Voltron Voltage Indicator Brightness Photostability
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Solution Overview
Problem
Current genetically encoded voltage indicators (GEVIs) face limitations in brightness and photostability, making it difficult to image fast spikes and sub-threshold voltage signals in neurons over extended periods and large fields of view, with existing fluorophores like microbial rhodopsins and fluorescent proteins lacking the necessary brightness and photostability for millisecond-timescale interrogation of neural circuits.
Innovation Solution
Development of a chemigenetic GEVI called Voltron, which uses bright and photostable synthetic dyes like Janelia Fluor dyes, and incorporates mutations to invert the direction of fluorescence change, allowing an increase in membrane potential to result in an increase in fluorescence, enabling rapid and sustained imaging of neuronal spiking and sub-threshold signals with improved photon yield and response time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If microbial rhodopsins or fluorescent proteins are used as fluorophores in GEVIs, then the indicator can be genetically encoded and targeted to specific neurons, but the brightness and photostability are insufficient for in vivo voltage imaging over large fields of view and extended periods
Solution Approach 1:
The patent combines a genetically encoded voltage-sensitive domain (opsin) with a synthetic fluorescent dye (Janelia Fluor dye) to create a hybrid chemigenetic indicator. The voltage-sensitive domain is expressed genetically and targeted to neurons, while the synthetic dye provides superior photophysical properties including high brightness and photostability. This merging resolves the contradiction by integrating the advantages of both genetic encoding and synthetic fluorophore performance.
Solution Approach 2:
The GEVI construct uses a composite approach combining biological components (opsin protein, genetic elements) with synthetic chemical components (Janelia Fluor dye). This composite material strategy allows the indicator to achieve both genetic targetability and the enhanced optical properties of synthetic dyes, overcoming the limitations of purely protein-based fluorophores.
2Productivity
If existing GEVIs are used for voltage imaging, then neuronal activity can be monitored, but the imaging duration and number of neurons imaged simultaneously are limited due to insufficient photon yield
Solution Approach 1:
The patent changes the photophysical parameters of the indicator by replacing protein-based fluorophores with synthetic Janelia Fluor dyes. These dyes exhibit higher quantum yields and photostability, resulting in increased photon yield per indicator molecule. This parameter change enables simultaneous imaging of more neurons over extended durations without signal degradation.
3Measurement precision
If traditional eFRET GEVIs are used, then fluorescence decreases with increasing membrane potential, but this downward-going signal shape reduces signal-to-noise ratio and increases photobleaching
Solution Approach 1:
The patent inverts the traditional eFRET signal direction by engineering the voltage indicator to produce an upward-going fluorescence signal. Instead of fluorescence decreasing with depolarization (downward-going), the designed indicator shows increased fluorescence with increasing membrane potential. This inversion improves signal-to-noise ratio against background fluorescence and reduces photobleaching by spending more time in the low-fluorescence state.
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
Voltron enables in vivo imaging of neuronal activity with an order-of-magnitude improvement in the number of neurons imaged simultaneously and duration, providing clear, rapid, and robust fluorescent signals in response to voltage changes, overcoming the limitations of existing GEVIs by enhancing brightness, photostability, and response speed.
Implementation Method 1
a voltage indicator including a polypeptide sequence comprising a voltage-sensitive opsin domain including one, two, three, or four amino acid mutations relative to a wild type polypeptide sequence, and a capture protein domain arranged and disposed to capture a fluorescent dye ligand. When the fluorescent dye ligand is captured and the voltage indicator is bound to a cell membrane, an increase in voltage across the cell membrane causes an increase in fluorescent emission.
Data Source
AI summary
A voltage indicator includes a polypeptide sequence comprising a voltage-sensitive opsin domain and a capture protein domain arranged and disposed to capture a fluorescent dye ligand. When the fluorescent dye ligand is captured and the voltage indicator is bound to a cell membrane, an increase in voltage across the cell membrane causes an increase in fluorescent emission.