Optogenetic Membrane Potential Probes via Microbial Rhodopsins
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Solution Overview
Problem
Current methods for measuring membrane potential are invasive, limited to specific cell types, and cannot access deeply buried or small tissues, making it difficult to study membrane potential in various biological contexts, especially in vivo and in small or motile cells.
Innovation Solution
The use of microbial rhodopsins as optical sensors to detect voltage across phospholipid layers by expressing them in cells, exposing them to light, and measuring fluorescence intensity, which reflects membrane potential, allowing for non-invasive, high-throughput measurement of membrane potential changes without the need for electrodes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If electrodes are used to measure membrane potential, then direct voltage recording is achieved, but the method is invasive, slow, and damages or kills the cell
Solution Approach 1:
The patent replaces the mechanical/electrical electrode system with an optical sensing system. Microbial rhodopsins are expressed in cells and detect membrane potential through optical signals (fluorescence emission) rather than electrical contact. This substitution eliminates the need for physical electrode insertion, thereby avoiding cell damage while maintaining measurement capability.
Solution Approach 2:
The patent introduces microbial rhodopsins as intermediary sensors between the membrane potential and the detection system. These rhodopsin proteins convert electrical potential differences into optical signals, serving as a mediator that translates the electrical parameter into a detectable optical form without requiring direct electrical contact with the cell.
2Measurement precision
If electrodes are positioned on both sides of the membrane, then direct voltage recording is achieved, but the setup is slow and can only be performed on one or a few cells at a time
Solution Approach 1:
The patent creates multiple copies of the measurement capability by expressing microbial rhodopsins throughout the cell membrane. Instead of using a single electrode pair, the rhodopsin proteins are distributed across the membrane, allowing simultaneous optical measurement of many cells in parallel, thereby dramatically increasing throughput.
Solution Approach 2:
The optical detection method replaces the slow mechanical electrode positioning and recording process. Optical signals can be captured simultaneously from multiple cells using fluorescence microscopy techniques, enabling high-throughput measurement without the time-consuming electrode setup required for each individual cell.
3Measurement precision
If electrodes are used, then membrane potential can be measured, but deeply buried tissues and small cells cannot be accessed
Solution Approach 1:
The patent replaces the mechanical electrode approach with an optical system that can penetrate tissues and access small cells. Optical signals can travel through tissues and detect fluorescence from rhodopsins in cells that would be inaccessible to electrodes, including deeply buried cells and those in hard-to-reach locations.
Solution Approach 2:
The microbial rhodopsin-based system provides universal applicability across different cell types and tissue locations. The same optical sensing principle can be applied to various cell types (neurons, cardiac cells, bacteria) and tissue depths, making the measurement method universally adaptable where electrode methods are limited.
4Measurement precision
If microbial rhodopsin with ion pump activity is used, then optical sensing is achieved, but the rhodopsin participates in altering voltage through establishing ionic gradients
Solution Approach 1:
The patent extracts or removes the ion pump function from the microbial rhodopsin while retaining its optical sensing capability. By using mutants or modified rhodopsins that lack proton pumping activity, the system eliminates the confounding effect of ionic gradient establishment, allowing the rhodopsin to function as a pure voltage sensor without altering the membrane potential.
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 the optical measurement of membrane potential in various cell types, including small and deeply buried tissues, with high spatial and temporal resolution, facilitating research and drug screening applications.
Implementation Method 1
exposing the cell to a light source and detecting the emitted fluorescence from the microbial rhodopsin, wherein the intensity of the emitted fluorescence reflects membrane potential
Implementation Method 2
microbial rhodopsin proteins... can be used as optical sensors to sense voltage across membranes in a cell
Data Source
AI summary
The invention provides methods, cells and constructs for optical measurement of membrane potential. These methods can be used in cells that are not accessible to presently available methods using electrodes. The methods can be directed to, for example, high-throughput drug screening assays to determine agents that can affect membrane potential of a target cell.