Multi-Depth Confocal Imaging With Oblique Sensor Alignment
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Solution Overview
Problem
Conventional fluorescent microscopy techniques, including confocal and light-sheet microscopy, face challenges such as high photodamage, complex workflows, and spatial requirements, limiting their throughput and applicability to samples that cannot use immersion oil.
Innovation Solution
A multi-depth confocal imaging system that uses a combination of excitation beams focused at multiple depths within the sample and an oblique-oriented image sensor, eliminating the need for high-NA immersion objectives, and enabling simultaneous imaging at multiple focal planes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional confocal microscopy uses high-NA immersion objectives to achieve high optical resolution, then measurement precision is improved, but device complexity and spatial requirements increase
Solution Approach 1:
The patent divides the imaging function into multiple independent depth channels, each with its own focal plane. Multiple excitation beams are focused at different depths simultaneously, allowing each beam to operate independently at optimal focus without requiring complex high-NA immersion objectives for the entire volume.
Solution Approach 2:
The patent transitions from single-plane imaging to multi-depth volumetric imaging by adding the depth dimension as a parallel imaging axis. The oblique-oriented image sensor captures multiple focal depths simultaneously, converting a three-dimensional imaging problem into multiple two-dimensional captures that can be processed independently.
2Productivity
If light sheet fluorescence microscopy scans in multiple axial directions for rapid 3D imaging, then productivity is improved, but photodamage and phototoxicity increase
Solution Approach 1:
The patent applies local quality by concentrating excitation energy only at the specific focal depths where imaging is required, rather than illuminating the entire sample volume. Each excitation beam is focused to a specific depth, providing high intensity only where needed and minimizing photodamage to out-of-focus regions.
Solution Approach 2:
The patent enables continuous simultaneous imaging at multiple focal planes without requiring sequential scanning. All depth channels capture images concurrently, eliminating the time delays and repeated exposures associated with sequential scanning methods.
3Object-affected harmful factors
If light sheet fluorescence microscopy uses orthogonal detection axis to minimize photodamage, then object-affected harmful factors are reduced, but device complexity increases
Solution Approach 1:
The patent makes the objective lens multi-functional by using it for both excitation and collection of light. The same optical path serves dual purposes: delivering excitation beams to multiple focal depths and collecting emissions from all depths, eliminating the need for separate illumination and detection objectives.
Solution Approach 2:
The patent inverts the traditional light sheet approach by using oblique illumination through a single objective rather than orthogonal illumination. The excitation beams enter through the same objective that collects emissions, reversing the conventional separation of illumination and detection paths.
4Measurement precision
If conventional microscopy illuminates the entire sample to capture multiple depths, then measurement precision is improved, but energy consumption increases
Solution Approach 1:
The patent applies local quality by concentrating excitation energy only at the specific focal depths where imaging is required, rather than illuminating the entire sample volume. Each excitation beam is focused to a specific depth, providing high intensity only where needed and minimizing photodamage to out-of-focus regions.
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 system significantly increases imaging throughput by concurrently capturing multiple focal depths, overcoming spatial limitations and reducing photodamage, while allowing imaging of a wider range of samples without the need for high-NA immersion lenses.
Implementation Method 1
illuminating a sample at a plurality of depths and detecting light from the sample (e.g., fluorescent excitation events, scattered light, transmitted light, or reflected light)
Implementation Method 2
the excitation beams are focused into a sample at a first plurality of focus depths (e.g., two or more different depths, such as 2-10 different depths, 4-12 different depths, 5-8 different depths) along an excitation direction through the objective lens
Implementation Method 3
the emissions define foci relative to the image sensor at a second plurality of focus depths
Data Source
AI summary
A multi-depth confocal imaging system includes at least one light source configured to provide excitation beams and an objective lens. The excitation beams are focused into a sample at a first plurality of focus depths along an excitation direction through the objective lens. An image sensor receives emissions from the sample via the objective lens, wherein the emissions define foci relative to the image sensor at a second plurality of focus depths.