Concurrent Fluorescence Sensing with Long-Stokes-Shift Reference
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Solution Overview
Problem
Current fiber photometry devices and 1-photon epifluorescence mesoscope systems are unable to image more than one biological parameter simultaneously with low biological noise, and lack a suitable method to account for biological artifacts when imaging two spectrally orthogonal fluorescent proteins.
Innovation Solution
A molecular strategy using long Stokes shift fluorescent compounds, implemented through fiber photometry and mesoscope imaging, allows for concurrent measurement of multiple biological parameters by employing a lock-in amplification technique and precise control over illumination and imaging frame acquisition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple fluorescent proteins are imaged simultaneously using conventional fiber photometry or mesoscope systems, then the capability to monitor multiple biological parameters is improved, but biological noise and artifacts increase making accurate measurement impossible
Solution Approach 1:
The patent segments the fluorescence detection into multiple spectral channels, each optimized for specific fluorophores. By dividing the detection spectrum into distinct bands and assigning each to specific fluorescent proteins, the system can simultaneously monitor multiple biological parameters while maintaining measurement precision through channel-specific optimization.
Solution Approach 2:
The patent introduces spectral unmixing algorithms as an intermediary processing step between fluorescence signal acquisition and biological parameter interpretation. This computational mediator separates overlapping spectral signatures of different fluorophores, enabling accurate simultaneous measurement of multiple parameters even when their emission spectra overlap.
2Productivity
If spectrally orthogonal fluorescent proteins are used to capture independent biological parameters, then the ability to measure multiple parameters concurrently is improved, but biological artifacts cannot be properly accounted for
Solution Approach 1:
The patent implements a universal reference channel design that can serve multiple functions: monitoring biological artifacts, calibrating spectral unmixing algorithms, and providing a stability reference for all detected fluorophores. This multi-functional reference channel enhances reliability by enabling artifact correction across all simultaneous measurements.
Solution Approach 2:
The patent employs real-time feedback through reference channel monitoring, where detected artifacts and drift are continuously fed back to correct the measurements of target fluorophores. This feedback mechanism dynamically compensates for biological artifacts during the measurement process, maintaining reliability across concurrent parameter measurements.
3Quantity of substance
If conventional fluorescence imaging systems are used to image multiple parameters, then the measurement capability is improved, but instrumental and biological noise cannot be reduced
Solution Approach 1:
The patent implements periodic illumination schemes where different fluorophores are excited in alternating time windows or at different duty cycles. This periodic action allows the system to measure multiple parameters sequentially within each measurement cycle while using the off-periods to characterize and subtract background noise and artifacts specific to each channel.
Solution Approach 2:
The patent employs dynamic measurement protocols that adapt illumination intensity, exposure timing, and detection sensitivity based on the specific fluorophore being measured and current signal-to-noise conditions. This dynamic optimization reduces instrumental noise by adjusting detection parameters in real-time and minimizes biological noise by adapting measurement timing to physiological rhythms.
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 enables artifact-free fluorescent sensing of multiple biological parameters, such as membrane voltage dynamics of different neuron types in awake mice, while reducing instrumentation and biological noise, and is applicable to various fluorescence sensing technologies.
Implementation Method 1
A molecular strategy using long Stokes shift fluorescent compounds
Implementation Method 2
fluorescence signals from a first genetically encoded fluorescent indicator (GEFI), a second GEFI, and a long-Stokes-shift (LSS) fluorescent compound
Data Source
AI summary
A method for concurrently measuring multiple biological parameters in an animal includes: a) illuminating using multiple illumination sources a region-of-interest of the animal that expresses a first genetically encoded fluorescent indicators (GEFI), a second GEFI, and a long-Stokes-shift (LSS) fluorescent compound that is insensitive to the multiple biological parameters; b) concurrently detecting fluorescence signals from the first GEFI, the second GEFI, and the LSS fluorescent compound using a multi-channel fluorescence sensing optical system; and c) processing the detected fluorescence signals to determine values of the multiple biological parameters, wherein the processing reduces instrument and/or biological artifacts in the values of the multiple biological parameters. The first and second GEFIs have distinct absorption spectra, where the first GEFI absorption spectrum overlaps the LSS fluorescent compound absorption spectrum, and the second GEFI emission spectrum overlaps the emission spectrum of the LSS fluorescent compound.