Single-Objective Multi-Depth Confocal Imaging for Higher Throughput
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Solution Overview
Problem
Conventional fluorescent microscopy techniques face challenges such as high photodamage, complex workflows, and spatial limitations due to the need for two objectives, especially in oblique-plane light-sheet microscopy, and low throughput in confocal microscopy.
Innovation Solution
A multi-depth confocal imaging system that uses a light source, objective lens, and image sensor array, allowing simultaneous imaging at multiple depths without high-NA immersion objectives, utilizing fiber optics and oblique orientation of the image sensor to capture multiple focal planes concurrently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If conventional fluorescent microscopy techniques are used, then imaging can be performed, but photodamage occurs due to high illumination intensities
Solution Approach 1:
The illumination is segmented into multiple discrete focal planes along the axial direction. Each light source in the array illuminates a specific depth plane, allowing the sample to be illuminated only at the required depths rather than uniformly across the entire sample volume, thereby reducing overall photodamage
Solution Approach 2:
The system transitions from single-plane illumination to multi-plane illumination by adding the axial dimension to the illumination pattern. The array of light sources is positioned at different axial heights to create multiple focal planes, enabling simultaneous imaging at multiple depths while reducing the illumination intensity required at each plane
2Measurement precision
If confocal microscopy is used to achieve optical sectioning, then imaging resolution is improved, but throughput decreases due to sequential scanning
Solution Approach 1:
The detection process is segmented into multiple independent detection channels, each corresponding to a specific focal plane. Multiple image sensors simultaneously detect fluorescence from different depths, eliminating the need for sequential scanning and thereby improving imaging throughput while maintaining optical sectioning capability
Solution Approach 2:
The system enables continuous simultaneous detection at multiple depths by using an array of light sources and image sensors that operate in parallel. This eliminates the intermittent nature of sequential scanning, maintaining continuous useful action across all focal planes
3Productivity
If oblique-plane light-sheet microscopy is used to improve imaging speed, then throughput increases, but spatial limitations occur due to the need for two objectives
Solution Approach 1:
The illumination and detection functions are merged into a single objective lens. The array of light sources positions illumination paths through the objective, and the same objective collects the emitted light, eliminating the need for separate illumination and detection objectives and their associated spatial requirements
Solution Approach 2:
The single objective lens serves multiple functions: it acts as both the illumination objective and the detection objective. The objective is used to focus excitation light at multiple depths and to collect emitted light from all focal planes, demonstrating multi-functionality that reduces device complexity
4Measurement precision
If high-NA immersion objectives are used to achieve high resolution, then measurement precision improves, but device complexity and spatial requirements increase
Solution Approach 1:
The high-resolution imaging function is segmented across multiple light sources and image sensors operating at different focal planes. This distributed approach achieves high overall resolution without requiring a single high-NA immersion objective, thereby reducing device complexity
Solution Approach 2:
The system changes the parameter of numerical aperture by using multiple lower-NA objectives or air objectives instead of a single high-NA immersion objective. By adjusting the illumination and detection parameters across multiple focal planes, the system achieves comparable or superior resolution without the complexity of immersion objectives
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
Enhances imaging throughput and reduces photodamage by enabling simultaneous multi-depth imaging, overcoming spatial limitations and eliminating the need for high-NA immersion objectives, thus improving efficiency and versatility.
Implementation Method 1
illuminating a sample at a plurality of depths to generate fluorescent events
Implementation Method 2
the excitation beams are focused into a sample at a first plurality of focus depths along an excitation direction through the objective lens
Implementation Method 3
an image sensor that receives emissions from the sample via the objective lens
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.