Dual-Objective Light-Sheet Microscopy for Focal Plane Shift Compensation
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Solution Overview
Problem
In microscopes with two imaging objectives, focal plane shifts due to refractive index differences between samples and the surrounding medium lead to non-overlapping focal planes, compromising image quality and 3D image reconstruction.
Innovation Solution
Adjust the illumination beam to align with individual focal planes of each imaging objective, using distinct adjustment settings to compensate for focal plane shifts, allowing sequential image acquisition and correction of shifts during 3D image reconstruction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If a single illumination setting is used for both imaging objectives, then the device operation is simple, but the focal plane shift causes image quality degradation
Solution Approach 1:
The patent divides the illumination adjustment into separate settings for each imaging objective. The control unit manages multiple illumination adjustment settings corresponding to different combinations of first and second imaging objectives, allowing each objective to have its focal plane shift compensated independently.
2Device complexity
If focal plane shifts are not compensated, then the device complexity is low, but the measurement precision of 3D image reconstruction deteriorates
Solution Approach 1:
The control unit stores calibration data that characterizes the focal plane shift for each imaging objective. During operation, the control unit automatically selects and applies the appropriate illumination adjustment setting based on which imaging objectives are active, effectively compensating for focal plane shifts without requiring real-time measurement feedback.
3Manufacturing precision
If separate illumination settings are used for each imaging objective, then image quality is improved, but the ease of operation decreases
Solution Approach 1:
The control unit automatically manages the complexity of coordinating multiple illumination adjustment settings. When an imaging objective is selected, the control unit autonomously applies the appropriate calibration data and adjusts the illumination parameters without requiring manual intervention from the user.
4Device complexity
If focal plane shifts are not corrected, then the device complexity remains low, but the reliability of image acquisition deteriorates
Solution Approach 1:
The patent performs preliminary calibration to determine focal plane shift characteristics for each imaging objective before actual image acquisition. The calibration data is stored in the control unit and used to pre-configure the illumination settings, ensuring reliable image acquisition from the start without requiring real-time corrections during imaging.
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 and enables efficient, precise 3D image reconstruction by compensating for focal plane misalignments caused by refractive index variations.
Implementation Method 1
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 (more specifically, on the difference between the refractive index of the sample and the one of the medium that was there in the absence of the sample)
Data Source
AI summary
A method for acquiring 2D images of a sample includes illuminating the sample with an illumination beam forming a light sheet, and detecting light emitted from the sample along two different imaging paths by using a first imaging objective and a second imaging objective, respectively. The method further includes obtaining a first stack of images using the first imaging objective by sequentially illuminating the sample by adjusting the light sheet according to a first adjustment setting, and obtaining a second stack of images using the second imaging objective by sequentially illuminating the sample by adjusting the light sheet according a second adjustment setting. Each of the first stack and the second stack of images represents multiple planes of the sample in a z-direction. The first stack of images and the second stack of images are characterized by a shift of the planes in the z-direction relative to each other.