Fluorescence Imaging Field Flattening via Pixel Remapping
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Solution Overview
Problem
Fluorescence imaging systems face challenges in achieving uniform image flattening across the entire field of view due to spatial variations in fluorescence and scatter signals, which are exacerbated by auto-fluorescence and scattered light, especially at longer exposure lengths where scattered light becomes dominant.
Innovation Solution
The method involves acquiring images of stable fluorescent and scattering targets to calculate a pixel remapping function, which is then used to adjust pixel intensities in sample images, correcting for both fluorescence and scatter spatial variations, and storing this data for later use in imaging systems to form a field-flattened image.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If longer exposure lengths are used to detect low fluorescence signals, then detectability of low fluorescence signals is improved, but scattered light becomes dominant and spatial variation increases
Solution Approach 1:
The patent segments the correction process into two distinct components: fluorescence spatial variation correction and scatter spatial variation correction. By acquiring separate images of fluorescent targets and scattering targets, the system independently characterizes and corrects each type of spatial variation, allowing optimal exposure times for detecting low fluorescence signals without being overwhelmed by scattered light
Solution Approach 2:
The patent uses calibration targets (fluorescent targets and scattering targets) as intermediaries to measure and characterize the spatial variations in the imaging system. These targets serve as reference objects that allow the system to determine correction factors for fluorescence and scatter separately, which are then applied to sample images to eliminate the harmful effects of scattered light
2Stability of the object's composition
If uniform excitation illumination is employed to minimize fluorescence spatial variation, then fluorescence uniformity is improved, but scatter spatial variation remains non-uniform and system-dependent
Solution Approach 1:
The patent separates the correction of fluorescence spatial variation from scatter spatial variation. Uniform excitation illumination addresses fluorescence uniformity, while a distinct scatter correction process using scattering targets handles the non-uniform scatter component. This segmentation allows each correction to be optimized independently without interfering with the other
Solution Approach 2:
The patent changes the parameters being measured by using different target types (fluorescent targets for fluorescence characterization, scattering targets for scatter characterization). By adjusting which parameter is being measured through target selection, the system can independently optimize correction for each type of spatial variation
3Stability of the object's composition
If scattered light is reduced through filtering to improve image uniformity, then spatial variation is reduced, but detection sensitivity may be compromised
Solution Approach 1:
The patent performs preliminary characterization of scatter spatial variation using scattering targets before analyzing sample images. By measuring the scatter pattern in advance and creating a correction map, the system can remove scattered light effects from sample images without applying aggressive filtering that would reduce detection sensitivity
Solution Approach 2:
The patent uses feedback from calibration target images to determine correction factors. The measured spatial variation patterns from fluorescent and scattering targets provide feedback that guides the correction process, allowing the system to adjust for scattered light in a way that preserves detection sensitivity while improving uniformity
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 improves image uniformity across the field of view, enhancing the detectability of low fluorescence signals and reducing the impact of scattered light, particularly at lower light levels, thereby maximizing the usable information from imaging systems.
Implementation Method 1
acquiring a first image of a stable fluorescent target located proximal to the sample platform using the detector, wherein the stable fluorescent target absorbs illumination at said first frequency and emits at said second frequency
Implementation Method 2
acquiring a second image of a scattering target located proximal to the sample platform using the detector, wherein the scattering target scatters illumination at said first frequency
Data Source
AI summary
Systems and methods for flattening the image across the entire field by correcting the image for both the fluorescence and scatter spatial variations. Images of a stable fluorescence target and a scattering target are separately acquired in an imaging system. From these target images, a pixel remapping function, e.g., including two correcting pixel slopes, is calculated for subsequent image pixel remapping. An image of a sample under investigation is then acquired by the imaging system and the sample image is remapped based on the pixel remapping function for the imaging system to form a corrected (field flattened) image. Which correction pixel slope to be used is determined based on whether a sample image pixel value is higher or lower than a threshold value.