Single particle detection device, single particle detection method, and computer program for single particle detection, using optical analysis

• US 9,488,578 B2
• Filed: 01/23/2014
• Issued: 11/08/2016
• Est. Priority Date: 08/26/2011
• Status: Active Grant

• Current Assignee: Olympus Corporation
• Original Assignee: Olympus Corporation
• Inventors: Hanashi, Takuya, Tanabe, Tetsuya
• Primary Examiner(s): Geisel, Kara E
• Assistant Examiner(s): MERLINO, AMANDA H
• Application Number:
  US14/162,142
• Publication Number:
  US 20140134608A1
• Time in Patent Office: 1,020 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/1429 Signal processing
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 21/51 inside a container, e.g. in an ampoule G01N21/53 takes precedence
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0032 Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/008 Details of detection or image processing, including general computer control

Abstract

There is provided a single particle detection technique based on a scanning molecule counting method, enabling individual detection of a single particle using light measurement with a confocal or multiphoton microscope, and quantitative observation of conditions or characteristics of the particle. The inventive technique of detecting a single particle in a sample solution detects light containing substantially constant background light from a light detection region with moving the position of the light detection region of the microscope in a sample solution to generate time series light intensity data; and detects individually a light intensity reduction occurred when a single particle which does not emit light (or a particle whose emitting light intensity in a detected wavelength band is lower than the background light) enters in the light detection region in the time series light intensity data as a signal indicating the existence of each single particle.

Claims

1. A single particle detection device which detects a single particle dispersed and moving at random in a sample solution using an optical system of a confocal microscope or a multiphoton microscope, comprising:

a light detection region mover which moves a position of a light detection region of the optical system in the sample solution;

a light detector which detects light from the light detection region; and

a signal processor which generates time series light intensity data of the light from the light detection region detected with the light detector with moving the position of the light detection region in the sample solution and detects a signal indicating an existence of each single particle individually in the time series light intensity data;

wherein the light from the light detection region includes substantially constant background light; and

the signal indicating an existence of each single particle is a reduction of the light intensity detected with the light detector, which reduction occurs when the single particle enters into the light detection region,

wherein the signal processor makes the time series light intensity data smoothed, and detects in the smoothed time series light intensity data a downwardly convex, bell-shaped pulse form signal whose intensity is lower than a predetermined threshold value measured from an intensity of the background light as the signal indicating the existence of the single particle.

2. The device of claim 1, wherein the background light is fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light owing to substances dispersed in the sample solution or illumination light.

3. The device of claim 1, wherein an emitting light intensity of the single particle is lower than the background light in the detected light wavelength band.

4. The device of claim 1, wherein an outer diameter of the single particle is not less than 15% of a diameter of the light detection region.

5. The device of claim 4, wherein an outer diameter of the single particle is not less than 35% of a diameter of the light detection region.

6. The device of claim 1, wherein the light detection region mover moves the position of the light detection region at a velocity quicker than a diffusion moving velocity of the single particle.

7. The device of claim 1, wherein the light detection region mover moves the position of the light detection region in the sample solution by changing an optical path of the optical system.

8. The device of claim 1, wherein the signal processor judges that one single particle has entered into the light detection region when a signal whose light intensity is lower than a predetermined threshold value measured from an intensity of the background light is detected.

9. The device of claim 1, wherein the signal processor counts a number of the signals indicating the existences of the single particles individually detected during moving the position of the light detection region to count a number of the single particles.

10. The device of claim 1, wherein the signal processor determines a number density or concentration of the single particle in the sample solution based on the number of the detected single particles.

11. The device of claim 1, wherein the moving of the position of the light detection region of the optical system by the light detection region mover, the detecting of the light from the light detection region by the light detector and the detecting of the signals indicating the existences of the single particles by the signal processor are repeated until the number of the signals indicating the existences of the single particles detected with the signal processor reaches a predetermined number;

and a concentration of the single particle in the sample solution is determined based on the time taken for the number of the signals indicating the existences of the single particles to reach the predetermined number.

12. A single particle detection method of detecting a single particle dispersed and moving at random in a sample solution using an optical system of a confocal microscope or a multiphoton microscope, comprising:

(a) moving a position of a light detection region of the optical system in the sample solution;

(b) detecting light including substantially constant background light from the light detection region with moving the position of the light detection region in the sample solution, and generating time series light intensity data; and

(c) detecting in the time series light intensity data individually a reduction of an light intensity occurring when the single particle has entered into the light detection region as a signal indicating an existence of each single particle,

wherein, in the step (c), the time series light intensity data is smoothed, and a downwardly convex, bell-shaped pulse form signal whose intensity is lower than a predetermined threshold value measured from an intensity of the background light in the smoothed time series light intensity data is detected as the signal indicating the existence of the single particle.

