Multi-Illumination Fluorescence Imaging for Faster Molecule Localization
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Solution Overview
Problem
Conventional optical microscopes have resolution limits due to light diffraction, and existing super-resolution methods fail to fully utilize the objective lens's numerical aperture and are affected by single molecule image shape and flickering, leading to poor localization accuracy and long imaging times.
Innovation Solution
A device and method utilizing multi-illumination excitation patterns at different phases and directions to measure fluorescence signals, synchronized with fluorescence imaging, enhancing localization accuracy and minimizing the influence of sample changes and single molecule flickering.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional image fitting method is used for single molecule localization, then the method is simple to implement, but the localization accuracy is limited due to not fully utilizing the numerical aperture of the objective lens and being affected by single molecule image shape
Solution Approach 1:
The patent divides the fluorescence image into multiple sub-images corresponding to different phase regions (0-2π divided into multiple segments). Each sub-image captures fluorescence signal from a specific phase range, allowing independent analysis and fitting. This segmentation enables more precise localization by analyzing phase-specific signals rather than treating the entire image as a single entity, thereby improving localization accuracy while managing system complexity through modular processing.
Solution Approach 2:
The patent employs periodic modulation of the excitation light phase to illuminate the sample at different phase angles sequentially. By periodically varying the illumination phase and capturing corresponding fluorescence sub-images at each phase state, the system collects comprehensive phase information that enhances localization precision. This periodic action allows the system to fully utilize the objective lens numerical aperture across different phase cycles.
2Productivity
If single molecule localization is performed using conventional methods, then the imaging process is straightforward, but the imaging time is long due to poor localization accuracy requiring multiple measurements
Solution Approach 1:
The patent performs preliminary phase separation by dividing the fluorescence signal into multiple phase-specific sub-images before localization analysis. By pre-organizing the fluorescence data according to phase information and preparing sub-images for each phase segment, the system eliminates the need for repeated measurements to achieve accurate localization. This preliminary action of phase-based signal separation significantly reduces imaging time while maintaining high localization accuracy.
Solution Approach 2:
The patent introduces a phase dimension to the traditional single-image localization approach. Instead of analyzing a single fluorescence image in two dimensions (x, y), the system adds a phase dimension by capturing and analyzing multiple sub-images corresponding to different phase angles. This dimensional expansion provides more information for localization, improving accuracy and reducing the number of measurements needed, thereby increasing imaging speed.
3Measurement precision
If continuous illumination is used for fluorescence imaging, then the signal intensity is high, but the single molecule exhibits flickering and bleaching phenomena leading to poor localization accuracy
Solution Approach 1:
The patent uses periodic phase-modulated illumination instead of continuous static illumination. By sequentially illuminating the sample at different phase angles and capturing fluorescence signals at each phase state, the system accumulates sufficient signal information while reducing the exposure time at each phase. This periodic illumination approach minimizes the cumulative damage to single molecules, reducing bleaching and flickering effects, thereby improving localization accuracy.
Solution Approach 2:
The patent maintains continuous useful action by rapidly cycling through different phase illumination states and continuously collecting phase-specific fluorescence signals. Instead of using long continuous illumination that causes bleaching, the system continuously switches between phase states, accumulating useful fluorescence information from multiple phase angles. This continuous phase-cycled collection achieves high signal intensity through temporal integration while minimizing damage to individual molecules at any given moment.
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
Improves localization accuracy and imaging speed by eliminating errors from single molecule brightness flicker and bleaching, achieving high signal-to-noise ratio and enhanced resolution in super-resolution microscopy.
Implementation Method 1
a light path on which an acousto-optic modulator, a first half-wave plate, an analyzer, a first electro-optic modulator
Implementation Method 2
a first electro-optic modulator, and a first polarizing beam splitter are sequentially provided
Implementation Method 3
a laser having a light path on which an acousto-optic modulator
Implementation Method 4
a fluorescence imaging system configured to collect and scan fluorescence sub-images
Implementation Method 5
one fluorescence image contains N fluorescence sub-images
Data Source
AI summary
Disclosed are a device and method for measuring fluorescence signal in multi-illumination mode and use of the method. An excitation light is modulated on an illumination excitation light path, to generates excitation illumination patterns at different phases on a sample after passing through an objective lens; a high-speed switching device is arranged on a fluorescence collection light path to switch the position of a fluorescence image of the sample on a target plane of a photoelectric sensor, and a plurality of sub-images can be simultaneously obtained after one exposure through synchronous operation of multi-illumination excitation light paths and fluorescence imaging light paths, which correspond to fluorescence signals in a plurality of illumination modes.