3D Localization Microscopy Using Spatial Light Modulator
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Solution Overview
Problem
Current 3D localization microscopy techniques face limitations in achieving high-resolution three-dimensional localization of fluorescence emitters due to inaccuracies in determining the z-coordinate, which restricts their ability to provide accurate depth information and requires extensive data processing and multiple image recordings.
Innovation Solution
A method utilizing a localization uncertainty table that accounts for the z-coordinate and the number of photons collected, allowing for accurate determination of localization uncertainty, either experimentally or through simulation, to enhance the accuracy of three-dimensional location information beyond the spatial resolution, thereby overcoming previous limitations in two-dimensional analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional wide-field illumination is used for 3D localization microscopy, then the method is simple and accessible, but the localization accuracy in the z-direction deteriorates due to insufficient isolation of fluorescent emitters
Solution Approach 1:
The patent segments the wide-field illumination into multiple focal planes using a spatial light modulator. By displaying different phase patterns on the SLM, the illumination is divided into distinct focal depths, allowing selective excitation of fluorescent emitters at different z-positions. This segmentation enables accurate 3D localization while maintaining the simplicity of wide-field microscopy.
Solution Approach 2:
The patent introduces a fourth dimension (temporal dimension) to the traditional wide-field illumination by modulating the phase patterns over time. The spatial light modulator dynamically changes the illumination profile across multiple time points, effectively adding a temporal dimension that enables 3D localization capability to the conventional 2D wide-field system.
2Measurement precision
If statistical effects are used to isolate individual labeling molecules, then localization accuracy improves, but the number of images required increases significantly
Solution Approach 1:
The patent performs preliminary action by pre-calculating and storing optimal phase patterns in a lookup table that correspond to different z-positions. Instead of using statistical isolation methods that require many images, the system proactively prepares the illumination patterns in advance, allowing direct addressing of specific focal planes and reducing the number of images needed for 3D localization.
Solution Approach 2:
The spatial light modulator acts as an intermediary between the light source and the sample, enabling precise control over the illumination focal plane. This intermediary device translates electrical control signals into spatially and temporally modulated light patterns, allowing selective excitation at different depths without requiring statistical isolation methods.
3Loss of information
If the focus is shifted to image fluorescent emitters at different depths, then three-dimensional information is captured, but the number of photons collected per emitter decreases
Solution Approach 1:
The patent employs periodic action by sequentially cycling through different phase patterns that correspond to different focal planes. The spatial light modulator periodically switches between pre-calculated phase patterns, allowing the system to scan through multiple z-positions and capture 3D information while maintaining sufficient photon collection at each focal plane through optimized illumination.
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 precise three-dimensional localization of fluorescence emitters with improved accuracy, reducing the need for extensive data processing and multiple image recordings, and provides a comprehensive uncertainty analysis for enhanced imaging capabilities.
Implementation Method 1
the fluorescent emitters in the sample are excited to emit fluorescence radiation
Data Source
Figure 1~2
Figure 3~4
Figure 5
AI summary
The method involves providing a sample (2) containing a fluorescence emitter. The fluorescence emitter in the sample is arranged for delivery of excited fluorescence radiation, and the sample is imaged with a spatial resolution in a wide field (4). A localization uncertainty table is provided along a Z-coordinate. A set of photons is collected during imaging in the wide field. A local data uncertainty is determined for the localization analysis so as to provide specific location information. An independent claim is also included for a high resolution microscope.