Vacuum Well Plate Holder for Uniform Focal Plane Imaging
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Solution Overview
Problem
Current methods for imaging biological samples in well plates are inefficient due to the need for frequent camera refocusing and inadequate illumination, especially when there is insufficient contrast between cells and their surroundings, leading to prolonged processing times and potential image quality issues.
Innovation Solution
A vacuum holder system that flattens the well plate to create a common focal plane, allowing for single-camera focusing across multiple wells, combined with an illumination method that directs light onto the well base without shadows by using a beam that illuminates all parts of the base in series, ensuring even illumination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the camera refocuses for each well in a well plate, then the imaging can be performed on multiple samples, but the processing time becomes very long (over an hour per well)
Solution Approach 1:
The system performs a preliminary action by establishing a common focal plane for the entire well plate before imaging begins. The autofocus system determines a focal length that allows the camera to focus on cells across multiple wells simultaneously, eliminating the need for refocusing during the imaging process. This preliminary focal plane establishment enables rapid sequential imaging of all wells without time-consuming refocusing operations.
Solution Approach 2:
The invention changes the focusing parameter from individual well focus to a unified focal length for the entire plate. By adjusting the camera's focal length parameter once to accommodate the common focal plane of all wells, the system transforms the imaging process from multiple individual focusing operations to a single parameter setting that enables imaging of all samples efficiently.
2Extent of automation
If standard autofocus system is used for imaging wells without stains or fluorescent tags, then the system can operate automatically, but the visual contrast is insufficient for efficient optical feedback focusing
Solution Approach 1:
The invention introduces an intermediary approach by using the well plate base itself as a reference surface for focusing. Instead of relying on contrast from stained cells or fluorescent tags, the system uses the known geometric structure of the well plate base as an intermediary reference point. The autofocus system focuses on the base surface, which provides sufficient optical feedback contrast, and then this focal length is applied to image the cells in the wells above the base.
3Illumination intensity
If the laser beam is focussed near the lower surface of the well base, then the reflected light reaches maximum intensity, but the focal point cannot be positioned accurately due to variations in base thickness
Solution Approach 1:
The system creates an optical copy or representation of the well base surface as the focusing target. Instead of focusing directly on the physical base surface where thickness variations occur, the system uses the reflected light pattern from the base as an intermediary to determine the focal length. The maximum reflected light intensity serves as a proxy indicator that allows the system to infer the correct focal plane without being directly affected by base thickness variations.
4Measurement precision
If the focussed spot is moved onto the upper surface of the well base, then the base thickness variation issue is avoided, but the reflected signal becomes weaker due to refractive index change
Solution Approach 1:
The invention converts the harmful effect of refractive index changes at the liquid-air interface into a beneficial focusing mechanism. The refractive index change, which normally reduces reflected signal intensity, is actually exploited to create a distinct optical signature at the liquid interface. The autofocus system uses this optical signature as a reliable indicator to determine the focal plane, transforming the signal weakness into a useful focusing reference that reliably indicates when the beam is focused at the correct depth.
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 significantly reduces the time required to image multiple wells by maintaining focus and ensures high-quality images without the need for frequent refocusing and improves illumination, reducing processing time and enhancing image clarity.
Implementation Method 1
an exhaust outlet for evacuating a volume defined by the vacuum bed, the perimeter portion and a biological sample container received on the seal so that the lower surface of the biological sample container is flattened against the planar surface of the vacuum bed
Data Source
AI summary
Disclosed herein is a holder for biological sample containers such as well plates. The holder comprises a flat vacuum bed surrounded by a seal. A container is placed within the seal and a vacuum is applied, pressing and flattening the lower surface of the sample container against the flat vacuum bed. Samples in all portions of the container may then be imaged without the need to refocus on each portion of the container. For imaging, a sample in a well can be illuminated by a beam of light arranged so that a part or all of the sample is illuminated by direct rays that have not passed through the well plate. The beam is redirected to other parts of the well if a single illumination does not cover the whole well, so that the sample to be imaged using a series of partial images.