Fluorescence Localization Microscopy Axial Resolution
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Solution Overview
Problem
Conventional light microscopy is limited by the diffraction limit, making it difficult to achieve sub-resolution spatial information and precise localization of fluorescent molecules, especially in living cells, as it struggles to overcome the Abbe limit of 200 nm laterally and 600 nm axially, which hinders the analysis of cellular nanostructures and active processes.
Innovation Solution
The method employs high-intensity illumination in the range of 1 kW/cm2 to 1 MW/cm2 to transfer fluorescent molecules between distinct states, allowing for efficient optical isolation and localization of single molecules, enabling sub-resolution spatial information acquisition by determining the barycenters of fluorescence emission distributions, and combining this with structured illumination for enhanced axial resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional light microscopy is used to observe fluorescent molecules, then the imaging process is non-invasive and suitable for living cells, but the spatial resolution is limited to about 200 nm laterally and 600 nm axially due to the diffraction limit
Solution Approach 1:
The patent segments the observation process into two distinct phases: a low-intensity scanning phase for navigation and a high-intensity localization phase for precise molecular positioning. This segmentation allows the system to achieve super-resolution (improving measurement precision) while limiting the duration and intensity of high-power illumination, thereby reducing photodamage to living cells.
Solution Approach 2:
The patent employs periodic switching between different illumination intensities and states. Fluorescent molecules are activated periodically, allowing them to emit photons that are detected and localized. This periodic activation pattern enables precise spatial measurement while controlling the total light exposure to living cells, balancing resolution improvement with harm reduction.
2Measurement precision
If high-intensity illumination is used to transfer fluorescent molecules between states for localization, then sub-resolution spatial information can be obtained, but the risk of photodamage and photobleaching increases
Solution Approach 1:
The patent applies preliminary low-intensity illumination to activate fluorescent molecules into a detectable state before applying high-intensity localization illumination. This preliminary activation ensures that molecules are in the appropriate fluorescent state ready for precise localization, while minimizing the duration of high-intensity exposure that would cause photobleaching, thus improving localization precision while maintaining photostability.
Solution Approach 2:
The system uses feedback from detected photon emissions to control the illumination intensity and duration. When fluorescent molecules are detected and localized, the system adjusts subsequent illumination to maintain the molecules in a stable fluorescent state, preventing over-illumination that would cause photobleaching. This feedback mechanism enables precise localization while preserving the reliability of fluorescent labels.
3Productivity
If fast data collection is implemented for in-vivo observations, then dynamic cellular processes can be captured, but the temporal resolution may compromise spatial precision
Solution Approach 1:
The patent applies partial illumination strategies where only specific regions of interest are illuminated with high intensity at any given time, rather than illuminating the entire field of view. This allows fast data collection from localized areas (improving productivity) while maintaining the photon statistics necessary for precise localization (maintaining measurement precision). The system collects data from multiple partial views to reconstruct the complete image.
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 allows for sub-resolution localization with improved axial resolution by a factor of 30 compared to conventional methods, enabling fast data collection within minutes, suitable for in-vivo observations, and utilizing conventional fluorescent labels, including proteins and non-protein based dyes, for precise determination of molecular positions and distances.
Implementation Method 1
The method employs high-intensity illumination in the range of 1 kW/cm2 to 1 MW/cm2 to transfer fluorescent molecules between distinct states
Implementation Method 2
detecting by an information acquiring sensor of at least a portion of the fluorescent light emitted by at least a portion of the fluorescent molecules of the at least one type fluorescent label upon illumination
Data Source
AI summary
A method and apparatus are provided for obtaining a sub-resolution spatial information of a sample labeled with at least one type fluorescent label. The sub-resolution spatial information has localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction. The method acquires localization image data by employing fluorescence localization microscopy. The acquired localization image data is processed to obtain the localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction. The step of processing includes determining in each of the detected images of the series the positions of the barycenters of the detected fluorescence emission distributions from the single fluorescent molecules of the one or more fluorescent labels in at least one spatial direction.