Fluorescent Image Acquisition Using Intensity Ratios for Single-Color Pixels

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Solution Overview

Problem

Conventional methods struggle to accurately discriminate single-color pixels in fluorescent images, making it difficult to efficiently observe and analyze samples with multiple substances.

Innovation Solution

A method involving irradiation with multiple wavelength excitation lights, acquisition of fluorescent images in different optical states, and calculation of intensity ratios between these images to discriminate single-color pixels, using a fluorescence filter unit with specific wavelength ranges and an inclined filter to enhance discrimination.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional fluorescent image acquisition methods are used, then fluorescent images can be obtained, but single-color pixels cannot be accurately discriminated in mixed dye environments

Engineering Contradiction:
Improvepixel discrimination accuracyVSAvoidsingle-color pixel identification
Core Design Contradiction:
Measurement precisionVSLoss of information

Solution Approach 1:

The patent segments the fluorescent image analysis into two distinct categories: single-color pixels and mixed-color pixels. By calculating intensity ratios across multiple excitation wavelengths and separating pixels based on whether their ratios match reference single-dye ratios, the method achieves accurate discrimination and classification of different pixel types in the fluorescent image

Inventive Principle:
Principle #1Segmentation

Solution Approach 2:

The patent changes the parameter of measurement by acquiring fluorescent images at multiple excitation wavelengths and calculating intensity ratios. This parameter transformation allows single-color pixels to be identified by their characteristic ratio values that match reference single-dye ratios, thereby improving discrimination accuracy in mixed dye environments

Inventive Principle:
Principle #35Parameter changes

2Measurement precision

If multiple excitation lights are used to improve discrimination, then single-color pixels can be identified, but the complexity of the acquisition system increases

Engineering Contradiction:
Improvesingle-color pixel discriminationVSAvoidfluorescent image acquisition system
Core Design Contradiction:
Measurement precisionVSDevice complexity

Solution Approach 1:

The patent employs a fluorescence filter unit with multiple reflection wavelength ranges and transmission wavelength ranges that can handle multiple excitation lights and multiple fluorescences simultaneously. This multi-functional component allows the system to acquire images under different excitation conditions without requiring separate acquisition systems for each wavelength

Inventive Principle:
Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent combines multiple fluorescent images acquired under different excitation lights into a unified analysis framework. By merging the images and calculating intensity ratios across all excitations, the system achieves accurate single-color pixel discrimination while using a single integrated acquisition device rather than multiple separate systems

Inventive Principle:
Principle #5Merging (Combining)

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 discrimination of single-color pixels in fluorescent images, even in mixed dye environments, allowing for effective analysis and separation of fluorescent signals.

Implementation Method 1

acquiring, with respect to each of a plurality of fluorescences corresponding to each of the plurality of excitation lights, a first fluorescent image in a first optical state and a second fluorescent image in a second optical state

Methodology Applied
Scientific EffectFluorescence: Fluorescence

Implementation Method 2

through a fluorescence filter unit having a plurality of reflection wavelength ranges and a plurality of transmission wavelength ranges

Methodology Applied
Scientific EffectReflection: Reflection

Data Source

PatentEP4653849A1Fluorescent image acquisition method, fluorescent image acquisition device, and fluorescent image acquisition program
Publication Date: 2025.11.26 HAMAMATSU PHOTONICS KK
  • EP4653849A1 patent drawingFigure 1
  • EP4653849A1 patent drawingFigure 2
  • EP4653849A1 patent drawingFigure 3

AI summary

A fluorescent image acquisition system 1 includes an irradiation device 2 which irradiates a sample with each of excitation lights of a plurality of wavelength distributions; an image acquisition device 3 which acquires, with respect to each of a plurality of fluorescences corresponding to each of the plurality of excitation lights, a first fluorescent image in a first optical state and a second fluorescent image in a second optical state in which fluorescence is measured at a wavelength characteristic different from that in the first optical state through a fluorescence filter unit having a plurality of reflection wavelength ranges and a plurality of transmission wavelength ranges; and an image processing device 4 which processes a plurality of the first fluorescent images and a plurality of the second fluorescent images, in which the image processing device 4 calculates, for the first fluorescent image and the second fluorescent image, an intensity ratio that is a ratio between an intensity value of a pixel of the first fluorescent image and an intensity value of a pixel of the second fluorescent image corresponding to the pixel, and calculates the intensity ratio for each of the plurality of excitation lights, and discriminates whether the pixel is a single-color pixel based on the intensity ratio for each of the plurality of excitation lights.