SPIM Microscope Image Stack Registration via Maximum Intensity Projection
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Solution Overview
Problem
Current methods for registering three-dimensional image stacks in Plane Illumination Microscopy (SPIM) are computationally intensive and often unreliable, requiring artificial references that can distort images or harm biological samples, and may not always produce accurate results due to the complexity of sample structures.
Innovation Solution
A manual registration process is implemented, where image stacks are aligned using maximum intensity projections along multiple axes, allowing for efficient alignment without the need for artificial markers, and enabling the use of various transformation types such as rigid, affine, or elastic transformations, which can be partially automated.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If automatic registration methods are used to merge image stacks, then alignment can be performed without manual intervention, but the computational burden increases and reliability decreases
Solution Approach 1:
The patent introduces artificial reference markers as intermediary objects that facilitate the registration process. These markers serve as reliable reference points that can be automatically detected and used to align image stacks, bridging the gap between automated processing and reliable alignment without requiring complex computational algorithms.
Solution Approach 2:
The method creates a simplified representation of the sample structure through maximum intensity projections, which serve as copies or summaries of the three-dimensional data. These projections are easier to process and compare, enabling more reliable automated registration while reducing computational complexity.
2Measurement precision
If artificial reference markers are introduced for registration, then alignment accuracy improves, but sample preparation time increases and biological samples may be harmed
Solution Approach 1:
The patent enables the sample itself to provide registration references through its natural structures. By using maximum intensity projections to identify prominent sample features as reference points, the system eliminates the need for separate artificial markers, allowing the sample to serve its own registration needs without additional preparation steps.
Solution Approach 2:
The method transforms the three-dimensional image data into two-dimensional maximum intensity projections, changing the dimensional parameter to create a simplified view that highlights prominent features. This parameter change enables automatic identification of reference points without requiring physical markers on the sample.
3Extent of automation
If complex computational algorithms are used for registration, then automated alignment is achieved, but processing time and computational resources increase
Solution Approach 1:
The patent extracts only the most essential information from the image stacks by creating maximum intensity projections. This extraction process removes unnecessary computational complexity while retaining the critical features needed for registration, enabling automated alignment with reduced processing requirements.
Solution Approach 2:
Instead of directly comparing complex three-dimensional image stacks, the patent inverts the approach by projecting them into two-dimensional maximum intensity views. This inversion simplifies the comparison process and enables more efficient automated registration algorithms to be applied.
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 approach allows for accurate and efficient merging of image stacks from different angles into a 3D image without the need for artificial markers, reducing computational burden and sample preparation time, while maintaining high image quality and avoiding biological interference.
Implementation Method 1
The illumination means comprise an illumination source, preferably emitting coherent light
Implementation Method 2
SPIM technology involves exciting fluorophores contained in or introduced into the sample with laser light
Data Source
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AI summary
Method for SPIM microscopy with a microscope comprising an illumination device comprising an illumination light source (L1,L2) and an illumination beam path for illuminating a sample (P) with a light sheet, a detection device for detecting light emitted by the sample (P), with an objective (O), wherein the light sheet is substantially planar at the focus of the objective (O) or at a defined plane near the focus of the imaging objective, and the objective has an optical axis (OA) that intersects the plane of the light sheet at a non-zero angle, preferably perpendicularly.wherein the sample (P) is moved by the light sheet in the direction of the optical axis (OA) of the objective to capture different sample planes, and the sample is illuminated at least at a first and a second illumination angle, and several sample planes are detected at each illumination angle and stored as at least first and second image stacks (ST1, ST2), wherein the image stacks are aligned to each other by aligning the coordinate systems of all image stacks to the coordinate system of the first image stack, combining the image stacks into one image stack (ST1+ST2), projecting the three-dimensional image stack into a two-dimensional representation (ST1+ST2 2D), and aligning sample features captured from different illumination directions to each other in their position.A coordinate transformation is determined from the alignment coordinates, and this coordinate transformation is applied to the combined image stack for alignment.