Point-spread-function measurement device and measurement method, image acquisition apparatus, and image acquisition method
A point-spread-function measurement device includes: a scanner that scans two illumination light beams emitted from a light source; an illumination optical system that radiates the two illumination light beams scanned by the scanner onto a sample; a relative-position adjustor that changes a relative irradiation position, in the sample, between the two illumination light beams radiated by the illumination optical system; a detection optical system that detects signal light generated at an overlapping position, in the sample, of the illumination light beams radiated by the illumination optical system; and a calculator that calculates a point spread function based on the signal light detected by the detection optical system and the relative irradiation position between the two illumination light beams when the signal light is detected.
1. A point-spread-function measurement device comprising:
a scanner configured to scan two illumination light beams emitted from a light source;
an illumination optical system configured to radiate the two illumination light beams scanned by the scanner onto a sample;
a relative-position adjustor configured to repeatedly change a relative irradiation position, in the sample, between the two illumination light beams radiated by the illumination optical system if an acquisition of a light intensity distribution is not completed;
a detection optical system configured to detect signal light generated at an overlapping position, in the sample, of the illumination light beams radiated by the illumination optical system in each time the relative illumination position radiated by the two illumination lights, in the sample, is repeatedly changed by the relative-position adjustor; and
a calculator configured to calculate a point spread function based on the light intensity distribution, the light intensity distribution being a distribution indicating an intensity of the signal light at each distance between the relative irradiation positions radiated by the two illumination lights in the sample.
2. The point-spread-function measurement device according to claim 1 ,
wherein the calculator is configured to calculate the point spread function by determining an autocorrelation waveform of the illumination light in two-dimensional space based on the light intensity distribution.
3. The point-spread-function measurement device according to claim 2 ,
wherein at least one signal light image having at least a one-dimensional size is acquired by using the signal light detected by the detection optical system, and
wherein the calculator is configured to calculate the autocorrelation waveform by using the signal light image.
4. The point-spread-function measurement device according to claim 2 ,
wherein the calculator is configured to calculate the point spread function by performing Fourier transformation of the autocorrelation waveform.
5. The point-spread-function measurement device according to claim 2 ,
wherein the calculator calculates the autocorrelation waveform of the illumination light in two-dimensional space by creating a rotationally symmetric image in which the light intensity distribution is rotated.
6. The point-spread-function measurement device according to claim 2 ,
wherein the relative-position adjustor repeatedly changes a relative irradiation position, in the sample, between the two illumination light beams in a two-dimensional direction, and
wherein the calculator calculates the autocorrelation waveform of the illumination light in two-dimensional space based on a two-dimensional light intensity distribution acquired by changing a relative irradiation position in the sample in a two-dimensional direction.
7. The point-spread-function measurement device according to claim 6 ,
wherein the illumination optical system is configured to set polarization states of the two illumination light beams such that the polarization states are orthogonal to each other.
8. The point-spread-function measurement device according to claim 1 ,
wherein the illumination optical system is configured to set polarization states of the two illumination light beams such that the polarization states are orthogonal to each other.
9. The point-spread-function measurement device according to claim 1 ,
wherein the signal light is generated in a nonlinear optical process in accordance with irradiation of the illumination light beams.
10. The point-spread-function measurement device according to claim 9 ,
wherein the illumination light beams are ultra-short pulsed laser beams, and wherein the signal light is fluorescence generated in accordance with a multiphoton absorption effect.
11. The point-spread-function measurement device according to claim 1 or 2 ,
wherein the illumination light beams are pulsed light beams,
wherein the illumination optical system includes a timing adjustor configured to switch the timing for radiating the two illumination light beams onto the sample between a simultaneous mode and a non-simultaneous mode, and
wherein the calculator is configured to calculate the point spread function by using a difference between signal light detected in the simultaneous mode switched by the timing adjustor and signal light detected in the non-simultaneous mode switched by the timing adjustor.
12. The point-spread-function measurement device according to claim 1 ,
wherein the illumination optical system includes a light modulator configured to modulate a spatial distribution or polarization states of the illumination light beams so that a high-frequency component is accentuated in a spatial frequency distribution of the point spread function.
