Microscope Calibration Targets for Sub-Diffraction Imaging
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Solution Overview
Problem
Conventional optical microscopy is limited by the diffraction limit, which restricts resolution to around 200 nm, making it challenging to image samples below this threshold, especially for live cells that require longer data collection times, potentially exposing them to harmful radiation and experimental constraints.
Innovation Solution
An optical microscope calibration device with features below the diffraction limit is used, allowing for image processing to combine separate color component images and adjust parameters to achieve focused, non-distorted images of samples at resolutions below the diffraction limit, overcoming chromatic aberration and diffraction limitations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If super-resolution techniques are used to image below the diffraction limit, then measurement precision is improved, but loss of time increases significantly due to the need to accumulate weak signals over long periods
Solution Approach 1:
The patent applies preliminary action by using a calibration target with known sub-diffraction features to pre-determine microscope parameters (magnification, focal plane, chromatic aberration) before actual sample imaging. This calibration step allows the system to achieve super-resolution capability without requiring long signal accumulation times during actual sample imaging, as the parameter optimization is performed in advance on the calibration target.
2Measurement precision
If super-resolution techniques are used to image below the diffraction limit, then measurement precision is improved, but object-affected harmful factors increase due to prolonged exposure to electromagnetic radiation
Solution Approach 1:
The calibration process is performed in advance on a non-living calibration target, separating the parameter optimization step from the actual sample imaging. This allows the microscope parameters to be optimized for super-resolution without exposing living samples to prolonged radiation, as the calibration is completed beforehand on a durable calibration target that can withstand repeated imaging.
3Ease of operation
If conventional optical microscopy is used, then ease of operation is maintained, but measurement precision is limited to the diffraction limit of approximately 200 nm
Solution Approach 1:
The patent introduces a calibration target as an intermediary element that bridges conventional and super-resolution imaging. The calibration target with known sub-diffraction features serves as a mediator to determine optimal imaging parameters, allowing the system to achieve super-resolution capability while maintaining operational simplicity through automated parameter application to subsequent sample images.
4Measurement precision
If live cells are imaged over long periods to accumulate sufficient signal, then measurement precision is improved, but reliability decreases due to cell movement and shape changes
Solution Approach 1:
The calibration parameters are determined in advance on a stable calibration target before imaging living cells. This preliminary calibration allows subsequent live cell imaging to use pre-optimized parameters, reducing the need for prolonged signal accumulation and minimizing the time cells are exposed to imaging conditions, thereby maintaining cell stability and reliability throughout the imaging process.
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
Enables high-resolution imaging below the diffraction limit, reducing the time required for data collection and minimizing exposure to harmful radiation, thus improving the imaging of live cells and other samples.
Implementation Method 1
overcoming chromatic aberration and diffraction limitations
Implementation Method 2
features with dimensions below the diffraction limit of a microscope objective
Data Source
AI summary
This disclosure is directed to optical microscope calibration devices that can be used with optical microscopes to adjust the microscope imaging parameters so that images of samples can be obtained below the diffraction limit. The microscope calibration devices include at least one calibration target. Each calibration target includes a number of features with dimensions below the diffraction limit of a microscope objective. Separate color component diffraction limited images of one of the calibration targets are obtained for a particular magnification. The color component images can be combined and image processed to obtain a focused and non-distorted image of the calibration target. The parameters used to obtain the focused and non-distorted image of the calibration target can be used to obtain focused and non-distorted images of a sample for the same magnification by using the same parameters.