SPIM Microscope Light Sheet Positioning Correction
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Solution Overview
Problem
Existing light sheet microscopy techniques face challenges in achieving precise adjustment of the light sheet relative to the sample, especially due to variations in refractive indices and sample structures, leading to incorrect evaluations and time-consuming manual adjustments.
Innovation Solution
Implementing a coupled movement of the light sheet and sample relative to the detection focal plane, calibrated via a control unit, to maintain the sample plane's identity and adjust the light sheet position based on refractive index variations, ensuring optimal overlap and contrast.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If manual adjustment of light sheet position is performed, then adjustment precision can be improved, but adjustment time and operational complexity increase significantly
Solution Approach 1:
The system uses the sample itself as the reference object for adjustment. The processor automatically analyzes sample images to determine the optimal light sheet position, eliminating the need for separate reference samples and manual adjustment procedures. The system self-calibrates by utilizing the sample's own optical properties.
Solution Approach 2:
The manual mechanical adjustment process is replaced with an automated computational system. The processor automatically calculates the optimal light sheet position based on image analysis, substituting the mechanical manual positioning with an automated control system that drives the positioning actuator.
2Measurement precision
If reference samples are used for adjustment, then adjustment accuracy is improved, but device complexity and operational steps increase
Solution Approach 1:
The sample serves multiple functions: it is both the object of study and the reference standard for adjustment. This eliminates the need for separate reference samples and simplifies the overall system, as the same sample performs dual roles in the adjustment and imaging processes.
Solution Approach 2:
The adjustment reference function is extracted from the sample and used directly on the sample itself, rather than requiring a separate reference sample. This eliminates the additional component of reference samples and simplifies the adjustment procedure.
3Speed
If light sheet position is adjusted without coupling to sample movement, then adjustment speed is improved, but image quality and sectioning precision deteriorate
Solution Approach 1:
The system implements dynamic coupling between light sheet positioning and sample stage movement. When the sample is moved in the Z-direction, the light sheet position is automatically adjusted to maintain optimal alignment. This dynamic adaptation ensures that the light sheet remains properly positioned relative to the moving sample, maintaining image quality and sectioning precision throughout the imaging process.
Solution Approach 2:
The system uses feedback from sample images to automatically adjust the light sheet position. The processor analyzes the images to determine the optimal light sheet position and automatically adjusts the positioning, creating a closed-loop control system that maintains optimal alignment without requiring manual intervention.
4Ease of operation
If automated adjustment using sample images is implemented, then operational ease is improved, but processing complexity and computational requirements increase
Solution Approach 1:
The system uses the sample itself as the reference object for adjustment. The processor automatically analyzes sample images to determine the optimal light sheet position, eliminating the need for separate reference samples and manual adjustment procedures. The system self-calibrates by utilizing the sample's own optical properties.
Solution Approach 2:
The manual mechanical adjustment process is replaced with an automated computational system. The processor automatically calculates the optimal light sheet position based on image analysis, substituting the mechanical manual positioning with an automated control system that drives the positioning actuator.
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 method ensures precise and automated light sheet adjustment, reducing image inhomogeneities and focus shifts, allowing for intuitive manual adjustments and accurate image reconstruction without the need for reference samples, thereby improving image quality and simplifying the adjustment process.
Implementation Method 1
an illumination source that preferably emits coherent light
Implementation Method 2
a microscope which includes an imaging objective for imaging a sample onto a detector and means for illuminating the sample with a light sheet in the focal plane of the imaging objective
Implementation Method 3
with SPIM technology, fluorophores that are contained in the sample or are introduced into it are excited with laser light
Data Source
Figure 1
Figure 2
Figure 3a)~3b)
AI summary
Method and microscope for SPIM microscopy, wherein, in a first step, a calibration is performed on the basis of a specimen to be examined, in that, in various planes of the specimen, the actual position of the light sheet is recorded in dependence on the position in the specimen and stored, and, in a second step, when viewing and/or detecting the specimen, the stored position of the light sheet is used for correcting the position of the light sheet in relation to the focal plane of the detection objective on the basis of the values stored in the first step, and/or, during the shifting of the specimen, an adjustment of the position of the light sheet in relation to the focal plane of the detection objective is performed in such a way that the light sheet performs a relative movement in at least one direction in relation to the specimen and/or the detection objective performs a relative movement in relation to the specimen.