SPRM 3D Single-Molecule Tracking via Axial Intensity
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Solution Overview
Problem
Current techniques for tracking single-molecule dynamics in biological systems lack precision, particularly in the axial direction, and are limited by photobleaching and the complexity of introducing additional optical components for 3D tracking.
Innovation Solution
The use of surface plasmon resonance microscopy (SPRM) for multiplexed 3D single-particle tracking with sub-nanometer axial resolution and kHz frame rate, which directly extracts axial information from scattering intensity, allowing for precise tracking of molecular dynamics in three dimensions without additional complexity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence-based tracking is used, then molecular dynamics can be tracked, but temporal resolution and tracking precision are compromised due to photobleaching and limited photons
Solution Approach 1:
The patent replaces fragile fluorescent dyes that suffer from photobleaching with robust nanoparticles that can withstand prolonged illumination. The nanoparticles serve as durable, reusable tracking labels that maintain their optical properties over extended periods, enabling long-duration single-molecule tracking experiments without signal degradation.
2Illumination intensity
If nanoparticles are used as labels, then strong optical signals are achieved, but only 2D projection tracking is possible leading to biased results
Solution Approach 1:
The patent transitions from 2D projection tracking to 3D tracking by utilizing the axial intensity variation of the evanescent field. The scattering intensity of nanoparticles changes predictably with their axial position, allowing the system to extract z-coordinate information from the same 2D image plane without requiring additional optical components or complex hardware modifications.
3Measurement precision
If additional optical components are added for 3D tracking, then axial movement can be determined, but system complexity increases
Solution Approach 1:
The patent leverages the inherent properties of the evanescent field and nanoparticle scattering interaction to provide self-contained 3D tracking capability. The system uses the same illumination and detection optics already present in the SPRM setup, allowing axial position information to be extracted from intensity variations in the existing 2D image channel without requiring separate detection paths or additional specialized components.
4Speed
If high-speed laser scanning is used for 3D tracking, then multiple focal planes can be localized, but sub-nanometer resolution at kHz frame rate remains challenging
Solution Approach 1:
The patent exploits the exponential decay parameter of the evanescent field intensity with axial distance to achieve high-precision axial measurement. By calibrating the relationship between scattering intensity and axial position, the system can determine z-coordinates with sub-nanometer precision at kHz frame rates using simple intensity measurements rather than complex multi-plane scanning procedures.
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-precision 3D tracking of single molecules with sub-nanometer axial and lateral precision and millisecond time resolution, providing insights into molecular interactions and dynamics, and distinguishing specific from non-specific interactions in biosensing applications.
Implementation Method 1
introducing an incident light toward a second surface of a substrate to induce a plasmonic wave at least proximal to a first surface of the substrate
Implementation Method 2
detecting a change in position of one or more of the particles in the population along at least three dimensions over a duration, which three dimensions comprise two substantially lateral dimensions and an axial dimension, from a change in intensity of the incident light reflected at an interface of the first surface of the substrate
Data Source
AI summary
Provided herein are methods of tracking molecular dynamics in three dimensions. In some embodiments, the methods include introducing an incident light toward a second surface of a substrate to induce a plasmonic wave at least proximal to a first surface of the substrate. A population of particles is connected to the first surface of the substrate via one or more first biomolecules. In some embodiments, the methods also include detecting a change in position of the particles in the population along at least three dimensions over a duration from a change in intensity of the incident light reflected at an interface of the first surface of the substrate. Related systems and computer readable media are also provided.