Confocal Fluorescence Image Processing for Focal-Dependent Quantification
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Solution Overview
Problem
Fluorescence images captured by confocal microscopes exhibit unique focal characteristics due to varying fluorescence intensity based on focal position, making it difficult to handle them similarly to general dark field microscope images, and existing methods do not adequately account for these focal characteristics.
Innovation Solution
An information processing method that analyzes fluorescence images using optical systems where intensity varies with focal position, derives fluorescence intensity characteristics, and collates this with reference standard data to determine fluorescent molecule concentration, employing Fourier transforms and smoothing processes to correct singular points.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If confocal microscope optical system is used to capture fluorescence images, then high-contrast and high-resolution images are obtained, but fluorescence intensity varies according to focal position making quantitative analysis difficult
Solution Approach 1:
The patent applies parameter changes by modifying the fluorescence intensity values through mathematical transformation. Specifically, it converts the focal-position-dependent fluorescence intensity into a standardized parameter that accounts for the confocal optical characteristics, enabling quantitative comparison across different focal positions while preserving the high-resolution imaging capability
Solution Approach 2:
The patent introduces an intermediary computational process that acts as a mediator between the raw confocal fluorescence images and quantitative analysis. This intermediary step involves deriving fluorescence intensity characteristics and applying correction based on reference standard data, thereby bridging the gap between the optical system's focal characteristics and accurate concentration measurement
2Reliability
If fluorescence intensity is corrected using existing methods, then brown color influence is reduced, but focal characteristics of confocal microscope are not adequately considered
Solution Approach 1:
The patent applies local quality by implementing focal-position-specific correction factors. Instead of applying a uniform correction method, it derives fluorescence intensity characteristics for each focal position and applies localized corrections based on reference standard data corresponding to each focal plane, thereby adapting the correction method to the specific characteristics of confocal imaging at different depths
Solution Approach 2:
The patent implements dynamics by making the correction method adaptive to different focal positions. The system dynamically selects and applies appropriate reference standard data based on the focal position of each image, allowing the correction approach to vary according to the specific imaging conditions rather than using a static correction factor
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 handling of fluorescence images by considering focal characteristics, allowing for precise determination of fluorescent molecule concentrations.
Implementation Method 1
a fluorescence microscope that irradiates a fluorescent-stained specimen (sample) stained with a fluorescent staining reagent with excitation light to generate fluorescence in the fluorescent-stained specimen, and captures an image of the fluorescence
Data Source
AI summary
To provide a technique of handling a fluorescence image captured and acquired by using an optical system in which fluorescence intensity in a captured image changes according to a focal position, in consideration of focal characteristics.An information processing method includes: a step of analyzing an observation fluorescence image captured and acquired by using an optical system in which fluorescence intensity in a captured image changes according to a focal position and acquiring an observation fluorescence intensity; and a step of deriving a fluorescent molecule concentration in an observation sample from the observation fluorescence intensity while collating with reference standard data that associates a standard fluorescent molecule concentration and a standard fluorescence intensity with each other. The reference standard data is obtained on the basis of the standard fluorescence intensity and the standard fluorescent molecule concentration, the standard fluorescence intensity being derived from a plurality of reference fluorescence images that is a plurality of fluorescence images of a reference sample captured and acquired by using an optical system in which fluorescence intensity in a captured image changes according to a focal position, the plurality of reference fluorescence images having different focal positions at the time of imaging from each other, and the standard fluorescent molecule concentration being obtained from a fluorescent molecule concentration in the reference sample.