MINFLUX Microscope Trajectory Tracking With Adaptive Termination
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Solution Overview
Problem
Existing methods for tracking individual particles in samples face limitations in observation period and temporal resolution, particularly in low-viscosity media, and often require immobilization, which restricts applications in native, living systems, and lack effective termination criteria for irrelevant trajectory tracking.
Innovation Solution
A method and light microscope that tracks particles using the MINFLUX principle, incorporating a second measured variable to define a termination criterion for trajectory recording, ensuring relevant information is captured while minimizing exposure to scanning light and measurement time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If point detector-based methods are used to achieve high temporal resolution, then temporal resolution is improved, but observation period is limited to dwell time in focus
Solution Approach 1:
The system dynamically adjusts the focus position using a piezoelectric stage to track the particle's axial position in real-time, maintaining the particle in focus throughout its trajectory rather than relying on a fixed focal plane. This dynamic focusing extends the observation period while preserving high temporal resolution through fast beam deflection.
Solution Approach 2:
The system uses feedback from the detected photon signal to continuously update the particle's position and adjust the focus position accordingly. This closed-loop control enables the system to track particles through large axial ranges while maintaining optimal focus conditions throughout the extended observation period.
2Duration of action of moving object
If particles are immobilized using optical tweezers to extend observation period, then observation period is improved, but applicability in native living systems is restricted
Solution Approach 1:
The patent extracts the focusing function from the sample preparation (removing the need for immobilization) and implements it in the detection system through dynamic focus adjustment. This allows freely diffusing particles in native environments to be tracked over extended periods without requiring coupling to carrier particles or immobilization.
Solution Approach 2:
The patent replaces the mechanical immobilization approach (optical tweezers, carrier particles) with an optical detection approach (dynamic focus tracking with piezoelectric stage and fast beam deflection). This substitution enables tracking of freely moving particles in their natural state while achieving extended observation periods.
3Measurement precision
If scanning light intensity is increased to improve localization precision, then measurement precision is improved, but photon budget is depleted faster
Solution Approach 1:
The system dynamically adjusts the scanning light intensity based on the particle's position and the current localization precision requirements. When the particle is well-localized, intensity is reduced to conserve photons. When precision needs improvement, intensity is temporarily increased. This dynamic adjustment optimizes the trade-off between precision and photon consumption throughout the trajectory tracking.
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 efficient and precise tracking of individual particles by terminating trajectories when irrelevant information is detected, reducing unnecessary scanning and exposure, and allowing for larger data sets to be acquired within a given time frame.
Implementation Method 1
Fluorescent particles or single molecules of a fluorescent dye are most commonly used. The particle is illuminated with a light distribution of a scanning light having an intensity minimum and the photons emitted by the particle are registered.
Implementation Method 2
Light-scattering particles, such as metallic nanoparticles, can be used for this purpose
Implementation Method 3
The piezoelectric-based stages used in this work for tracking the sample
Implementation Method 4
fast beam deflection means, in particular electro-optical or acousto optical deflectors
Implementation Method 5
fast beam deflection means, in particular electro-optical or acousto optical deflectors
Implementation Method 6
galvanometer mirrors, which allow (slower) pre-positioning of the focused excitation light in a large area of the sample
Data Source
AI summary
The invention is directed to a method for recording a motion trajectory of an individual particle in a sample and to a light microscope performing this method. Starting from an at least approximately known initial position, the particle is scanned with an intensity distribution of a scanning light comprising a local intensity minimum. When illuminated with the scanning light, the particle to be tracked generates a detectable light signal, from the intensity of which updated coordinates of the particle are calculated. According to the invention, the scanning is terminated when a second measured variable detected in parallel satisfies a termination criterion.