13. The method of claim 12, wherein the background light is fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light owing to substances dispersed in the sample solution or illumination light.

14. The method of claim 12, wherein an emitting light intensity of the single particle is lower than the background light in the detected light wavelength band.

15. The method of claim 12, wherein an outer diameter of the single particle is not less than 15% of a diameter of the light detection region.

16. The method of claim 15, wherein an outer diameter of the single particle is not less than 35% of a diameter of the light detection region.

17. The method of claim 12, wherein, in the step (a), the position of the light detection region is moved at a velocity quicker than a diffusion moving velocity of the single particle.

18. The method of claim 12, wherein, in the step (a), the position of the light detection region in the sample solution is moved by changing an optical path of the optical system.

19. The method of claim 12, wherein, in the step (c), it is judged that one single particle has entered into the light detection region when a signal whose light intensity is lower than a predetermined threshold value measured from an intensity of the background light is detected.

20. The method of claim 12, further comprising (d) counting a number of the signals indicating the existences of the single particles individually detected during moving the position of the light detection region to count a number of the single particles.

21. The method of claim 12, further comprising (e) determining a number density or concentration of the single particle in the sample solution based on the number of the detected single particles.

22. The method of claim 12, repeating until the number of the signals indicating the existences of the single particles reaches a predetermined number the moving of the position of the light detection region of the optical system, the detecting of the light from the light detection region and the detecting of the signals indicating the existences of the single particle;

and determining a concentration of the single particle in the sample solution based on the time taken for the number of the signals indicating the existences of the single particles to reach the predetermined number.

23. A computer readable storage device having a computer program product including programmed instructions for single particle detection of detecting light from a single particle dispersed and moving at random in a sample solution using an optical system of a confocal microscope or a multiphoton microscope, said programmed instructions causing a computer to perform steps of:

moving a position of a light detection region of the optical system in the sample solution;

detecting light including substantially constant background light from the light detection region with moving the position of the light detection region in the sample solution, and generating time series light intensity data; and

detecting in the time series light intensity data individually a reduction of a light intensity occurring when the single particle has entered into the light detection region as a signal indicating an existence of each single particle,

wherein, in the procedure of detecting the signal indicating the existence of the single particle individually, the time series light intensity data is smoothed, and a downwardly convex, bell-shaped pulse form signal whose intensity is lower than a predetermined threshold value measured from an intensity of the background light in the smoothed time series light intensity data is detected as the signal indicating the existence of the single particle.

24. The computer readable storage device of claim 23, wherein the background light is fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light owing to substances dispersed in the sample solution or illumination light.

25. The computer readable storage device of claim 24, wherein an emitting light intensity of the single particle is lower than the background light in the detected light wavelength band.

26. The computer readable storage device of claims 23, wherein an outer diameter of the single particle is not less than 15% of a diameter of the light detection region.

27. The computer readable storage device of claim 26, wherein an outer diameter of the single particle is not less than 35% of a diameter of the light detection region.

28. The computer readable storage device of claim 23, wherein, in the procedure of moving the position of the light detection region, the position of the light detection region is moved at a velocity quicker than a diffusion moving velocity of the single particle.

29. The computer readable storage device of claim 23, wherein, in the procedure of moving the position of the light detection region, the position of the light detection region in the sample solution is moved by changing an optical path of the optical system.

30. The computer program of claim 23, wherein, in the procedure of detecting the signal indicating the existence of the single particle individually, it is judged that one single particle has entered into the light detection region when a signal whose light intensity is lower than a predetermined threshold value measured from an intensity of the background light is detected.

31. The computer readable storage device of claim 23, further comprising a procedure of counting a number of the signals indicating the existences of the single particles individually detected during moving the position of the light detection region to count a number of the single particles.

32. The computer readable storage device of claim 23, further comprising a procedure of determining a number density or concentration of the single particle in the sample solution based on the number of the detected single particles.

33. The computer readable storage device of claim 23, repeating until the number of the signals indicating the existences of the single particles reaches a predetermined number the moving of the position of the light detection region of the optical system, the detecting of the light from the light detection region and the detecting of the signals indicating the existences of the single particle;

and determining a concentration of the single particle in the sample solution based on the time taken for the number of the signals indicating the existences of the single particles to reach the predetermined number.