Microscope Field Inhomogeneity Correction by Lateral Scanning
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Solution Overview
Problem
Modern microscopes face challenges in accurately correcting field inhomogeneities such as vignetting and specimen-induced shading effects, which are not adequately addressed by existing prospective and retrospective methods, leading to inefficiencies in image processing and data integrity.
Innovation Solution
A method and microscope configuration that involves setting the mechanical drive to multiple lateral positions, collecting measurement data, and using an iterative double-blind estimation to model and correct field inhomogeneities independently of the lateral position, allowing for precise extraction of both illumination and sample information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If prospective correction methods using calibration targets are used to measure illumination inhomogeneity, then field inhomogeneity correction is achieved, but specimen-induced shading effects cannot be corrected and additional costs and complexity are introduced
Solution Approach 1:
The system uses the specimen itself to perform the calibration function. By acquiring multiple images at different lateral positions and using the specimen's own signal variations to determine the illumination profile, the method eliminates the need for separate calibration targets. The specimen serves dual purposes: as the object of study and as the calibration reference.
Solution Approach 2:
The illumination profile is determined in advance through a systematic scanning process that captures images at multiple lateral positions. This preliminary measurement of the illumination field allows for subsequent correction of both illumination inhomogeneity and specimen-induced shading effects without requiring additional calibration steps for each specimen.
2Measurement precision
If retrospective methods with nonlinear blending are used to mitigate shading between tiles, then perceptional image quality is improved, but linearity between fluorescence channels is broken
Solution Approach 1:
The method changes the approach from nonlinear pixel blending to linear correction by modifying the illumination profile parameter. Instead of applying nonlinear weights to pixels, the solution transforms the illumination map into a linear correction factor that can be applied multiplicatively to maintain linearity across fluorescence channels while still achieving effective shading correction.
3Measurement precision
If calibration targets are used for shading correction, then field inhomogeneity is measured, but calibration targets degrade over time and cause additional costs
Solution Approach 1:
The system eliminates dependence on external calibration targets by using the specimen itself for calibration. The specimen's inherent signal variations across different lateral positions provide the necessary information to determine the illumination profile, making the calibration process self-contained and eliminating degradation issues associated with physical calibration targets.
Solution Approach 2:
Instead of using a physical calibration target that can degrade, the method creates a virtual calibration reference by capturing and processing images of the specimen at multiple positions. This digital copy of the illumination field replaces the need for physical calibration artifacts.
4Manufacturing precision
If immersion objectives are used to achieve high resolution, then spatial resolution is improved, but specimen-induced shading effects cannot be captured during calibration
Solution Approach 1:
The method performs a preliminary systematic scanning measurement that captures the complete illumination field including specimen-induced shading effects before actual imaging. By measuring the illumination profile at multiple lateral positions in advance, the system accounts for meniscus effects and other specimen-induced variations that would otherwise be missed during standard calibration with immersion objectives.
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 high-accuracy correction of field inhomogeneities without requiring specific test samples, reduces reliance on a priori assumptions, and maintains linearity across fluorescence channels, enhancing image quality and data integrity.
Implementation Method 1
a light source for supplying excitation light, an illumination beam path for guiding the excitation light to a sample space
Implementation Method 2
a detector for detecting emission light emitted by a sample in the sample space
Data Source
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AI summary
The present invention is concerned with a microscope comprising light source, an illumination beam path for guiding excitation light to a sample, a detector for detecting emission light emitted by a sample, a detection beam path comprising a microscope objective for guiding the emission light to the detector, a mechanical drive for setting a relative lateral position between the sample and the microscope objective, and a control unit. The microscope is characterized in that the control unit is configured for carrying out a setting step wherein the mechanical drive is sequentially set to at least three different relative lateral positions, a collecting step wherein measurement data are collected from the detector at least for a subset of points in the sample in a field of view of the detection beam path, wherein for each of the points of the subset measurement data are collected for at least two different lateral positions of the mechanical drive, and an evaluation step wherein a field inhomogeneity in the field of view and microscopic sample information extracted from the measurement data. In further aspects, the invention relates to a method for determining a field inhomogeneity in a field of view of a microscope and a microscopy method.