Fluorescence Localization Microscopy High-Intensity Illumination
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Solution Overview
Problem
Current localization microscopy techniques are limited in precision and speed, particularly for in-vivo observations, due to the diffraction limit and the complexity of achieving sub-resolution spatial information of fluorescent molecules in living systems.
Innovation Solution
A method utilizing high-intensity illumination (1kW/cm² to 1MW/cm²) to transfer fluorescent molecules between distinct states, allowing for efficient optical isolation and sub-resolution imaging, enabling the use of conventional fluorescent labels and combining with structured illumination for enhanced axial resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence localization microscopy is used, then fluorescent molecules can be detected, but the localization precision is limited by the diffraction limit to about 200 nm laterally and 600 nm axially
Solution Approach 1:
The patent changes the illumination intensity parameter to extremely high values (1 kW/cm² to 1 MW/cm²), which fundamentally alters the interaction with fluorescent molecules. This high-intensity illumination enables the transfer of molecules between distinct states, allowing optical isolation and sub-resolution imaging beyond the diffraction limit without requiring complex additional optical components.
Solution Approach 2:
The patent utilizes periodic switching between distinct states of fluorescent molecules through high-intensity illumination. By cycling molecules between fluorescent and non-fluorescent states, the method achieves temporal separation of signals, enabling precise localization without the need for complex spatial filtering or multiple objective lenses.
2Measurement precision
If high-intensity illumination (1kW/cm² to 1MW/cm²) is used to transfer fluorescent molecules between states, then sub-resolution localization precision is achieved, but the illumination intensity requirement becomes extremely high
Solution Approach 1:
The patent creates an optical copy of the molecular position through the transfer of molecules between states. By detecting the state transitions induced by high-intensity illumination, the system reconstructs sub-resolution positional information without requiring the illumination itself to be resolved at that precision, thus achieving localization precision without proportionally increasing optical resolution requirements.
Solution Approach 2:
The patent fundamentally changes the illumination parameter to extreme intensity values (1 kW/cm² to 1 MW/cm²), which enables non-linear optical effects and state transfers in fluorescent molecules. This parameter change allows the system to overcome the diffraction limit by utilizing the quantum states of molecules rather than relying on classical optical resolution.
3Adaptability or versatility
If conventional fluorescence microscopy is used, then multiple types of labeled molecules can be identified in intact cells, but the spatial resolution is insufficient to resolve cellular nanostructures
Solution Approach 1:
The patent segments the detection process into discrete temporal phases using high-intensity illumination cycles. By separating molecular signals in time through state transitions, the method resolves spatially overlapping fluorescent molecules that would normally be indistinguishable, while maintaining the ability to identify multiple molecule types through their spectral signatures.
Solution Approach 2:
The patent employs periodic high-intensity illumination to cycle fluorescent molecules between states, creating temporal separation of signals from different molecular types. This periodic action allows multiple labeled molecules to be resolved spatially while maintaining their spectral identification capabilities, thus achieving both high resolution and multi-type identification.
4Measurement precision
If localization microscopy is performed to obtain sub-resolution spatial information, then the speed of observation is reduced, but the precision is improved
Solution Approach 1:
The patent uses periodic high-intensity illumination to rapidly cycle fluorescent molecules between states, enabling fast temporal separation of signals. This periodic action allows sub-resolution localization to be achieved at high speeds by utilizing the rapid state transitions rather than requiring slow scanning or sequential observation, thus improving both precision and productivity.
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 achieves sub-resolution spatial information with improved localization precision and speed, allowing for in-vivo observations of small structures and overcoming the diffraction limit, with axial resolution enhanced by a factor of 30 compared to conventional methods.
Implementation Method 1
The first state is a fluorescent state. Upon illumination with illumination light having intensity in the range of approximately 1kW/cm2, a portion of the fluorescent molecules is transferred from the first state to a second state.
Implementation Method 2
a portion of the fluorescent molecules is transferred from the first state to a second state, which is a semi-stable, or respectively semi-long lasting dark (for example a non-fluorescent) state. In particular, the second state may be a reversibly bleached state.
Data Source
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AI summary
The invention concerns a method and a corresponding apparatus for obtaining a sub-resolution spatial information of a sample labeled with at least one type fluorescent label, said sub-resolution spatial information comprising localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction. The method comprises: - acquiring localization image data by employing fluorescence localization microscopy, wherein said localization image data comprises a series of images obtained by - illuminating a region of interest of the sample with illumination light having intensity in the range of approximately 1 kW/cm2 to approximately 1 MW/cm2, - detecting by an information acquiring sensor 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, thereby obtaining an image of the region of interest; - repeating the steps of illuminating and detecting of the emitted fluorescent light a plurality of times, thereby obtaining the series of images, each image being taken at a different time step; and - processing the acquired localization image data to thereby obtain said localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction, wherein the step of processing comprises 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.