Confocal Microscopy Observation Volume Design for High Throughput
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Solution Overview
Problem
Conventional confocal microscopy faces limitations in achieving high resolution and throughput at a commercially viable cost due to the need for high numerical aperture objectives, pinhole-based observation volumes, and complex scanning mechanisms, which restrict its application in cost-effective and high-speed imaging of biological samples.
Innovation Solution
The solution involves defining the observation volume by the intersection of the focal width of the illumination and detection optics, eliminating the need for a pinhole, and using a rotating sample container to pass the sample through the observation volume, allowing for parallel detection of multiple points and high-speed imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a pinhole is used to limit the observation volume in confocal microscopy, then the spatial resolution and contrast are improved, but the imaging speed and throughput deteriorate because only one point can be imaged at a time
Solution Approach 1:
The patent segments the observation volume into multiple discrete points arranged in a grid pattern across the field of view. Instead of imaging one point at a time through scanning, multiple points are simultaneously illuminated and detected, enabling parallel acquisition of multiple spatial locations while maintaining the pinhole-based optical sectioning for each point.
Solution Approach 2:
The patent transitions from sequential one-dimensional scanning to simultaneous two-dimensional parallel detection by arranging multiple pinholes in a grid pattern. This dimensional expansion allows multiple observation points to be imaged concurrently, dramatically increasing throughput while preserving the confocal optical sectioning capability in each channel.
2Measurement precision
If high numerical aperture objectives are used to achieve high resolution, then the spatial resolution is improved, but the cost and complexity of the system increase due to chromatic aberration compensation requirements
Solution Approach 1:
The patent employs a single objective lens that serves multiple functions: it provides high numerical aperture for spatial resolution, accepts illumination from multiple angles for parallel point detection, and works across the broad spectral range of fluorophores. The multi-point detection architecture eliminates the need for multiple specialized optical paths or complex chromatic correction systems.
Solution Approach 2:
The patent changes the operational parameters by using a single objective lens with fixed numerical aperture and focal length, but varies the illumination angles and detection positions to achieve multiple observation points. This parameter variation approach maintains optical simplicity while achieving high resolution through the inherent capabilities of the single high-NA objective.
3Measurement precision
If scanning mechanisms are used to move the sample or observation volume through the focal plane, then the spatial resolution is maintained, but the imaging speed and throughput are reduced
Solution Approach 1:
The patent segments the field of view into multiple discrete observation points that are simultaneously active. Instead of mechanically scanning through the sample to build an image point-by-point, the system divides the imaging task into parallel segments that are acquired concurrently, eliminating the time penalty of sequential scanning while maintaining the optical sectioning that provides spatial resolution.
Solution Approach 2:
The patent enables continuous imaging by simultaneously detecting multiple points across the field of view without requiring mechanical interruption for scanning. The parallel detection architecture allows the system to continuously accumulate image data from multiple locations, achieving high throughput while the optical sectioning maintains the spatial resolution characteristics of confocal microscopy.
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 high spatial resolution and high frame rate imaging at a lower cost by removing the pinhole constraint, allowing for efficient imaging of biological samples with improved signal-to-noise ratio and reduced photobleaching, while maintaining mechanical stability and precision.
Implementation Method 1
an illumination means for illuminating a sample volume with illumination light from a light source, wherein said illumination means defines an observation plane by focusing said illumination light at a specific depth in said sample
Implementation Method 2
very often, confocal microscopy is combined with fluorescence spectroscopy, where specific particles, e.g. proteins, are marked with fluorophores and thus can be discerned from the background
Implementation Method 3
the said illumination and said detecting are automatically aligned... scattered or fluorescent light emanating from illuminated parts of the sample
Data Source
AI summary
A method and apparatus are provided, in which an observation volume is defined by a volume where light from illumination means and a field of view of detection means overlap. The central axes of said light from the illumination means and said field of view of the detection means are non-parallel, and the sample is transported through the observation volume during imaging, preferably by rotation of a sample container holding the sample.