Confocal Microscope Scanning for Particle Diffusion Measurement
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Solution Overview
Problem
Current optical analysis techniques, such as FCS and FIDA, require high concentrations of fluorescent molecules to detect individual particles, limiting their ability to analyze particles at lower concentrations and failing to quantify diffusion constants of particles moving randomly in solutions.
Innovation Solution
A method using a confocal or multiphoton microscope to detect light from a micro region in a sample solution by moving a light detection region periodically, allowing for the individual detection and quantification of light-emitting particles and computation of their diffusion characteristics, even at lower concentrations, by analyzing the deviation times of signal intervals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional optical analysis techniques (FCS, FIDA) are used to detect fluorescent molecules, then measurement of fluorescence intensity and molecular characteristics is achieved, but the required fluorescent molecule concentration is extremely high and sample amount is large
Solution Approach 1:
The invention divides the measurement into two independent stages: first detecting individual fluorescent molecules one by one using single-molecule sensitivity, then statistically analyzing their diffusion characteristics. This segmentation allows detection at extremely low concentrations while maintaining measurement precision through statistical accumulation of individual molecule data.
Solution Approach 2:
The invention transitions from conventional bulk fluorescence measurement to single-molecule tracking in the temporal dimension. By recording the arrival times of individual photons from single molecules and analyzing their diffusion trajectories over time, the method achieves both high detection sensitivity and ability to measure molecular characteristics at extremely low concentrations.
2Measurement precision
If high concentration of fluorescent molecules is used to ensure sufficient signal for analysis, then measurement accuracy is improved, but the ability to analyze rare particles and expensive samples is reduced
Solution Approach 1:
The method segments the analysis into individual molecule detection and statistical evaluation. Each rare fluorescent molecule is detected and tracked individually, then their collective diffusion characteristics are analyzed statistically. This allows accurate measurement of rare particles without requiring high concentration, as each molecule contributes to the statistical analysis.
Solution Approach 2:
The system automatically identifies and tracks individual fluorescent molecules based on their photon emission patterns, without requiring external manipulation or high concentration input. The method self-adapts to the actual concentration level by statistically accumulating data from the detected molecules, making it versatile for analyzing both rare and abundant species.
3Measurement precision
If conventional fluorescence measurement techniques are used, then average molecular characteristics can be measured, but diffusion constant quantification of individual particles cannot be achieved
Solution Approach 1:
The invention performs preliminary tracking of individual molecule positions and arrival times before statistical analysis. By first recording the temporal sequence of individual photon arrivals and calculating mean square displacements, the method prepares data that directly enables diffusion constant quantification, transforming qualitative fluorescence detection into quantitative diffusion measurement.
Solution Approach 2:
The method replaces conventional bulk fluorescence measurement with single-molecule photon counting and trajectory analysis. By substituting the mechanical averaging process with individual molecule tracking and statistical evaluation of their diffusion paths, the system achieves both molecular characteristic measurement and diffusion constant quantification.
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 the detection and analysis of light-emitting particles and their diffusion constants at lower concentrations than previously possible, reducing the required sample amount and measurement time while providing accurate diffusion characteristic values.
Implementation Method 1
a particle which emits light (hereafter, referred to as a 'light-emitting particle') may be any of a particle which itself emits light and a particle to which an arbitrary light-emitting label has been attached, and the light emitted from a light-emitting particle may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, etc.
Implementation Method 2
the light emitted from a light-emitting particle may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, etc.
Implementation Method 3
the light emitted from a light-emitting particle may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, etc.
Implementation Method 4
the light emitted from a light-emitting particle may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, etc.
Implementation Method 5
the light emitted from a light-emitting particle may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, etc.
Implementation Method 6
a light-emitting particle dispersed and moving at random in a sample solution
Data Source
AI summary
There is provided a method of measuring a diffusion characteristic value (for example, a diffusion constant) of a light-emitting particle using the scanning molecule counting method using the optical measurement with a confocal microscope or a multiphoton microscope. The inventive method of measuring a diffusion characteristic value of a light-emitting particle is characterized to measure light intensity from the light detection region with moving the position of the light detection region in the sample solution by changing an optical path of the optical system to generate light intensity data and to compute a diffusion characteristic value of the light-emitting particle based on a deviation time from a moving cycle time of the light detection region in an interval of generation times of two or more signals corresponding to a same light-emitting particle on the light intensity data.