Microscope Field Inhomogeneity Mapping by Lateral Position Shifts
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Solution Overview
Problem
Modern microscopes face challenges in correcting field inhomogeneities due to vignetting and field obstacles, which existing methods struggle to accurately model and remove, especially when using large field of views and aberration-corrected objectives, leading to issues like non-linear blending and inefficient averaging strategies.
Innovation Solution
A method and microscope configuration that involves setting the mechanical drive to multiple lateral positions, collecting measurement data, and using an iterative model to explicitly extract field inhomogeneity independent of lateral position, allowing precise determination of illumination inhomogeneity without a priori assumptions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If prospective correction strategies using calibration targets are used to measure shading, then illumination inhomogeneity can be measured, but specimen-induced shading effects cannot be corrected and calibration targets degrade over time
Solution Approach 1:
The method performs preliminary measurement of illumination inhomogeneity by acquiring images of a uniform fluorescent sample at multiple lateral positions before correcting the actual specimen images. This preliminary action captures the illumination profile under the same optical conditions that will be used for the specimen, enabling accurate correction of specimen-induced shading effects.
Solution Approach 2:
The system uses the microscope's own imaging capabilities and the specimen's fluorescence to measure illumination inhomogeneity, rather than requiring external calibration targets. The uniform fluorescent sample serves as both the measurement medium and the reference for correction, making the system self-sufficient and eliminating target degradation issues.
2Ease of operation
If nonlinear blending is used to mitigate shading between adjacent tiles, then perceptionally appealing images are achieved, but linearity between fluorescence channels is broken
Solution Approach 1:
The illumination inhomogeneity profile is measured in advance using a uniform fluorescent sample, creating a correction map that is then applied to all subsequent images. This preliminary measurement approach allows for perceptually appealing shading correction while maintaining the linear relationship between fluorescence channels, because the correction is based on a multiplicative factor derived from the illumination profile rather than nonlinear blending.
3Measurement precision
If averaging many tiles is used to estimate field inhomogeneity, then statistical convergence is achieved, but hundreds of samples are required and the method is inefficient
Solution Approach 1:
The method extracts the illumination inhomogeneity profile directly from images of a uniform fluorescent sample by identifying regions with constant fluorescence intensity. By taking out the illumination information from the uniform sample images, the system obtains an accurate field inhomogeneity estimate without requiring statistical averaging of multiple specimen tiles, thus dramatically reducing the number of samples needed.
4Area of stationary object
If large field of views are used to increase space-bandwidth product, then field of view size increases, but field inhomogeneity and vignetting become more pronounced
Solution Approach 1:
The system performs preliminary measurement of the illumination profile across the entire large field of view using a uniform fluorescent sample. This pre-acquired illumination map captures the spatial variation of illumination intensity, including vignetting effects at the edges. The stored illumination profile is then used to correct all subsequent images, enabling the use of large field of views while maintaining illumination uniformity through computational correction.
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
Accurately captures vignetting and other inhomogeneities with high precision, applicable to any sample, and can be performed separately from sample investigation, improving image quality by removing shading artefacts.
Implementation Method 1
a light source 10, e.g., laser, for supplying excitation light 12... illuminating a sample through an illumination beam path of the microscope with excitation light... guiding emission light emitted by the sample through a detection beam path
Data Source
AI summary
A microscope comprising 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 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 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.