Near-Infrared Fluorogen Activating Proteins for Deep Tissue Imaging
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Solution Overview
Problem
Current fluorogen-activating peptide (FAP) technology is limited by the longest wavelength of fluoromodule excitation/emission maxima at 633/667 nm, which restricts its application in tissue slices and whole animals due to light absorption and scattering, and is confounded by fluorescent drug candidates and biological background fluorescence.
Innovation Solution
Development of new fluorogens and cognate FAPs that absorb and emit at longer wavelengths, specifically up to 733 nm, allowing for near-infrared (NIR) fluorescence, enabling deeper tissue penetration and reduced interference from biological background fluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If conventional fluorogens with excitation/emission maxima at 633/667 nm are used, then the fluorogen-activating peptide technology can be applied to detect cellular proteins, but the application is limited in tissue slices and whole animals due to light absorption and scattering
Solution Approach 1:
The patent applies parameter changes by extending the fluorescence emission wavelength from the conventional 633/667 nm range to the near-infrared region (700-900 nm). This wavelength shift fundamentally changes the optical parameters of the fluorogen, allowing it to penetrate deeper into tissues by avoiding absorption and scattering from hemoglobin and other biological chromophores that dominate the visible light spectrum.
2Measurement precision
If conventional fluorescence-based assays are used, then detection can be performed, but the assays are confounded by fluorescent drug candidates and biological background fluorescence
Solution Approach 1:
The patent converts the harmful effect of biological background fluorescence and fluorescent drug interference into a benefit by operating at near-infrared wavelengths where these interfering substances have minimal absorption or emission. The near-infrared fluorogen emits at wavelengths (700-900 nm) that fall outside the typical fluorescence ranges of most drug candidates and biological molecules, thereby converting the problematic spectral overlap into a clean detection window with minimal background interference.
3Volume of moving object
If the fluorogen emission wavelength is extended to near-infrared region, then deeper tissue penetration is achieved, but the fluorogen structure becomes more complex
Solution Approach 1:
The patent employs composite materials by combining a fluorogen with extended near-infrared emission properties with a fluorogen-activating peptide (FAP) component. This composite fluorogen-FAP system achieves deep tissue penetration through the near-infrared fluorogen while maintaining the biological targeting capability of the FAP. The composite approach allows the fluorogen to be engineered for optimal optical properties without compromising its ability to bind specifically to target proteins on cell surfaces or within cells.
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
The new fluorogen structures facilitate more efficient excitation and detection of fluorescent structures deeper in tissues, reduce false-positive/false-negative events, and enhance detection sensitivity, enabling multi-parameter detection and physiological indicator development.
Implementation Method 1
new fluorogens and cognate FAPs that absorb and emit at longer wavelengths, specifically up to 733 nm, allowing for near-infrared (NIR) fluorescence
Implementation Method 2
The new fluorogen structures facilitate more efficient excitation and detection of fluorescent structures deeper in tissues
Data Source
AI summary
Tissue slices and whole organisms offer substantial challenges to fluorescence imaging. Autofluorescence and absorption via intrinsic chromophores, such as flavins, melanin, and hemoglobins, confound and degrade output from all fluorescent tags. An “optical window,” farther red than most autofluorescence sources and in a region of low hemoglobin and water absorbance, lies between 650 and 900 nm. This valley of relative optical clarity is an attractive target for fluorescence-based studies within tissues, intact organs, and living organisms. Novel fluorescent tags were developed herein, based upon a genetically targeted fluorogen activating protein and cognate fluorogenic dye that yields emission with a peak at 733 nm exclusively when complexed as a “fluoromodule”. This tool improves substantially over previously described far-red/NIR fluorescent proteins in terms of brightness, wavelength, and flexibility by leveraging the flexibility of synthetic chemistry to produce novel chromophores.