Autofocus via Spatially Resolved Light Pattern Reflections
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Solution Overview
Problem
Existing autofocus methods for microscopy are inadequate for high magnification and small depth of field applications, as they struggle to accurately locate and focus on specimens due to mechanical tolerances and require excessive time, especially when dealing with weak or missing sample signals.
Innovation Solution
A method and apparatus that emit a light pattern varying along the optical axis, detect spatially resolved reflections, and adjust the focus based on positioning signals to accurately measure the Z-position of the sample, rejecting strong reflections from air-glass interfaces and enhancing the detection of weak reflections from glass-specimen interfaces, while optionally using a phase plate to extend the depth of field and a pre-calibrated 3D contour map for prediction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If position measurement using reflection from sample interfaces is used, then the position of the specimen can be located, but the strong reflection from air-glass interfaces overwhelms the weak reflections from glass-specimen interfaces
Solution Approach 1:
The detection of reflected light is segmented into multiple spatially resolved components. The detector array captures reflections from different interfaces (air-glass, glass-specimen) at different positions, allowing selective processing of signals from the glass-specimen interface while rejecting stronger air-glass reflections.
Solution Approach 2:
The evaluation function applies different weighting factors to different spatial components of the reflected light. By assigning higher weights to signals from the glass-specimen interface region and lower weights to air-glass interface regions, the system enhances local quality of the measurement while suppressing harmful reflections.
2Measurement precision
If contrast based autofocus is used, then focus quality can be assessed, but excessive time is spent at each X,Y-position and weak or missing sample signal becomes a problem
Solution Approach 1:
The system replaces mechanical scanning autofocus methods with an optical field-based evaluation. By using spatially resolved detection of reflected light patterns and computing an evaluation function, the system achieves focus assessment without mechanical movement, significantly reducing measurement time.
Solution Approach 2:
The system performs preliminary spatial resolution of the reflected light pattern to identify characteristic features before computing the final evaluation function. This preliminary action allows for faster processing and reduces the time required for focus assessment at each position.
3Manufacturing precision
If high magnification microscopy is used, then detailed imaging is achieved, but the depth of field becomes very small making focus difficult
Solution Approach 1:
The system transitions from two-dimensional image plane analysis to three-dimensional spatial analysis of reflected light patterns. By detecting the axial position information encoded in the reflected light pattern and using an evaluation function that incorporates depth information, the system achieves precise focus control despite the limited depth of field at high magnification.
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 fast, accurate, and robust autofocus with millisecond reaction times, reducing the need for multiple Z-axis acquisitions and improving robustness by combining reflection and content-based focusing methods, allowing reliable detection of the glass-specimen interface and maintaining focus even in the presence of disturbances.
Implementation Method 1
focusing the light on a sample via an objective resulting in a generation of a light pattern in the sample
Implementation Method 2
detecting a reflection of the light pattern spatially resolved via a detector
Data Source
AI summary
An autofocus apparatus and a method achieve a higher level of speed and robustness, and are particularly suited for fluorescence microscopy of biological samples, automated microscopy and scanning microscopy. A high speed is achieved via a light pattern in the sample, detected spatially resolved by a detector generating at least two signals corresponding to a reflex pattern of the light pattern. The two signals are subtracted generating a positioning signal and the focus of the objective in the sample is adjusted depending on the positioning signal.