Adaptive Sample-Fluid Control for Single-Molecule Localization Imaging
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Solution Overview
Problem
Existing super-resolution microscopy techniques face challenges in controlling photo-physical and photo-chemical properties of light-emitting molecules in real-time during experiments, which affects the efficiency and accuracy of single molecule localization and imaging.
Innovation Solution
A method and microscope system that adjusts the composition of the sample fluid in real-time by using a fluidic device, based on detected light properties, to optimize parameters such as blinking rate and emission lifetime of light-emitting molecules, thereby enhancing the accuracy of super-resolution imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If photo-activatable or photo-switchable fluorophores are used to enable single molecule localization, then super-resolution imaging below the diffraction limit is achieved, but real-time control of photo-physical and photo-chemical properties during experiments becomes difficult
Solution Approach 1:
The system continuously monitors the actual number of emitting molecules during imaging and compares it to the target value, then automatically adjusts buffer composition (oxidizing/reducing agent ratios) to maintain optimal conditions. This closed-loop feedback enables real-time adaptation of photo-chemical properties while preserving super-resolution localization precision.
Solution Approach 2:
The patent transforms static buffer conditions into dynamic, adjustable parameters. By implementing real-time modification of buffer composition through fluidic devices, the system adapts photo-physical properties (blinking rate, emission lifetime) during experiments, allowing optimization for different imaging stages and conditions.
2Reliability
If buffer composition is manually optimized to control blinking rate and emission lifetime, then imaging quality is improved, but experimental complexity and time consumption increase
Solution Approach 1:
The system performs self-optimization by automatically monitoring imaging quality metrics and adjusting buffer composition without manual intervention. The control device autonomously determines optimal buffer conditions based on real-time data, eliminating the need for manual trial-and-error optimization while maintaining high imaging quality.
Solution Approach 2:
The patent implements automated adjustment of chemical parameters (buffer composition, oxidizing/reducing agent ratios) to control photo-physical properties. This replaces manual optimization with systematic, real-time parameter modification, reducing experimental complexity while improving reliability.
3Measurement precision
If the number of emitting molecules is kept low for single molecule localization, then resolution below diffraction limit is achieved, but imaging speed decreases
Solution Approach 1:
The system employs periodic switching of molecules between dark and bright states through controlled buffer composition changes. This allows different cohorts of molecules to emit photons at different times, effectively multiplying the information collected per unit time while maintaining low instantaneous emitter density for super-resolution.
Solution Approach 2:
By dynamically adjusting buffer conditions to maintain optimal blinking rates, the system ensures continuous useful signal generation. The automated control prevents periods of suboptimal imaging by continuously adapting conditions, thereby maximizing imaging speed without sacrificing resolution.
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 precise control of imaging conditions to maintain a desired number of molecules in the bright state, improving the resolution and speed of super-resolution microscopy by ensuring optimal photo-physical and photo-chemical conditions.
Implementation Method 1
a light source (2) for illuminating a sample (S)
Implementation Method 2
a detector (3) for detecting light emitted by the light-emitting molecules in the sample (S)
Implementation Method 3
a fluidic device (7) for adjusting a composition of a sample fluid (6)
Data Source
Figure 1~2
Figure 3~4
AI summary
The invention relates to a method, a microscope (1) and a computer program for localizing and/or imaging light-emitting molecules (M) in a sample (S) contained in a sample reservoir (6) comprising illuminating the sample (S) by a light source (2) comprised in or connected to a microscope (100), detecting light emitted by the light-emitting molecules (M) in the sample (S) by a detector (3) comprised in or connected to the microscope (100), wherein the light-emitting molecules (M) comprise a bright state (B) and a dark state (D), determining, based on the detected light (F), a current value of one or more parameters, wherein at least one or the parameters co-depends on the photo-physical or photo-chemical properties of the light-emitting molecules (M) other than their average photon emission rate, and adjusting a composition of a fluid in the sample reservoir (6) to optimize the one or more parameters during the localization or imaging of the light-emitting molecules.