Confocal Microscope Scanning for Single Particle Detection
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Solution Overview
Problem
Current optical analysis techniques using confocal microscopes struggle to detect and quantify light-emitting particles at low concentrations, as they require statistically significant numbers of particles within the confocal volume for analysis, limiting their ability to identify individual particles and determine their characteristics accurately.
Innovation Solution
An optical analysis device and method that uses a confocal or multiphoton microscope with a moving light detection region to detect light from individual light-emitting particles across multiple wavelength bands, allowing for the identification of particle types based on their emission wavelength characteristics, even at low concentrations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional optical analysis techniques (FCS, FIDA) are used to detect fluorescent molecules in a confocal volume, then statistical average characteristics can be obtained, but individual particle detection and identification is not possible and low concentration samples cannot be analyzed
Solution Approach 1:
The patent segments the detection process by separating individual particle detection from statistical averaging. The confocal volume is used to isolate single particles, and the system records time-resolved fluorescence intensity signals for each individual particle event, enabling identification of individual particles rather than just statistical averages of populations
Solution Approach 2:
The patent employs periodic scanning of the confocal volume through sample movement or stage scanning, allowing repeated detection opportunities for individual particles. This periodic sampling enables the system to detect low concentration particles by accumulating detection events over multiple scanning cycles
2Loss of information
If statistical procedures are used to analyze fluorescence intensity data, then average characteristics of fluorescent molecules can be determined, but the signal from individual fluorescent molecules cannot be seen or analyzed
Solution Approach 1:
The patent performs preliminary action by recording and preserving individual particle fluorescence signals before applying any statistical analysis. The system captures time-resolved intensity data for each individual particle event, maintaining the integrity of individual particle information while enabling subsequent statistical processing if needed
Solution Approach 2:
The patent introduces dynamics by analyzing the temporal characteristics of fluorescence signals. By examining the time course of fluorescence intensity for individual particles (including dwell time in the confocal volume), the system can identify and characterize individual particles while preserving their unique temporal signatures
3Productivity
If a confocal microscope with fixed detection region is used, then fluorescence intensity can be measured, but the detection region cannot be moved to scan the sample solution for individual particles
Solution Approach 1:
The patent makes the confocal microscope system multi-functional by integrating sample scanning capability with the existing fluorescence detection system. The optical path is modified to accommodate movable mirrors or galvanometric scanners that enable both fixed-point measurement and systematic scanning of the sample solution, allowing the same device to perform multiple detection modes
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 identification of individual light-emitting particles and determination of their concentration and characteristics in solutions with low particle densities, enhancing the analysis of biological and non-biological particles by reducing the required sample amount and measurement time.
Implementation Method 1
the light emitted from a light-emitting particle may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, etc.
Implementation Method 2
the light emitted from a light-emitting particle may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, etc.
Implementation Method 3
the light emitted from a light-emitting particle may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, etc.
Implementation Method 4
the light emitted from a light-emitting particle may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, etc.
Implementation Method 5
the focal region to which the laser light of the microscope is condensed, called a 'confocal volume'
Data Source
AI summary
The inventive technique of detecting and analyzing light from a light-emitting particle in accordance with the scanning molecule counting method using an optical measurement with a confocal microscope or a multiphoton microscope is characterized by detecting intensities of components of two or more wavelength bands of light from a light detection region of an optical system with moving the position of the light detection region in a sample solution by changing the optical path of the optical system of the microscope; detecting individually signals of the light from each light-emitting particle in the intensities of the components of the two or more wavelength bands of the detected light; and identifying a kind of light-emitting particle based on the intensities of the components of the two or more wavelength bands of the signals of the light of the detected light-emitting particle.