Light Sheet Microscopy Resolution via Three-Objective Lens Fusion
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Solution Overview
Problem
Light sheet microscopy systems suffer from relatively poor spatial resolution due to the need for low numerical aperture objectives, and existing resolution enhancement techniques like multi-view acquisition and structured illumination either fail to improve resolution sufficiently or compromise the speed and signal quality of the imaging process.
Innovation Solution
A depth-of-focus optical arrangement using three objective lenses and a processor to combine images from different lenses, with an extended depth-of-focus optical system such as a cubic phase mask or incoherent 'layer cake' optical arrangement, which preserves lateral resolution while extending the depth of field, allowing for improved fluorescence emission collection and enhanced image resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If perpendicular geometry is used between excitation and detection, then optically sectioned imaging with reduced photo-damage is achieved, but spatial resolution deteriorates due to low numerical aperture objectives
Solution Approach 1:
The patent introduces a third dimension by adding a third objective lens perpendicular to both the illumination and detection paths. This creates a new detection dimension that collects fluorescence emissions from a different angular perspective, effectively increasing the numerical aperture without compromising the perpendicular geometry that reduces photo-damage.
Solution Approach 2:
The patent merges images from three different objective lenses (illumination objective and two detection objectives) into a single composite image. This combination of multiple viewing angles allows the system to achieve higher spatial resolution while maintaining the low photo-damage benefits of light sheet microscopy.
2Measurement precision
If multi-view acquisition or structured illumination is used, then resolution is improved, but signal/photon budget is sacrificed due to increased illumination exposure
Solution Approach 1:
The patent uses a light sheet to pre-illuminate the entire sample volume before detection, creating fluorescence emissions throughout the sample. This preliminary illumination action allows the third objective lens to collect emissions from multiple depths simultaneously without requiring additional illumination exposure during the detection phase.
Solution Approach 2:
The patent maintains continuous fluorescence emission generation throughout the sample volume by keeping the light sheet continuously illuminated. This continuous useful action ensures that fluorescence emissions are constantly available for collection by all three objective lenses without requiring repeated illumination cycles that would deplete the photon budget.
3Measurement precision
If multiple images are captured for multi-view acquisition, then resolution is improved, but imaging speed deteriorates
Solution Approach 1:
The patent employs periodic scanning of the light sheet through the sample while the third objective lens continuously detects fluorescence emissions. This periodic illumination scanning combined with continuous detection from multiple angles allows the system to acquire high-resolution data from all three objectives simultaneously at each scan position, maintaining imaging speed while improving resolution.
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 significantly enhances lateral resolution in light sheet microscopy systems while maintaining high signal-to-noise ratio and speed, overcoming the limitations of conventional light sheet microscopy by effectively capturing more fluorescence emissions and reducing out-of-focus light issues.
Implementation Method 1
A depth-of-focus optical arrangement using three objective lenses and a processor to combine images from different lenses, with an extended depth-of-focus optical system such as a cubic phase mask or incoherent 'layer cake' optical arrangement
Implementation Method 2
an extended depth-of-focus optical system such as a cubic phase mask or incoherent 'layer cake' optical arrangement, which preserves lateral resolution while extending the depth of field
Implementation Method 3
The illuminated sample emits fluorescence emissions 20 scattered at substantially a ninety degree angle relative to the light sheet 18 illuminating the sample and detected by a second objective lens 14
Implementation Method 4
The illuminated sample emits fluorescence emissions 20 scattered at substantially a ninety degree angle relative to the light sheet 18
Implementation Method 5
A depth-of-focus optical arrangement using three objective lenses and a processor to combine images from different lenses
Data Source
AI summary
Embodiments of a resolution enhancement technique for a light sheet microscopy system having a three objective lens arrangement in which one objective lens illuminates a sample and the second and third objective lenses collect the fluorescence emissions emitted by the sample are disclosed. The second objective lens focuses a first portion of the fluorescence emissions for detection by a second detection component, while the third objective lens focuses a second portion of the fluorescence emissions through a diffractive or refractive optic component for detection by a first detector component. A processor combines the images resulting from the first and second portions of the fluorescence emissions for generating composite images with increased axial and lateral resolution.