conventional sequence and avoiding PSF distortion
2Object-generated harmful factors
If STED light intensity is increased to improve background suppression, then background flux is reduced, but fluorophore saturation and phototoxicity increase
Solution Approach 1:
The patent applies STED light selectively to specific regions or time points where background suppression is needed, rather than continuously applying high-intensity STED light throughout the entire imaging process. This localized application reduces overall phototoxicity while maintaining effective background suppression where required
Solution Approach 2:
The patent uses partial STED illumination - applying STED light only to the extent necessary for background suppression in specific regions, rather than using excessive STED light intensity across the entire field of view. This partial action achieves the needed background reduction while minimizing photodamage
3Measurement precision
If conventional MINFLUX method is used for single molecule localization, then localization accuracy is high, but background fluorescence from surrounding molecules reduces detection efficiency
Solution Approach 1:
The patent extracts and removes background fluorescence signals from the detection process by using STED light to selectively suppress emission from molecules that are not in the immediate vicinity of the targeted fluorophore. This extraction of background noise improves the signal-to-noise ratio and maintains high detection efficiency
Solution Approach 2:
The patent introduces STED light as an intermediary mechanism that mediates between the excitation light and the fluorescence detection. The STED light acts as a selective gatekeeper, allowing signal from the target molecule while blocking background fluorescence from surrounding molecules, thereby maintaining both accuracy and efficiency
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 effectively reduces background flux and enhances detection efficiency, allowing for precise localization of molecules with minimal impact on the detection point spread function, thereby improving the accuracy and reliability of molecular positioning.
Implementation Method 1
a sample is scanned with fluorophores with an excitation light intensity distribution... an emission signal is measured for each position, preferably by counting individual fluorescence photons
Implementation Method 2
STED microscopy is based on the depletion of fluorescence by stimulated emission: By irradiating STED light of a suitable wavelength, fluorophores can be specifically (re)converted from the excited state to the ground state
Data Source
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AI summary
The invention relates to a method for determining positions of mutually spaced molecules (M) in a sample (20), having the steps of generating (101) a plurality of light distributions, each light distribution having a local intensity minimum (110, 310) and adjacent regions (120, 320) of increasing intensity, comprising an excitation light distribution (100) and a deactivation light distribution (300); illuminating (102) the sample (20) with the excitation light distribution (100) and the deactivation light distribution (300); detecting (103) photons emitted by the molecule (M) for different positions of the excitation light distribution (100); and deriving (104) the position of the molecule (M) on the basis of the photons detected for the different positions of the excitation light distribution (100), wherein the local minimum (110) of the excitation light distribution (100) is arranged at a plurality of scanning positions (201) one after the other within a scanning region (200), and the light intensity of the deactivation light in a catching region (210), which is paired with the scanning region (200) and in which the position of the molecule (M) can be unambiguously derived from the scanning positions (201) and the paired detected photons, corresponds maximally to three times the saturation intensity of the deactivation light. The invention further relates to a device for carrying out said method.