Autofocus with Variable Angle Illumination for Microscopy
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Solution Overview
Problem
Current autofocus techniques in microscopy face limitations in accuracy and reliability due to interference structures and weakly contrasting samples, which hinder precise Z-positioning of the sample stage.
Innovation Solution
A method that involves acquiring images under multiple angle-variable illumination geometries, separating the measurement object from interference structures using a priori knowledge, and determining object displacement to adjust the Z-position of the sample stage for precise focusing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If angle-variable illumination is used for autofocus, then Z-positioning capability is provided, but reliability and accuracy are limited due to interfering structures and low-contrast samples
Solution Approach 1:
The image is segmented into multiple components: the measurement object, interfering structures, and background. This is achieved by capturing images at multiple illumination angles and using the fact that the measurement object exhibits angle-dependent displacement while interfering structures remain relatively static. The segmentation allows selective analysis of object displacement without interference from other structures.
Solution Approach 2:
The illumination angle is varied as a parameter to induce differential displacement between the measurement object and interfering structures. By changing the illumination angle, the measurement object's image position changes predictably, while interfering structures remain stationary or change minimally, enabling reliable separation and accurate displacement measurement.
2Measurement precision
If traditional autofocus with angle-variable illumination is used, then Z-positioning is achieved, but accuracy deteriorates due to interfering structures captured alongside the measurement object
Solution Approach 1:
The measurement object's signal is extracted from the composite image by analyzing its unique response to angle-variable illumination. The object's image displacement pattern with respect to illumination angle changes is distinct from interfering structures, allowing selective extraction of object-related information while eliminating interference from other structures.
Solution Approach 2:
The illumination angle serves as an intermediary parameter that differentiates between the measurement object and interfering structures. By using angle-variable illumination as a mediator, the system can selectively enhance the measurement object's signal while suppressing interfering structures, improving defocus position determination accuracy.
3Measurement precision
If autofocus is performed on low-contrast samples with little absorption and phase shift, then Z-positioning is attempted, but signal separation becomes insufficient preventing accurate object displacement detection
Solution Approach 1:
The system introduces dynamic angle-variable illumination to create temporal and spatial variations in the image signal. Even low-contrast samples exhibit dynamic displacement patterns when illuminated from different angles, transforming a static low-contrast problem into a dynamic high-contrast problem that can be reliably detected and measured.
Data Source
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AI summary
Several aspects of the invention relate to techniques for enabling autofocus techniques for a measurement object (499) using angle-variable illumination. Images are captured under a variety of angle-variable illumination geometries (301, 302). The captured images are then evaluated to determine the Z-position.