Microscopy Focal Positioning Using Off-Axis Laser Reflection
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Solution Overview
Problem
High-content screening microscopy imaging systems face challenges in accurately determining the focal position of the objective due to variations in sample holder thickness and curvature, leading to inaccurate focus adjustments and increased processing time, especially when dealing with multiple reflections from both the upper and lower surfaces of the sample holder.
Innovation Solution
A method and system that condition the upper surface of the sample holder with a reflective coating to enhance the detection of reflected laser beams, allowing the microscopy imaging system to determine the focal position based on the intensity or position of reflected light beams, thereby improving focus accuracy and reducing the complexity of the focal position determination process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If laser beams are used to automatically adjust the objective position by detecting reflections from the sample holder, then focus adjustment automation is improved, but multiple reflections from upper and lower surfaces cause measurement precision to deteriorate
Solution Approach 1:
The patent extracts the harmful lower surface reflection from the detection system by using an off-axis laser beam geometry. The laser beam is directed at an angle such that only the upper surface reflection enters the detection path, while the lower surface reflection is excluded. This selective extraction of the desired reflection signal resolves the contradiction by maintaining automation while eliminating the source of measurement error.
Solution Approach 2:
The patent introduces an off-axis beam geometry as an intermediary mechanism to differentiate between upper and lower surface reflections. By using a detection path that is offset from the laser source axis, the system creates a geometric filter that allows only specific reflections to reach the detector, thereby enabling precise focal position measurement while maintaining automated operation.
2Speed
If the objective is offset from the focal position to compensate for sample holder thickness, then focus adjustment speed is improved, but focus accuracy deteriorates due to thickness variations
Solution Approach 1:
The patent implements a feedback mechanism where the actual focal position is continuously measured by detecting the upper surface reflection. The system uses this measurement information to dynamically adjust the objective position, replacing fixed offset compensation with adaptive feedback control. This resolves the contradiction by maintaining fast adjustment through automation while achieving high accuracy through real-time measurement and correction.
Solution Approach 2:
The patent performs preliminary measurement of the upper surface position using laser reflection detection before final image acquisition. This preliminary action provides accurate focal position information that guides subsequent objective positioning, enabling both rapid adjustment and high precision by preparing the system in advance with accurate positional data.
3Productivity
If multiple laser beams are used to scan across the sample holder, then productivity is improved, but device complexity increases due to need to distinguish multiple reflections
Solution Approach 1:
The patent applies local quality by making each measurement location have a distinctive geometric relationship with the laser source and detector. The off-axis geometry creates unique reflection paths for each well position, allowing the system to distinguish reflections from different locations without additional complexity. Each location's local geometric configuration serves as its identifier, enabling high throughput while maintaining simple reflection discrimination.
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
The solution enables more accurate and efficient determination of the focal position, reducing errors and processing time by clearly distinguishing reflections from the upper and lower surfaces, resulting in sharper images across multiple measurement locations.
Implementation Method 1
A reflected light beam resulting from reflection of the focusing light beam off the conditioned upper surface is observed
Data Source
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Figure 2A~2B
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AI summary
The invention relates to a method of determining a focal position for an objective (702) positioned at a measurement location of a sample holder (704, 718) in a microscopy imaging system (100), the method comprising: moving the objective (702) to a position corresponding to a distance between the objective and the sample holder (704, 718), wherein the position of the objective is a z-position along a Z-axis relative to the sample holder; projecting a focusing light beam (700) onto the sample holder (704, 718) when the objective (702) is located at the position, wherein the objective focuses the focusing light beam on the sample holder; observing a reflected light beam resulting from reflection of the focusing light beam off an upper surface (706, 720) of the sample holder (704, 718); determining the focal position for the objective (702) based on the reflected light beam such that the objective produces an in focus image of a microscopy sample residing at the measurement location when the objective is located at the focal position; directing the reflected light beam (710, 712, 714; 724, 726, 728) onto a linear detector (124) such that the reflected light beam strikes a location on the linear detector; comparing the location on the linear detector (124) to a recorded linear detector location, the recorded linear detector location corresponding to a focused z-position that results in an in focus image when the objective (702) is located at the focused z-position; determining whether the location on the linear detector (124) has shifted away from the recorded linear detector location; and in response to a determination that the location on the linear detector (124) has shifted away from the recorded linear detector location, determining a new z-position for the objective based on the comparison of the location on the linear detector to the recorded linear detector location and identifying the new z-position as the focal position for the objective (702).