# US 8,785,886 B2 · Optical analysis method using the light intensity of a single light-emitting particle

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- Number: US 8,785,886 B2
- Document ID: US8785886B2
- Status: Granted
- Kind: B2
- Application number: 13/788,972
- Filed: 2013-03-07
- Published: 2013-09-05
- Granted: 2014-07-22
- Earliest claimed: 2010-09-10
- Assignees: Olympus Corporation
- Applicants: Olympus Corporation
- Inventors: Kazutaka Nishikawa; Tetsuya Tanabe; Mitsushiro Yamaguchi
- Web page: https://patentgrep.com/patent/US8785886B2

## CPC

- G01N 21/64: Fluorescence; Phosphorescence
- G01N 15/1433: using image recognition
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates}
- G01N 2015/1493: {Particle size}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

There is provided a scanning molecule counting method using an optical measurement with a confocal microscope or a multiphoton microscope, enabling characterization of a light-emitting particle or identification of a light-emitting particle with emitted light intensity of a single light-emitting particle measured individually. In the inventive optical analysis technique, with reference to the ratio of the intensities of simultaneously generated signals of the lights of at least two light-emitting sites having mutually different emission wavelengths, possessed by a light-emitting particle contained in a sample solution, the intensities being measured with moving the position of the light detection region of an optical system by changing the optical path of the optical system, a single light-emitting particle corresponding to the signals is identified, and the kind, the size, etc. of the light-emitting particle is identified.

## Claims

### Claim 1

A method of detecting and analyzing light from a light-emitting particle dispersed and moving at random in a sample solution using an optical system of a confocal microscope or a multiphoton microscope, the method comprising steps of:
preparing the sample solution containing a light-emitting particle dispersed and moving at random in the sample solution, the light-emitting particle having a first light-emitting site and a second light-emitting site which has a different emission wavelength from that of the first light-emitting site;
moving a position of a light detection region of the optical system in the sample solution by changing an optical path of the optical system;
measuring individually and simultaneously light intensity of the first light-emitting site and light intensity of the second light-emitting site from the light detection region during the moving of the position of the light detection region in the sample solution;
generating a first light intensity data of the light intensity of the first light-emitting site and a second light intensity data of the light intensity of the second light-emitting site;
detecting individually in the first and the second light intensity data a signal indicating light of a single light-emitting particle based on a variation in time of the light intensity, the signal being detected when the variation in time of the light intensity has a bell shaped profile which is expected from the single light-emitting particle moving relatively inside the light detection region;
identifying the single light-emitting particle with a ratio of the light intensity of the first light-emitting site and the light intensity of the second light-emitting site in respective signals simultaneously generated on the first light intensity data and the second light intensity data.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first light-emitting site is a common light-emitting site which at least a part of light-emitting particles in the sample solution have in common, and the second light-emitting site is a specific light-emitting site characterizing the respective light-emitting particles.

### Claim 3 (depends on 1)

The method of claim 1, wherein a kind of the single light-emitting particle is identified with the ratio of the light intensity of the first light-emitting site and the light intensity of the second light-emitting site.

### Claim 4 (depends on 1)

The method of claim 1, wherein a size of the single light-emitting particle is determined with the ratio of the light intensity of the first light-emitting site and the light intensity of the second light-emitting site.

### Claim 5 (depends on 1)

The method of claim 1, wherein a kind of the second light-emitting site differs depending upon a kind of light-emitting particle.

### Claim 6 (depends on 1)

The method of claim 1, wherein a number of the second light-emitting sites differs depending upon a kind of light-emitting particle.

### Claim 7 (depends on 6)

The method of claim 6, wherein the number of the second light-emitting sites which the single light-emitting particle possesses increases as a size of the light-emitting particle increases.

### Claim 8 (depends on 1)

The method of claim 1, further comprising a step of counting a number of the identified single light-emitting particles.

### Claim 9 (depends on 1)

The method of claim 1, moving the position of the light detection region at a velocity quicker than a diffusion moving velocity of the light-emitting particle in the sample solution.

### Claim 10 (depends on 8)

The method of claim 8, further comprising a step of determining a number density or concentration of the light-emitting particle based on the number of the identified single light-emitting particles.