Autofluorescence Removal in Biological Microscopy via Reference Dye Subtraction
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Solution Overview
Problem
Current methods for removing inherent autofluorescence from biological tissues in microscopy applications are inefficient, leading to reduced signal detection sensitivity and inaccurate detection of fluorescent dye signals, as they often require expensive hardware or incomplete software-based corrections.
Innovation Solution
A two-step image acquisition process involving a reference image with a low autofluorescence dye and subsequent images with additional dyes, followed by coregistration and robust estimation to separate and remove autofluorescence signals, enhancing signal-to-noise ratio without needing costly instrumentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multi-spectral imaging hardware with spectral un-mixing is used, then autofluorescence removal capability is improved, but device cost and complexity increase significantly
Solution Approach 1:
The patent creates a computational copy of the autofluorescence signal by acquiring a reference image with a low-AF dye that mimics the tissue's autofluorescence characteristics. This copy is then subtracted from the original fluorescent image to remove AF, replacing the need for complex multi-spectral hardware with a simpler imaging system combined with image processing.
Solution Approach 2:
The patent replaces the mechanical complexity of multi-spectral imaging hardware with a digital image processing approach. Instead of using multiple filter cubes and spectral un-mixing algorithms requiring expensive instrumentation, the system uses a single fluorescent image acquisition combined with computational subtraction of a reference AF image.
2Device complexity
If software-based AF removal methods are used, then device cost is reduced, but measurement precision and completeness of AF removal are insufficient
Solution Approach 1:
The patent performs preliminary action by acquiring a reference image with a low-AF dye before the actual fluorescent imaging. This reference image captures the tissue's autofluorescence characteristics in advance, allowing for precise subtraction during the main imaging process. The reference image is obtained under identical imaging conditions to ensure accurate AF modeling.
Solution Approach 2:
The patent introduces an intermediary substance - a low-AF dye - that serves as a mediator to capture the tissue's autofluorescence signal. This intermediary dye has minimal interfering fluorescence but sufficient signal to model the AF characteristics, enabling accurate removal without directly measuring the problematic high-AF dyes.
3Measurement precision
If fluorescent dyes with high signal intensity are used, then detection sensitivity is improved, but autofluorescence interference increases
Solution Approach 1:
The patent converts the harmful autofluorescence interference into a beneficial reference signal. By using a low-AF dye to acquire a reference image, the system captures the AF characteristics that would otherwise interfere with detection. This reference AF image is then used to subtract and remove the interference, turning the problematic AF into a correctable signal pattern.
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 effectively increases the signal-to-autofluorescence ratio, improving detection sensitivity and accuracy in fluorescence imaging, making it adaptable for various tissue types and imaging applications.
Implementation Method 1
a reference image of the biological material is acquired. The reference image may be acquired by staining the biological material first with a reference dye, such as one or more low AF dyes (e.g. ultra-violet or infra-red), and then a first reference image is acquired using a filter corresponding to the low AF dye
Implementation Method 2
a first reference image is acquired using a filter corresponding to the low AF dye
Data Source
Figure 1(a)~1(b)
Figure 2
Figure 3
AI summary
Method and systems for removing any inherent autofluorescence associated with a biological material, comprising acquiring a first reference image of the biological material; acquiring a first set of one or more images of the biological matepal using one or more filters corresponding to one or more information dyes; applying the one or more additional dyes to the biological material and then acquiring a second set of one or more images comprising a separate image of the biological material with each of the filters corresponding to the information dyes and a second reference image of the biological material; co registering the first and second reference images, and then removing any inherent autofluorescence exhibited in the informational images acquired