Photoswitchable Molecules Sub-Diffraction Imaging
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Solution Overview
Problem
Standard fluorescence microscopy is limited by the diffraction limit, preventing ultra-resolution imaging of biological samples, and electron microscopy is cumbersome due to its requirements, necessitating new techniques to harness fluorescence microscopy for high-resolution imaging.
Innovation Solution
A method involving light-emitting entities that can be selectively activated and deactivated to determine their positions with sub-diffraction limit resolution, using photoswitchable molecules and time-modulated light sources to achieve high-resolution imaging by cycling through fluorescent and dark states.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard fluorescence microscopy is used, then non-invasive time-resolved imaging is achieved, but resolution is limited by diffraction
Solution Approach 1:
The patent applies preliminary action by pre-activating only a sparse subset of fluorophores before imaging. This allows individual fluorophores to be resolved despite being closer than the diffraction limit, because they are activated in a temporally controlled sequence rather than all simultaneously. The sparse activation pattern enables precise localization of each fluorophore position.
Solution Approach 2:
The patent employs periodic action through repeated cycles of sparse activation, imaging, and deactivation. Each cycle activates a different sparse subset of fluorophores, images their positions, then deactivates them. This periodic cycling continues until all fluorophores have been imaged, building up a complete super-resolution image over time while maintaining the ability to resolve sub-diffraction features.
2Measurement precision
If electron microscopy is used for high resolution imaging, then ultra-structural detail is achieved, but sample preparation becomes cumbersome
Solution Approach 1:
The patent replaces the mechanical/electrical system of electron microscopy with an optical system using fluorescence microscopy. Instead of using electrons and complex vacuum-based imaging, the invention uses light-emitting fluorophores that can be activated and imaged with standard optical microscopes, thereby achieving super-resolution without the cumbersome sample preparation required for electron microscopy.
3Measurement precision
If multiple fluorophores are activated simultaneously, then complete image data is obtained, but resolution is lost due to overlapping signals
Solution Approach 1:
The patent applies partial action by activating only a sparse subset of fluorophores at any given time rather than all fluorophores simultaneously. This sparse activation ensures that the number of emitting fluorophores is less than the resolution limit would normally allow, preventing signal overlap and enabling precise position determination. The process is repeated multiple times with different subsets to eventually image all fluorophores.
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 sub-diffraction limit resolution imaging, allowing for precise localization of entities separated by distances less than the wavelength of light, improving imaging resolution beyond conventional fluorescence microscopy and simplifying sample preparation compared to electron microscopy.
Implementation Method 1
using photoswitchable molecules and time-modulated light sources to achieve high-resolution imaging by cycling through fluorescent and dark states
Data Source
Figure 1A
Figure 1B
Figure 2A~2D
AI summary
The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. For example, the entities may be separated by a distance of less than about 1000 run, or less than about 300 run for visible light. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. A first entity may be activated and determined (e.g., by determining light emitted by the entity), then a second entity may be activated and determined. The entities may be immobilized relative to each other and/or to a common entity. The emitted light may be used to determine the positions of the first and second entities, for example, using Gaussian fitting or other mathematical techniques, and in some cases, with sub-diffraction limit resolution. The methods may thus be used, for example, to determine the locations of two or more entities immobilized relative to a common entity, for example, a surface, or a biological entity such as DNA, a protein, a cell, a tissue, etc. The entities may also be determined with respect to time, for example, to determine a time-varying reaction. Other aspects of the invention relate to systems for sub-diffraction limit image resolution, computer programs and techniques for sub-diffraction limit image resolution, methods for promoting sub-diffraction limit image resolution, methods for producing photoswitchable entities, and the like.