Scanning Molecule Counting for Low-Concentration Fluorescent Particle Detection
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Solution Overview
Problem
Current optical analysis technologies, such as FCS and FIDA, require high concentrations of fluorescent molecules for accurate analysis, limiting their effectiveness when concentrations are below 1 nM, and existing methods for detecting low concentrations are either insensitive or require large sample volumes and complex procedures.
Innovation Solution
The scanning molecule counting method, which involves detecting fluorescent particles using a photodetection region moved at a speed faster than their diffusion, in the presence of a triplet excited state quenching agent, allowing for sensitive detection of low-concentration particles without statistical processing of fluorescence intensity fluctuations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional optical analysis technologies (FCS, FIDA) are used to detect fluorescent molecules, then measurement can be performed with small sample volumes and short measurement times, but detection sensitivity is insufficient when fluorescent molecule concentration is below 1 nM
Solution Approach 1:
The invention segments the continuous fluorescence intensity signal into discrete photon counting events. By detecting individual photons and their arrival times, the method transforms a continuous analog measurement into discrete digital counts, enabling precise measurement of extremely low concentration fluorescent molecules that would be indistinguishable from noise in conventional continuous intensity measurement.
Solution Approach 2:
The invention employs periodic scanning of the photodetection region through the sample solution. The confocal microscope systematically moves the detection volume through the sample in a periodic manner, allowing repeated measurements and statistical accumulation of photon events. This periodic scanning enables detection of rare fluorescent molecules at concentrations below 1 nM by accumulating sufficient photon counts over multiple scan cycles.
2Measurement precision
If the photodetection region is moved slowly to allow fluorescent particles to diffuse into it, then detection efficiency increases, but measurement time increases and particles may diffuse out before detection
Solution Approach 1:
The invention replaces mechanical movement of the sample with optical scanning of the detection region. Instead of physically moving or stirring the sample solution, the confocal microscope uses galvanometer mirrors to rapidly scan the focused laser beam and photodetection volume through the sample. This optical scanning system achieves high-speed coverage of the sample volume without mechanical disturbance, enabling fast detection while maintaining high detection efficiency through rapid repeated sampling of different regions.
3Measurement precision
If statistical processing of fluorescence intensity fluctuations is used to detect low-concentration particles, then detection capability improves, but complex arithmetic processing is required
Solution Approach 1:
The invention extracts only the essential detection information (photon arrival times and counts) from the fluorescence signal, discarding unnecessary continuous intensity data. By focusing solely on photon counting events and their temporal distribution, the method achieves effective detection of low-concentration particles while avoiding complex continuous signal processing. The analysis extracts key statistical parameters (photon counts, correlation functions) from the simplified photon event data rather than processing the entire continuous intensity waveform.
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 of fluorescent particles with high sensitivity and accuracy at extremely low concentrations, using minimal sample volumes and avoiding complex procedures, thereby improving analysis efficiency and reducing sample requirements.
Implementation Method 1
detecting fluorescent particles using an optical system capable of detecting light from a microregion
Implementation Method 2
ultra-high-sensitivity photodetection technology capable of performing photon counting (detecting a single photon)
Implementation Method 3
detecting fluorescent particles using a photodetection region moved at a speed faster than their diffusion, in the presence of a triplet excited state quenching agent
Data Source
Figure 1A~1B
Figure 1C~2B
Figure 3A~4B
AI summary
A method for detecting a fluorescent particle comprises the preparation of a sample solution containing fluorescent particles and a substance that promotes transition of the fluorescent particles from a triplet excited state to a singlet ground state, and calculation of the number of molecules of fluorescent particles present in the prepared sample solution. Calculation of the number of molecules of the fluorescent particles comprises moving the location of a photodetection region of an optical system in the sample solution using the optical system of a confocal microscope or multi-photon microscope, individually detecting fluorescent particles by detecting a light signal from the fluorescent particles present in the photodetection region while moving the location of the photodetection region in the sample solution, and counting the number of fluorescent particles detected during movement of the location of the photodetection region by counting the number of individually detected fluorescent particles.