Fluorescence Measurement Device Optical Thickness Normalization
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Solution Overview
Problem
Conventional fluorescence measurement techniques face challenges in accurately discriminating cells based on fluorescence intensity due to factors like cell thickness, leading to erroneous determinations of fluorescence expression rates, especially in mixed cell populations.
Innovation Solution
A fluorescence measurement apparatus and method that acquire both fluorescence and interference images to determine optical thickness, allowing for accurate calculation of fluorescence expression rates by integrating fluorescence intensity with optical thickness in a region of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If only fluorescence intensity is used as the index for cell discrimination, then the measurement process is simple, but accurate discrimination is difficult due to variations in cell thickness
Solution Approach 1:
The patent introduces optical thickness as an intermediary parameter to mediate between the fluorescence intensity measurement and the cell's actual fluorescent protein expression level. By measuring optical thickness separately and using it to normalize fluorescence intensity, the patent eliminates the confounding effect of cell thickness variations, thereby improving measurement precision without significantly complicating the overall measurement process
Solution Approach 2:
The patent changes the measurement approach from using only fluorescence intensity to using a combined parameter that includes both fluorescence intensity and optical thickness. This parameter change allows the system to account for variations in cell thickness and achieve accurate discrimination of cell populations based on genuine fluorescent protein expression levels
2Quantity of substance
If fluorescence intensity is integrated along the optical axis for thick cells, then the total fluorescence signal is captured, but erroneous determination occurs where thick negative cells are classified as positive cells
Solution Approach 1:
Optical thickness serves as a mediator that allows the system to distinguish between true positive cells (high fluorescent protein expression) and false positive cells (thick cells with low expression). By normalizing fluorescence intensity with optical thickness, the system can accurately determine cell positivity regardless of cell thickness variations
Solution Approach 2:
The patent changes the evaluation parameter from raw fluorescence intensity to fluorescence intensity normalized by optical thickness. This parameter transformation ensures that cells are correctly classified based on their actual fluorescent protein expression rather than being misled by variations in cell thickness
3Quantity of substance
If fluorescence intensity is integrated along the optical axis for thin cells, then the measurement captures available signal, but positive cells with high expression are erroneously determined as negative cells
Solution Approach 1:
The patent transforms the measurement approach by introducing optical thickness as a normalizing factor. This parameter change ensures that thin cells with high fluorescent protein expression are not misclassified as negative cells, while still capturing the available fluorescence signal accurately
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 enables precise determination of fluorescence expression rates, reducing errors associated with cell thickness and allowing for accurate assessment of mixed cell populations without individual cell segmentation.
Implementation Method 1
an interference image acquisition unit for acquiring an interference image containing the object
Implementation Method 2
a fluorescence image acquisition unit for acquiring a fluorescence image containing an object
Data Source
AI summary
A fluorescence measurement apparatus includes a fluorescence image acquisition unit that acquires a fluorescence image containing an object, an interference image acquisition unit that acquires an interference image containing the object, and an operation unit. The operation unit determines an optical thickness image based on the interference image acquired by the interference image acquisition unit, and determines, in a region of interest set in common in both of the fluorescence image acquired by the fluorescence image acquisition unit and the optical thickness image, a fluorescence expression rate of the object based on an integrated value of a fluorescence intensity in the fluorescence image and an integrated value of an optical thickness in the optical thickness image.