Light-Sheet Microscope Imaging for Focal Plane Shift Compensation
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Solution Overview
Problem
In microscopes with overlapping imaging focal planes, the presence of a sample can cause focal plane shifts due to refractive index differences, leading to suboptimal image quality and 3D image reconstruction issues.
Innovation Solution
Adjust the illumination beam to align with individual focal planes of each imaging objective, allowing sequential imaging and subsequent correction of focal plane shifts during 2D image acquisition, followed by reconstruction of a 3D image.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the imaging focal planes of two imaging objectives are arranged to overlap, then simultaneous dual-view imaging of the same plane is achieved, but the focal planes shift relative to each other when a sample is placed between them due to refractive index differences
Solution Approach 1:
The system performs preliminary characterization of the sample's refractive index properties before dual-view imaging. Based on this preliminary information, the illumination beam position is pre-adjusted to compensate for the expected focal plane shift, ensuring that both imaging objectives remain focused on the same sample plane despite the refractive index mismatch.
Solution Approach 2:
The system dynamically adjusts the position of the illumination beam along the optical axis based on the measured or estimated refractive index of the sample. By changing the illumination plane position parameter, the system compensates for the focal plane shift caused by the sample, maintaining optimal focus for both imaging objectives simultaneously.
2Device complexity
If the focal plane shift is not compensated for, then the system structure remains simple, but the quality of recorded views and 3D image reconstruction deteriorates
Solution Approach 1:
Instead of using mechanical adjustments of the imaging objectives or tube lenses to compensate for focal plane shifts, the system uses software-based control to adjust the illumination beam position. This replaces complex mechanical adjustment mechanisms with a simpler optical control approach, maintaining image quality without increasing mechanical system complexity.
Solution Approach 2:
The illumination beam acts as an intermediary element that mediates between the two imaging objectives with different focal planes. By positioning the illumination beam at an optimized location, the system ensures that both objectives receive light from the same sample plane, effectively bridging the focal plane mismatch without requiring direct mechanical coupling between the objectives.
3Manufacturing precision
If the illumination beam is adjusted for each individual focal plane, then image quality is improved, but the time required for image acquisition increases due to sequential imaging
Solution Approach 1:
The system performs preliminary determination of the optimal illumination beam position based on sample characteristics before actual image acquisition. This pre-calculation allows the system to set the illumination parameters in advance, avoiding the need for time-consuming iterative adjustments during the imaging process and reducing overall acquisition time while maintaining image quality.
Solution Approach 2:
The system implements dynamic switching between different illumination configurations optimized for each imaging objective. Rather than using a fixed illumination setup, the system can rapidly switch between illumination modes adapted to the specific requirements of each objective, maintaining optimal image quality for both views while minimizing the time spent in each configuration state.
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
Improves image quality by compensating for focal plane shifts, enabling precise and efficient 2D and 3D image acquisition and reconstruction.
Implementation Method 1
illuminating the sample with an illumination beam (running along the illumination path(s)) forming at least one light sheet intersecting the sample
Implementation Method 2
detecting light emitted from the sample along two different imaging/detection paths (i.e. first and second detection paths) by means of first and second imaging objectives having respective first and second imaging focal planes
Implementation Method 3
the imaging focal planes of the two (e.g. opposing) imaging objectives overlap, i.e. are identical. Illumination (e.g. a sheet of light) may be set to illuminate such a plane such that the image of this same plane may be recorded simultaneously through the two imaging objectives... when an object/sample (e.g., a cell, a hydrogel) is placed between an imaging objective and its focal plane, such focal plane may translate/shift depending on the refractive index of the sample
Data Source
Figure 1
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AI summary
The present disclosure concerns a method for acquiring images of a sample, comprising the steps of illuminating the sample with an illumination beam forming at least one light sheet intersecting the sample; at multiple positions of the sample in a z-direction, detecting light emitted from the sample along two different imaging paths by means of first and second imaging objectives having respective first and second imaging focal planes shifted relative to each other in the z-direction and intersecting the sample, and obtaining corresponding first and second stacks of images, each stack representing multiple planes of the sample in the z-direction, and each image representing a plane of the sample in the z-direction.