Fluorescence Imaging Contrast via Patterned Illumination and Background Subtraction
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Solution Overview
Problem
Fluorescence imaging is hindered by a poor signal-to-background ratio due to reflection of excitation light and strong fluorescence signals from the surface, limiting the effectiveness of methods like FLIM and multi-spectral imaging, especially when the concentration of bio-markers is low.
Innovation Solution
A method and system utilizing patterned illumination to enhance contrast in fluorescence imaging, where a patterned illumination source is used to illuminate a subject, and image frames are processed to deduct background fluorescence, allowing for deeper penetration and improved signal detection from subsurface targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of stationary object
If conventional fluorescence imaging is used to image deep tissue targets, then the imaging depth can be achieved, but the signal-to-background ratio deteriorates due to surface reflection and near-surface fluorescence
Solution Approach 1:
The imaging process is segmented into multiple sequential steps: first acquiring a reference image without the target, then acquiring an image with the target, and finally subtracting the reference from the target image. This segmentation allows separate measurement of background fluorescence and total signal, enabling effective background removal and improvement of signal-to-background ratio for deep tissue targets
Solution Approach 2:
A reference image is acquired beforehand (before introducing the target) to capture the background fluorescence distribution. This preliminary measurement is then used to subtract the background from subsequent target images, eliminating the need for simultaneous background measurement and enabling accurate deep tissue target detection
2Measurement precision
If FLIM is used to differentiate fluorescent markers from tissue, then background auto-fluorescence is reduced, but the utility is limited when lifetime difference is small
Solution Approach 1:
The method changes the measurement parameter from fluorescence lifetime (used in FLIM) to spatial-temporal signal characteristics during washout. By monitoring how fluorescence signal decays over time as contrast agent washes out, the system can differentiate targets regardless of their lifetime properties, making it compatible with existing fluorescent markers that have small lifetime differences from tissue
3Measurement precision
If multi-spectral imaging with chemometric analysis is used to distinguish fluorescence signals, then background and markers can be differentiated, but signal loss occurs and real-time analysis is compromised
Solution Approach 1:
The method extracts only the necessary information (background fluorescence distribution from reference image) and removes it from the target image through simple subtraction. This avoids the complex chemometric analysis of full spectral data, reducing computational burden while maintaining the ability to differentiate signals, thus enabling real-time imaging
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 significantly enhances the signal-to-background ratio, improving the detection of fluorescent markers by reducing noise from surface reflections and near-surface fluorescence, enabling more accurate and sensitive imaging, particularly in real-time applications.
Implementation Method 1
The tissue inside the subject fluoresces either endogenously or exogenously in response to the excitation illumination
Implementation Method 2
the resulting emission is imaged to obtain information about the internal composition of the subject
Data Source
AI summary
A method for enhancing contrast in fluorescence imaging is provided. The method comprises providing a patterned illumination source for illuminating one or more regions corresponding to a scan step, scanning at least a portion of a surface of a subject using a plurality of scan steps, acquiring image frames corresponding to two or more scan steps, deducting a background fluorescence from the image frames corresponding to the two or more scan steps to form one or more processed image frames, and reconstructing an image using one or more of the processed image frames.