Structured Illumination Imaging Calibration Without Emission Pinhole
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Solution Overview
Problem
Conventional fluorescence imaging systems face challenges with photobleaching and the need for complex synchronization in multi-point confocal systems, as well as the requirement of a pinhole mask in the emission path, which can lead to artifacts and reduced image quality.
Innovation Solution
A fluorescence imaging system that calibrates and captures images without a pinhole mask in the emission path by using a pinhole mask in the excitation path, identifying pinhole locations, and adjusting capture settings to compensate for photobleaching, allowing for the generation of composite confocal images with improved resolution and reduced artifacts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a pinhole mask is used in the emission path of a confocal imaging system, then optical resolution and signal-to-noise ratio are improved, but the system complexity increases and artifacts are introduced
Solution Approach 1:
The patent removes the pinhole mask from the emission path, extracting the problematic component that caused system complexity and artifacts. Instead, it uses a pinhole mask only in the excitation path combined with structured illumination patterns to achieve confocal imaging without the emission path pinhole, thereby simplifying the system while maintaining resolution.
Solution Approach 2:
The patent inverts the conventional confocal approach by placing the pinhole mask in the excitation path rather than the emission path. This inversion, combined with structured illumination, allows the system to achieve optical sectioning and improved resolution without requiring a pinhole in the emission path, thus reducing system complexity and artifacts.
2Measurement precision
If multiple images are captured at discrete intervals to construct a composite image, then optical resolution and signal-to-noise ratio are improved, but the capture time increases and photobleaching artifacts worsen
Solution Approach 1:
The patent uses periodic structured illumination patterns that sweep across the sample in a systematic sequence. By capturing images at discrete intervals during this periodic sweep and then computationally reconstructing the full image, the system achieves high signal-to-noise ratio and optical resolution while reducing total capture time compared to traditional point-by-point scanning.
Solution Approach 2:
The patent performs preliminary structured illumination sweeps across the entire sample before final image reconstruction. This preliminary action allows the system to gather all necessary data efficiently, reducing the need for repeated scanning and thereby minimizing photobleaching while maintaining high signal-to-noise ratio in the final composite image.
3Measurement precision
If the excitation light is swept over the sample to illuminate only a portion at a time, then optical resolution is improved, but photobleaching occurs in illuminated areas
Solution Approach 1:
The patent uses structured illumination patterns that illuminate only specific portions of the sample at any given time, rather than illuminating the entire sample. By carefully designing these partial illumination patterns and sweeping them systematically, the system achieves high optical resolution while minimizing the total amount of excitation light exposure, thereby reducing photobleaching in illuminated areas.
4Device complexity
If a pinhole mask is used in the excitation path with structured illumination, then the need for an emission path pinhole is eliminated, but calibration complexity increases
Solution Approach 1:
The patent implements a self-calibration mechanism where the system automatically determines the relative positions of the pinhole mask and sensor using the captured structured illumination patterns. This self-service calibration process eliminates the need for complex manual alignment procedures, making the system easier to manufacture and deploy while maintaining the simplification of removing the emission path pinhole.
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 faster image capture with reduced photobleaching artifacts and eliminates the need for complex synchronization, resulting in higher quality images with improved signal-to-noise ratio and optical resolution.
Implementation Method 1
A fluorescence imaging system calibrates and captures images without a pinhole mask in the emission path by using a pinhole mask in the excitation path
Implementation Method 2
A basic widefield fluorescence microscope that is well known in the art includes a light source and several filters that correspond to a wavelength matching a fluorescence stain
Implementation Method 3
capturing an image of the sample using a sensor
Data Source
AI summary
A method for calibrating an imaging system can include at least the following method acts: illuminating a sample through a pinhole mask using an excitation light; capturing an image of the sample using a sensor; converting the image into data; in a processing module: filtering the data using a known spacing of pinholes in the pinhole mask to obtain filtered data that corresponds to the known spacing, using a threshold to identify regions of the filtered data that are bright enough to be associated with a pinhole, calculating the centroids of the regions, and fitting a known pattern for the pinhole mask to the regions in order to identify the best fit data for the filtered data; and storing, in a storage medium, the best fit data for use in a subsequent confocal capture routine.