Composite Counterstains for Dual Brightfield Fluorescence Imaging
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Solution Overview
Problem
Current methods require separate slides for brightfield and fluorescence illumination of biological samples, leading to errors and increased time and cost due to the need for mental reconstruction of images, as existing counterstains are incompatible and cause quenching, background interference, or auto-fluorescence.
Innovation Solution
Development of counterstains that combine fluorescent and brightfield capabilities, such as Hoechst 33342 and Celestine Blue, allowing for simultaneous analysis of morphological and molecular details on a single slide using both illumination methods without interference or background fluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If separate slides are used for brightfield and fluorescence staining, then morphological and molecular details can be obtained, but the process becomes more time-consuming and expensive
Solution Approach 1:
The patent combines brightfield and fluorescence stains into a single composite stain formulation that can be applied to one slide. The stain contains multiple fluorescent dyes with different excitation/emission wavelengths along with brightfield chromogenic components, allowing simultaneous visualization of both morphological and molecular features on a single slide rather than requiring separate serial sections.
Solution Approach 2:
The composite stain serves multiple functions simultaneously: it provides brightfield counterstaining for morphological context and multiple fluorescent signals for molecular characterization. This multi-functional stain eliminates the need for separate staining protocols and slides, reducing processing time while maintaining diagnostic accuracy.
2Adaptability or versatility
If separate slides are used for brightfield and fluorescence staining, then both types of illumination can be performed, but the complexity and cost increase
Solution Approach 1:
The patent merges brightfield and fluorescence staining capabilities into a single composite formulation applied to one slide. This reduces the number of slides and staining steps required, simplifying the overall workflow while maintaining compatibility with both brightfield and fluorescence illumination methods.
Solution Approach 2:
The composite stain functions as a multi-component material system containing brightfield chromogens and multiple fluorescent dyes with non-overlapping spectral properties. This composite formulation allows the single slide to respond appropriately to both brightfield and fluorescence illumination, eliminating the need for multiple specialized slides.
3Illumination intensity
If conventional counterstains are used with fluorescence stains, then brightfield illumination is possible, but quenching and background interference occur
Solution Approach 1:
The patent applies local quality by assigning specific functional components to different parts of the composite stain formulation. Brightfield chromogens provide localized colorimetric contrast for morphological features, while fluorescent dyes provide localized fluorescence signals for molecular targets. Each component is optimized for its specific function with minimal interference to the other modality.
Solution Approach 2:
The patent changes the chemical parameters of the counterstain by selecting fluorescent dyes with excitation/emission wavelengths that do not overlap with the brightfield illumination spectrum. This parameter optimization allows the brightfield chromogens to function without quenching fluorescent signals, and vice versa, eliminating the harmful interference observed with conventional counterstains.
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 and efficient analysis of biological samples by providing a single counterstain that functions under both brightfield and fluorescence illumination, reducing errors and costs associated with preparing multiple slides.
Implementation Method 1
fluorescence illumination methods utilize stains specifically developed to provide information at the molecular level
Implementation Method 2
Brightfield illumination utilizes stains specifically developed to provide morphological details
Data Source
AI summary
The present disclosure relates to counterstains for staining a biological sample on a single slide in preparation for microscopic examination. The counterstains are used to analyze the sample on the single slide using both brightfield and fluorescent illumination. The counterstains can identify both morphological details and molecular structures within the cells contained in the sample. The counterstains can be used in conjunction with other molecular stains.