Super-resolution 3D Imaging via Temporal Segmentation of Fluorescent Probes
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Solution Overview
Problem
Standard fluorescence microscopy is limited by the diffraction limit, making it unsuitable for ultra-structural imaging, and electron microscopy is difficult to use with biological samples due to its requirements, necessitating new non-invasive techniques for sub-diffraction limit image resolution in three dimensions.
Innovation Solution
A method involving entities that emit light, where the positions in three dimensions are determined using the light emitted by these entities, allowing for sub-diffraction limit image resolution by activating and deactivating entities to achieve precise localization of light-emitting entities even at distances below the diffraction limit, using techniques such as astigmatism imaging and multi-focal-plane imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard fluorescence microscopy is used, then imaging is non-invasive with molecular specificity, but resolution is limited by diffraction to about 200-300 nm
Solution Approach 1:
The patent segments the imaging process into multiple temporal phases: activation phase where only sparse entities are activated, imaging phase where positions are determined, and deactivation phase. This temporal segmentation allows super-resolution by ensuring that at any given moment, activated entities are sufficiently separated to be resolved individually, even when densely packed in the sample
Solution Approach 2:
The patent employs periodic activation and deactivation of light-emitting entities in cycles. During each cycle, a subset of entities is activated for a brief period to emit light for positioning, then deactivated. This periodic action enables repeated measurements and statistical determination of positions with precision far below the diffraction limit
2Measurement precision
If electron microscopy is used to achieve high resolution, then ultra-structural imaging is possible, but sample preparation is complex and invasive
Solution Approach 1:
The patent replaces the mechanical/electronic system of electron microscopy with an optical system using fluorescence microscopy. By substituting electron beams with light and using fluorescently labeled biological molecules, the method achieves comparable ultra-resolution while maintaining sample integrity and enabling imaging of live biological samples without invasive preparation
Solution Approach 2:
The patent changes the fundamental parameter of light interaction by using photoactivatable and photoswitchable fluorescent probes. These probes can be switched between emitting and non-emitting states by light, enabling precise temporal and spatial control that allows super-resolution imaging while keeping samples in their native biological state
3Productivity
If multiple entities are activated simultaneously, then imaging speed increases, but position determination becomes inaccurate due to overlapping diffraction patterns
Solution Approach 1:
The patent introduces dynamic control of entity activation states. Entities transition between dark and emitting states in a controlled temporal sequence, creating a dynamic imaging process where the set of active entities changes over time. This dynamics allows multiple entities to be imaged efficiently while maintaining resolution by ensuring sparse activation at any instant
Solution Approach 2:
The patent performs preliminary selection and activation of specific entity subsets before imaging. By pre-activating only a sparse, non-overlapping subset of entities and determining their positions before activating the next subset, the method accumulates position information for all entities while avoiding the resolution problems that would arise from simultaneous activation of all entities
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 high-resolution imaging of biological samples with molecular specificity, achieving resolutions on the order of 100 nm or less in three dimensions, comparable to electron microscopy but using light, and allowing for three-dimensional visualization of nanoscale features in cells.
Implementation Method 1
The entities may be photoactivatable or photoswitchable, i.e., able to be switched between a first state in which the entity produces light having a desired wavelength and a second state in which the entity does not produce light
Data Source
AI summary
The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques, including imaging in three dimensions. 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 nm, or less than about 300 nm for visible light. In some cases, the position of the entities can be determined in all three spatial dimensions (i.e., in the x, y, and z directions), and in certain cases, the positions in all three dimensions can be determined to an accuracy of less than about 1000 nm. 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 emitted light may be used to determine the x and y positions of the first and second entities, for example, by determining the positions of the images of these entities, and in some cases, with sub-diffraction limit resolution. In some cases, the z positions may be determined using one of a variety of techniques that uses intensity information or focal information (e.g., a lack of focus) to determine the z position. Non-limiting examples of such techniques include astigmatism imaging, off-focus imaging, or multi-focal-plane imaging. 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, and the like.