Structured Excitation Beam for Super-Resolution Fluorescence Microscopy
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Solution Overview
Problem
Current methods for determining the location of individual molecules in a sample with high spatial resolution are limited by the Abbe diffraction limit and require high photon numbers or specific molecular properties, such as switchability, which can be challenging to achieve with conventional fluorescent dyes.
Innovation Solution
A method involving excitation light with a local minimum intensity distribution, where the intensity of fluorescent light from individual molecules decreases significantly at the minimum, allowing for precise localization of molecules beyond the diffraction limit by analyzing the intensity progression across these minima, and adjusting the excitation light to ensure molecules are at distances smaller than the minimum value d = λ/(2nsinα √(1 + I/IS)), where λ is the wavelength, n is the refractive index, α is the opening angle, I is the maximum intensity, and IS is the fluorescence excitation saturation intensity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence microscopy is used to determine molecule locations, then the method is simple and direct, but the spatial resolution is limited by the Abbe diffraction limit
Solution Approach 1:
The patent divides the excitation light beam into multiple partial beams that interfere with each other to create a structured intensity distribution with multiple minima. This segmentation of the excitation field allows simultaneous localization of multiple molecules with super-resolution, overcoming the Abbe diffraction limit while maintaining a relatively simple implementation approach
Solution Approach 2:
The patent creates regions of different excitation intensity (minima and maxima) within the excitation field. Molecules located at intensity minima experience reduced excitation and thus reduced fluorescence emission, while molecules at maxima are strongly excited. This local variation in excitation quality enables precise localization by detecting the position-dependent fluorescence intensity variations
2Measurement precision
If localization methods are used to achieve super-resolution, then spatial accuracy beyond the diffraction limit is achieved, but a significant number of photons are required
Solution Approach 1:
By creating intensity minima in the excitation field, the patent reduces the number of photons emitted by molecules located at these minima. This allows for more efficient use of photons from molecules that are strongly excited at maxima, improving localization accuracy while reducing the total photon budget required compared to uniform excitation methods
3Measurement precision
If molecules with specific properties (switchability) are used to achieve super-resolution, then high spatial resolution is achieved, but the requirement for specific molecular properties increases
Solution Approach 1:
The patent achieves super-resolution by exploiting the spatial structure of the excitation field rather than requiring special molecular properties. The intensity minima and maxima in the structured excitation beam enable localization based on position-dependent fluorescence intensity, which works with conventional fluorescent molecules that do not require photo-switchability or other special properties
Solution Approach 2:
The patent changes the excitation light parameters (intensity distribution, spatial structure) rather than requiring changes in molecular properties. By structuring the excitation field with multiple minima and maxima, the method achieves super-resolution with standard fluorescent molecules, increasing versatility and reducing the need for specialized probes
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 enables high-resolution imaging of molecule distributions by accurately determining the locations of individual molecules with increased spatial accuracy, independent of molecular orientation, and allows for continuous or intermittent registration of fluorescent light, enhancing the precision of molecular localization.
Implementation Method 1
The individual molecules of the substance are in a fluorescent state in which they can be excited by excitation light to emit fluorescent light
Implementation Method 2
an intensity distribution of the excitation light has at least one local minimum
Data Source
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AI summary
In order to determine the location (XM) of individual molecules of a substance in a sample, wherein the individual molecules of the substance are in a fluorescent state in which they are excitable with excitation light for emission of fluorescent light, and wherein distances between the individual molecules of the substance in a region of interest in the sample comply with a minimum value d = λ/(2nsinα √(1 + l/ls)), the individual molecules of the substance are excited with the excitation light for emission of fluorescent light, wherein an intensity distribution of the excitation light has at least one zero. The fluorescent light from the excited individual molecules of the substance is registered for different positions (XN) of the at least one zero of the intensity distribution of the excitation light in the region of interest in the sample. In this case, distances between closest adjacent positions (XN) of the at least one zero of the intensity distribution of the excitation light in which the fluorescent light from the excited individual molecules of the substance is registered are not greater than half the minimum value d. The locations (XM) of the individual molecules of the substance are then derived from the profile of the intensity (I) of the fluorescent light from the respective molecule against the positions (XN) of the at least one zero of the intensity distribution of the excitation light in the region of interest in the sample.