Time-Resolved Fluorescence Imaging for Sub-10 Nanometer Localization
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Solution Overview
Problem
Existing super-resolution fluorescence imaging methods, such as single-molecule localization microscopy (SMLM), struggle to achieve true molecular resolution below 10 nm due to issues like anisotropic photon emission and low localization probability of fluorophores separated by a few nanometers, which are not adequately explained by current models.
Innovation Solution
A method and system for imaging cells using plasmid transfection, dye labeling, pulsed excitation, and time-resolved fluorescence detection with photoswitching fingerprint analysis to track independently emitting quantum systems, enabling sub-10 nm resolution by distinguishing emission energy from multiple fluorophores.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-molecule localization microscopy (SMLM) methods are used to achieve high localization precision (1-5 nm), then spatial resolution is improved, but detection probability of fluorophores separated by a few nanometers deteriorates
Solution Approach 1:
The patent introduces time-resolved fluorescence detection as an additional dimension to spatial localization. By analyzing fluorescence trajectories and emission energy over time, the system can distinguish between closely spaced fluorophores that appear overlapping in spatial domain, thereby maintaining high localization precision while improving detection probability for sub-10 nm separated fluorophores.
Solution Approach 2:
The patent segments the fluorescence signal analysis into multiple components: spatial position, temporal trajectory, and emission energy characteristics. This segmentation allows independent optimization of each parameter, enabling the system to achieve both precise localization and reliable detection by analyzing different aspects of the fluorescence signal separately.
2Measurement precision
If fluorophores are labeled at sub-10 nm distances to achieve molecular resolution, then imaging resolution is improved, but anisotropic photon emission and localization bias increase
Solution Approach 1:
The patent employs feedback mechanisms through real-time analysis of fluorescence trajectories and emission energy patterns. By continuously monitoring temporal variations in photon emission and comparing them against expected patterns, the system can compensate for anisotropic emission effects and localization bias, maintaining accurate positioning even when fluorophores are separated by sub-10 nm distances.
Solution Approach 2:
The patent utilizes temporal parameters (fluorescence lifetime, emission energy distribution over time) in addition to spatial parameters. By analyzing changes in emission characteristics over time rather than relying solely on spatial position, the system can distinguish between fluorophores that would otherwise appear indistinguishable due to anisotropic emission or localization bias.
3Measurement precision
If conventional SMLM methods are used to image sub-10 nm structures, then localization precision is improved, but current models fail to explain observed behavior
Solution Approach 1:
The patent adds temporal dimension to the existing spatial model by analyzing fluorescence trajectories and time-resolved emission energy. This extended model can account for dynamic effects such as fluorophore blinking, photobleaching, and anisotropic emission that conventional static models cannot explain, thereby improving model accuracy while maintaining high localization precision.
Solution Approach 2:
The patent creates a comprehensive digital model that copies and analyzes multiple aspects of fluorescence behavior (spatial position, temporal trajectory, emission energy spectrum) to better represent actual fluorophore behavior. This multi-parameter modeling approach allows the system to explain observed behaviors that simpler models cannot account for.
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
The method and system enhance imaging resolution to the sub-10 nm level by accurately tracking fluorophore locations and emission trajectories, overcoming limitations in previous methods and providing reliable molecular resolution.
Implementation Method 1
labeling the transfected one or more cells with a dye; exciting the dye with an energy source, wherein the dye is configured to emit an emission light
Implementation Method 2
imaging one or more portions of the transfected one or more cells with a sensor
Data Source
AI summary
The disclosure includes a system and method for imaging cells. The method includes: labeling transfected one or more cells with a dye; exciting the dye with an energy source, wherein the dye is configured to emit an emission light; imaging one or more portions of the transfected one or more cells with a sensor, for a predetermined exposure time, a plurality of times to form a plurality of images; classifying each image of the plurality of images into a first group of images above a predetermined threshold for a number of photons per image or a second group of images below the predetermined threshold the number of photons per image; and determining whether the emission energy is from one or more independently emitting quantum systems based on the measured emission, the tracked locations of the one or more portions and the fluorescence trajectory.