Method of detecting sparse particles in a solution using a light-emitting probe

1. An optical analysis method of detecting light from a light-emitting probe binding to a particle dispersed and moving at random in a sample solution to detect the particle by using an optical system of a confocal microscope or a multiphoton microscope, said method comprising steps of:

preparing the sample solution containing the particle and the light-emitting probe;

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;

detecting light from the light detection region while moving the position of the light detection region in the sample solution, over a plurality of units of time;

smoothing data values of light detected over the plurality of units of time, said smoothing being conducted until a gap in a light intensity variation over the plurality of units of time of a light signal from the light-emitting probe bound or having been previously bound to each particle can be disregarded; and

detecting the particle individually based on smoothed data values by detecting, as a light signal of the particle, a light intensity variation over consecutive units among the plurality of units of time which has a predetermined profile which is expected from a single light-emitting probe moving relatively inside the light detection region, while disregarding a light intensity variation over consecutive units of time among the plurality of units of time which does not have the predetermined profile as noise.

2. The method of claim 1 , further comprising a step of: counting the number of the individually detected particle(s) to count the number of the particle(s) detected during moving the position of the light detection region.

3. The method of claim 1 , wherein, in the step of moving the position of the light detection region, the position of the light detection region is moved at a predetermined speed.

4. The method of claim 1 , wherein, in the step of moving the position of the light detection region, the position of the light detection region is moved at a velocity quicker than a diffusional moving velocity of the light-emitting probe having bound to the particle.

5. The method of claim 1 , wherein, in the step of detecting individually the light signal from the light-emitting probe having bound to each particle to detect individually the particle(s), the entering of one particle into the light detection region is detected based upon a shape of a time series light signal detected by the light detection portion.

6. The method of claim 1 , wherein the step of preparing the sample solution comprises the step of separating the light-emitting probe having not bound to the particle from the sample solution.

7. The method of claim 1 , wherein the step of preparing the sample solution comprises the step of making an acceptor which absorbs light from the light-emitting probe bind to the light-emitting probe having not bound to the particle.

8. The method of claim 1 , wherein the light-emitting probe is a substance whose light-emitting characteristic changes when the light-emitting probe binds to the particle, and the detected light is light emitted from the light-emitting probe having bound to the particle.

9. The method of claim 1 , wherein the light-emitting probe is a substance having an energy donor site and an energy acceptor site which cause a fluorescence energy transfer phenomenon when they are mutually close, wherein a distance of the energy donor site and the energy acceptor site is different between a condition where the substance has bound to the particle and a condition where the substance has not bound to the particle, and a wavelength characteristic of light emitted from the substance is different between a condition where the substance has bound to the particle and a condition where the substance has not bound to the particle; and that the detected light is light emitted from the light-emitting probe having bound to the particle.

10. The method of claim 1 , wherein the light-emitting probe has an energy donor site and an energy acceptor site which cause a fluorescence energy transfer phenomenon; the step of preparing the sample solution includes the step of carrying out a reaction to decompose the light-emitting probe having bound to the particle; and the detected light is light emitted from the light-emitting probe decomposed through the reaction.

11. The method of claim 1 , wherein the light-emitting probe is a probe pair comprising

a first probe which is an energy donor in a fluorescence energy transfer phenomenon, and

a second probe which is an energy acceptor in the fluorescence energy transfer phenomenon; and

wherein the detected light is light of the second probe emitted through the fluorescence energy transfer phenomenon which occurs under a condition that both the first and second probes have bound to the particle.

12. The method of claim 1 , wherein the particle has a site to be an energy acceptor of light emitted by the light-emitting probe; and the detected light is light emitted from the energy acceptor site of the particle through a fluorescence energy transfer phenomenon which occurs when the light-emitting probe binds to the particle.

13. The method of claim 1 , wherein the particle has a light-emitting site; the light-emitting probe has a site to be an energy acceptor of the light which the light-emitting site of the particle emits; and the detected light is light emitted from the light-emitting probe through a fluorescence energy transfer phenomenon which occurs when the light-emitting probe binds to the particle.

14. The method of claim 1 , wherein the particle is nucleic acid and the light-emitting probe is a nucleic acid binding protein.

15. The method of claim 1 , wherein the light-emitting probe is a substance which consists of at least two components and emits fluorescence when the substance binds to the particle and the mutual position of the at least two components changes.

16. The method of claim 1 , wherein the predetermined profile which is expected from a single light-emitting probe moving relatively inside the light detection region has an approximately bell shape.

17. The method of claim 1 ,

wherein, after the smoothing of the data values, a pulse existing region is determined by referring to the time differential value sequentially, and

wherein after the pulse existing region is determined, an individual particle is detected by fitting the predetermined profile to the smoothed data values in the pulse existing region.