Fluorescence Microscope Layer Alignment for 3D Super-Resolution Imaging
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing microscopes face challenges in aligning and integrating three-dimensional information across multiple layers of a specimen effectively.
Innovation Solution
A microscope system utilizing single-molecule localization microscopy techniques like STORM and PALM, combined with image processing algorithms, to generate three-dimensional super-resolution images by correcting positional shifts between layers using overlapping regions and centroid calculations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple layers of fluorescence images are acquired to obtain three-dimensional information, then the three-dimensional structure of the specimen is improved, but the alignment accuracy between layers deteriorates due to positional shifts
Solution Approach 1:
The patent introduces a reference marker as an intermediary object that is present across multiple layers. This reference marker serves as a common reference point to calculate and correct positional shifts between layers, enabling accurate alignment without directly comparing all fluorescent substance positions across layers.
Solution Approach 2:
The patent implements a feedback mechanism where the positional shift between layers is calculated based on the reference marker, and this calculated shift information is fed back to correct the positions of fluorescent substances in subsequent layers. This continuous correction process ensures accurate three-dimensional reconstruction.
2Productivity
If the position of fluorescent substances is determined for each layer independently, then the processing speed is improved, but the integration accuracy across layers deteriorates
Solution Approach 1:
The reference marker acts as a mediator that connects independent layer processing with accurate integration. Each layer can be processed independently to determine fluorescent substance positions, but the reference marker provides the linking information needed to accurately integrate these independent results into a coherent three-dimensional structure.
3Measurement precision
If the number of layers is increased to improve three-dimensional resolution, then the depth information is improved, but the complexity of alignment and integration increases
Solution Approach 1:
The reference marker serves as a universal intermediary that simplifies the alignment and integration process regardless of the number of layers. Instead of performing complex pairwise alignments between all layers, the reference marker provides a common reference framework that reduces the complexity of integrating multiple layers into the three-dimensional reconstruction.
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
Enables accurate alignment and integration of three-dimensional information across multiple layers, resulting in high-resolution, stereoscopic images of specimens.
Implementation Method 1
STORM activates a fluorescent substance at low density and causes only the activated fluorescent substance to emit light by irradiation of excitation light
Implementation Method 2
forming, by an observation optical system, an image of light from the specimen
Data Source
Figure 1
Figure 2
Figure 3(A)~3(D)
AI summary
[Problem] When information on a specimen is three-dimensionally acquired for each of a plurality of layers, the information is matched among the layers. [Solving means] Provided is a microscope including: an illumination optical system that irradiates a specimen including a fluorescent substance with illumination light; an observation optical system including an objective lens; an imager that takes an image formed by the observation optical system; an image processor that performs image processing by using a result of the image taking; and a controller that moves at least one of a stage and the objective lens in the optical axis direction of the objective lens. The image processor uses position information on the fluorescent substance in at least a part of an overlapping region between a first region and a second region from position information on the fluorescent substance in the first region obtained from an imaging result when a focus position of the objective lens is located at a first position and position information on the fluorescent substance in the second region obtained from an imaging result when the focus position of the objective lens is located at a second position, to correct the position information on the fluorescent substance in the second region.