Scanning Microscope PSF Determination via Self-Calibration
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Solution Overview
Problem
Current methods for determining point spread functions in scanning microscopes are limited by requiring extensive sample preparation and are prone to errors due to manufacturing fluctuations and environmental factors, leading to inaccurate quantitative analysis of sample volumes.
Innovation Solution
A method and scanning microscope system that uses sub-Airy scanning with detector elements closer than a diffraction disc to record multiple sample images, calculating illumination and detection point spread functions, which are shifted based on scanning movement, allowing precise determination of point spread functions without additional sample measurements or preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If reference measurement with beads is used to determine PSF, then PSF can be determined with high accuracy for the objective lens, but elements related to the actual sample (immersion medium, temperature, coverslip variations) are not taken into account, leading to significant error in the actual sample examination
Solution Approach 1:
The system uses the sample itself (or a region of the sample) to determine the PSF through self-calibration. The evaluation unit calculates the PSF from the sample image data without requiring separate reference measurements, allowing the PSF to automatically account for actual sample conditions including immersion medium, temperature, and coverslip variations.
Solution Approach 2:
The invention introduces an evaluation unit that acts as an intermediary between the detector device and the image analysis. This evaluation unit performs blind deconvolution to calculate the PSF from the sample images themselves, serving as a mediator that adapts the PSF determination to actual sample conditions rather than relying on theoretical or reference-based PSFs.
2Ease of operation
If PSF is determined from reference measurement or calculation, then the process is simple, but the sample volume examined can deviate from calculated volume by a factor of two or more, requiring extensive sample preparation
Solution Approach 1:
The system performs self-calibration by determining the PSF from the sample images themselves. The evaluation unit calculates the PSF that actually applies to the sample examination, eliminating the need for separate reference measurements with beads and the associated extensive sample preparation while achieving accurate sample volume determination.
Solution Approach 2:
The invention extracts the PSF information directly from the sample images through blind deconvolution, rather than relying on external reference measurements. This extraction approach removes the need for additional sample preparation steps while providing PSF values that accurately reflect the actual sample conditions.
3Ease of manufacture
If theoretical PSF calculation is used, then the process is straightforward, but manufacturing variations, use variations, and environmental factors cause significant deviation from the actual PSF
Solution Approach 1:
The system performs self-calibration by determining the PSF from actual sample images rather than relying on theoretical calculations. The evaluation unit calculates the PSF that actually applies to the specific microscope setup and environmental conditions, automatically compensating for manufacturing variations, use variations, and environmental factors without requiring manual adjustment.
Solution Approach 2:
The invention implements a feedback mechanism where the PSF is determined from actual sample images and then used to improve the accuracy of sample analysis. The evaluation unit continuously refines the PSF calculation based on the actual imaging conditions, creating a feedback loop that adapts to real-world variations in the microscope system and environment.
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 provides precise calculation of point spread functions, reducing user effort and eliminating the need for extensive sample preparation, resulting in more accurate and quantitative analysis of sample volumes with improved image quality and resolution.
Implementation Method 1
a scanning device (104) for generating a scanning movement of the at least one illumination beam (108, 118) over a sample (102)
Implementation Method 2
a detector device (112, 122) for recording at least one sample image with a plurality of detector elements during scanning by the illumination beam (108, 118)
Implementation Method 3
calculating, by electronic control and evaluation means (128), the point spread function from the at least one sample image
Data Source
AI summary
The invention relates to a method for operating a scanning microscope and for determining point spread functions with which sample images are recorded by the scanning microscope. In such a method it is provided that a sample is scanned by at least one illumination light beam, that at least one sample image is recorded by a detector device of the scanning microscope during the scanning by the illumination light beam, and that the point spread function with which a sample image is recorded by the scanning microscope is calculated from the at least one sample image. Use is made of a detector device having receiving elements, the distance between which is smaller than a diffraction disk that generates a sample point on the detector device. Detector signals generated by means of the receiving elements are in each case read out for different positions of the illumination light beam on the sample, as a result of which, by means of the scanning of the sample, the detector signals read out yield a plurality of sample images. The point spread functions with respect to the different detector signals are defined here in each case by means of an illumination point spread function and a detection point spread function. With respect to all the detector signals, a matching illumination point spread function is assumed which is shifted in accordance with the scanning movement for different detector signals. In addition, with respect to all the detector signals, a matching detection point spread function is assumed which takes account of a spatial offset between the detector elements. The plurality of sample images are used to calculate the illumination point spread function and the detection point spread function, and these are used to calculate the point spread functions with respect to the different detector signals. The invention further relates to a corresponding scanning microscope.