Optical microscope device, microscopic observation method and computer program for microscopic observation using single light-emitting particle detection technique
There is provided a microscopic observation technique capable of detecting a light-emitting object or a light-emitting particle moving in a thick sample by the scanning molecule counting method. In the inventive technique, the light from a light detection region of is detected the optical system of a confocal or multiphoton microscope is detected with while moving the light detection region in each observed subregion obtained by dividing a region to be observed into plural regions; the signal of the light from a light-emitting particle is individually detected; and the position of the light-emitting particle corresponding to the detected signal is determined in the region to be observed. The moving of the position of the light detection region in each observed subregion is performed continuously in at least two directions or and/or continuously multiple times in each observed subregion.
1. An optical microscope device which detects light from a light-emitting particle in a sample liquid to detect the light-emitting particle, using an optical system of a confocal microscope or a multiphoton microscope, the device comprising:
a light detection region mover which moves a position of a light detection region multiple times continuously within each observed subregion, the respective observed subregions being obtained by dividing a region to be observed in a field of view of the microscope into plural regions;
a light detector which detects the 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 by the light detector while moving the position of the light detection region in each observed subregion, detects a signal having a profile of a light intensity variation indicating light from the light-emitting particle in the time series light intensity data, and determines a position of the light-emitting particle corresponding to the detected signal in the region to be observed, the position being at least an X coordinate and a Y coordinate of a subregion in the region to be observed, in which the light-emitting particle is detected,
wherein the light detection region mover is a stage position changing apparatus or a galvano mirror device.
2. The device of claim 1 , wherein the moving of the position of the light detection region in each observed subregion is conducted continuously in at least two directions for each observed subregion.
3. The device of claim 1 , wherein the moving of the position of the light detection region in each observed subregion is conducted multiple times in one same direction for each observed subregion.
4. The device of claim 1 , wherein the size of the observed subregion is determined based on the size of the light detection region.
5. The device of claim 1 , wherein a size of the observed subregion is set such that a moving length of a light-emitting particle to be detected within a time in which the position of the light detection region is moved in the observed subregion becomes smaller than the size of the observed subregion.
6. The device of claim 1 , wherein a length of one side edge of the Observed subregion is almost equal to a diameter of the light detection region; and one time of the moving of the position of the light detection region in each observed subregions is carried out from when a front edge in a moving direction of the light detection region passes through one side edge of the observed subregion and until a rear edge in the moving direction of the light detection region arrives at another side edge of the observed subregion.
7. The device of Claim 1 , wherein the device produces a plot image obtained by plotting a position of the light-emitting particle whose position in the region to be observed has been determined in a microscopic image of the region to be observed generated by an arbitrary way.
8. The device of claim 1 , wherein the signal processor determines information about a size of the light-emitting particle by using a characteristic of signals of one same light-emitting particle obtained through the multiple times of moving of the light detection region in each observed subregion.
9. The device of claim 8 , wherein the characteristic of the signals of the light-emitting particle used for determining the information about the size of the light-emitting particle is an index value expressing a translational diffusional characteristic of the light-emitting particle or an index value expressing a rotational diffusion characteristic of the light-emitting particle.
10. The device of claim 1 , wherein the signal processor determines the number of the light-emitting particle in the region to be observed or a concentration of the light-emitting particle in the liquid based on the number of the detected light-emitting particle.
11. An optical microscopic observation method of detecting light from a light-emitting particle in a sample liquid to detect the light-emitting particle, using an optical system of a confocal microscope or a multiphoton microscope, comprising the steps of:
(a) moving a position of a light detection region continuously multiple times within each observed subregion obtained by dividing a region to be observed within a field of view of the microscope into plural regions;
(b) detecting light from the light detection region by a light detector; and
(c) generating time series light intensity data of the light from the light detection region detected by the light detector while moving the position of the light detection region in each observed subregion, detecting a signal having a profile of a light intensity variation indicating light from the light-emitting particle in the time series light intensity data, and determining a position of the light-emitting particle corresponding to the detected signal in the region to be observed, the position being at least an X coordinate and a Y coordinate of a subregion, in the region to be observed, in which the light-emitting particle is detected.
12. A computer readable storage device having a computer program product including programmed instructions for observation with an optical microscope for detecting light from a light-emitting particle in a sample liquid to detect the light-emitting particle, using an optical system of a confocal microscope or a multiphoton microscope, said programmed instructions causing a computer to perform the steps of:
moving a position of a light detection region continuously multiple times within each observed subregion obtained by dividing a region to be observed within a field of view of the microscope into plural regions;
detecting light from the light detection region by a light detector; and
generating time series light intensity data of the light from the light detection region detected by the light detector while moving the position of the light detection region in each observed subregion, detecting a signal having a profile of a light intensity variation indicating light from the light-emitting particle in the time series light intensity data, and determining a position of the light-emitting particle corresponding to the detected signal in the region to be observed, the position being at least an X coordinate and a Y coordinate of a subregion, in the region to be observed, in which the light-emitting particle is detected.