Autofocus Apparatus for Fluorescence Microscopy
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Solution Overview
Problem
Existing autofocus methods for microscopy, particularly at high magnifications, struggle with accurately focusing on small biological samples due to strong reflections from air-glass interfaces overpowering weak reflections from glass-specimen interfaces, leading to mechanical tolerance issues and increased acquisition time.
Innovation Solution
A spatially filtered light source pattern with varying intensity maxima is used to detect reflections from the glass-specimen interface, while blocking strong reflections from the air-glass interface, enabling fast and accurate autofocus through a closed-loop control system, and optionally employing a phase plate for extended depth of field and a pre-calibrated 3D contour map for precise focus prediction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If position measurement is performed using reflection from sample interfaces, then focus position can be determined, but strong reflections from air-glass interfaces overpower weak reflections from glass-specimen interfaces
Solution Approach 1:
The patent extracts and isolates the weak reflection signal from the glass-specimen interface by spatially filtering out the strong reflection from the air-glass interface. This is achieved through a detection scheme that selectively captures reflections at specific angles and positions, separating the desired signal from the overwhelming background reflection.
Solution Approach 2:
The patent applies local quality by creating a non-uniform illumination pattern with varying intensity across different regions of the sample. This spatially varying light pattern ensures that the illumination intensity at the glass-specimen interface is optimized for detection, while the air-glass interface reflection remains suppressed through angular and spatial filtering.
2Measurement precision
If contrast based autofocus is used, then focus quality can be assessed, but time is spent at each X,Y-position and weak or missing sample signal is a problem
Solution Approach 1:
The patent replaces the mechanical scanning approach with an optical field-based solution. Instead of mechanically moving the sample or detector to assess focus, the invention uses a spatially varying light pattern and optical detection to simultaneously assess focus quality across the field of view, dramatically reducing acquisition time.
Solution Approach 2:
The patent employs periodic modulation of the illumination pattern in the optical axis direction. By varying the light pattern periodically and detecting the reflected light at different phases, the system can rapidly determine focus position without mechanical scanning, achieving fast focus assessment at each X,Y position.
3Measurement precision
If high magnification is used for thin specimen, then resolution is improved, but mechanical tolerances prevent accurate location and focusing of the specimen
Solution Approach 1:
The patent implements a feedback-based autofocus system that continuously monitors the reflection pattern from the glass-specimen interface and adjusts the focus position accordingly. This closed-loop feedback mechanism compensates for mechanical tolerances and positioning errors, enabling accurate focusing of thin specimens at high magnification despite mechanical imperfections.
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 allows for rapid, robust, and precise autofocus with millisecond reaction times, reducing the need for multiple Z-axis acquisitions and enhancing image quality by isolating weak reflections from the glass-specimen interface, thus improving the speed and reliability of image acquisition.
Implementation Method 1
the light being focused on a sample by an objective generating a light pattern in the sample
Implementation Method 2
reflection of the light pattern is detected spatially resolved by a detector (generating at least two signals)
Data Source
Figure 1~2
Figure 3~4
Figure 5
AI summary
The present invention applies to an autofocus apparatus (2) and methods which achieves a higher level of speed and robustness, and are particularly suited for fluorescence microscopy of biological samples (6), automated microscopy and scanning microscopy. High speed is achieved via a light pattern (62) in the sample (6), detected spatially resolved by a detector (36) generating at least two signals corresponding to a reflex pattern of the light pattern (62). The two signals are subtracted generating a positioning signal (80) and the focus of the objective (30) in the sample (6) is adjusted depending on the positioning signal (80).