13. An image acquisition apparatus comprising:
the point-spread-function measurement device according to claim 1 ; and
an image processor that generates an image of the sample by using the point spread function measured by the measurement device.
14. The image acquisition apparatus according to claim 13 ,
wherein the image processor generates a sample image acquired as a result of the illumination optical system radiating the illumination light beams emitted from the light source and scanned by the scanner onto the sample and the detection optical system detecting the signal light generated at the irradiation positions in the sample, and reconfigures the generated sample image by using the point spread function measured by the measurement device.
15. The image acquisition apparatus according to claim 14 ,
wherein the image processor reconfigures the sample image by performing deconvolution on the sample image by using the point spread function.
16. The point-spread-function measurement device according to claim 1 ,
wherein the calculator integrates the signal light detected by the detection optical system to obtain the light intensity distribution.
17. A point-spread-function measurement method comprising:
scanning two illumination light beams emitted from a light source over a sample while repeatedly changing a relative irradiation position in the sample if an acquisition of a light intensity distribution is not completed;
detecting signal light generated at an overlapping position, in the sample, of the illumination light beams radiated in each time the relative illumination position radiated by the two illumination lights, in the sample, is repeatedly changed; and
calculating a point spread function based on the light intensity distribution, the light intensity distribution being a distribution indicating an intensity of the signal light at each distance between the relative irradiation positions radiated by the two illumination lights in the sample.
18. The point-spread-function measurement method according to claim 17 ,
wherein the calculating of the point spread function includes calculating the point spread function by determining an autocorrelation waveform of the illumination light in two-dimensional space based on the light intensity distribution.
19. The point-spread-function measurement method according to claim 18 ,
wherein the detecting of the signal light generated at an overlapping position includes acquiring at least one signal light image having at least a one-dimensional size by using the detected signal light, and
wherein the calculating of the point spread function includes calculating the autocorrelation waveform by using the signal light image.
20. The point-spread-function measurement method according to claim 18 ,
wherein the calculating of the point spread function includes calculating the point spread function by performing Fourier transformation of the autocorrelation waveform.
21. The point-spread-function measurement method according to claim 18 ,
wherein the calculating of the point spread function includes calculating the autocorrelation waveform of the illumination light in two-dimensional space by creating a rotationally symmetric image in which the light intensity distribution is rotated.
22. The point-spread-function measurement method according to claim 18 ,
wherein the relative-position adjusting of the point spread function includes repeatedly changing a relative irradiation position, in the sample, between the two illumination light beams in a two-dimensional direction, and
wherein the calculating of the point spread function includes calculating the autocorrelation waveform of the illumination light in two-dimensional space based on a two-dimensional light intensity distribution acquired by changing a relative irradiation position in the sample in a two-dimensional direction.
23. The point-spread-function measurement method according to claim 22 ,
wherein the scanning of the two illumination light beams includes setting polarization states of the two illumination light beams such that the polarization states are orthogonal to each other and performing the scanning.
24. The point-spread-function measurement method according to claim 17 ,
wherein the scanning of the two illumination light beams includes setting polarization states of the two illumination light beams such that the polarization states are orthogonal to each other and performing the scanning.
25. The point-spread-function measurement method according to claim 17 ,
wherein the signal light is generated in a nonlinear optical process in accordance with irradiation of the illumination light beams.
26. The point-spread-function measurement method according to claim 25 ,
wherein the illumination light beams are ultra-short pulsed laser beams, and
wherein the signal light is fluorescence generated in accordance with a multiphoton absorption effect.
27. The point-spread-function measurement method according to claim 17 ,
wherein the illumination light beams are pulsed light beams,
wherein the scanning of the two illumination light beams includes performing the scanning by switching the timing for radiating the two illumination light beams onto the sample between a simultaneous mode and a non-simultaneous mode, and
wherein the calculating of the point spread function includes calculating the point spread function by using a difference between signal light detected in the simultaneous mode switched and signal light detected in the non-simultaneous mode switched.
28. The point-spread-function measurement method according to claim 17 ,
wherein the scanning of the two illumination light beams includes modulating a spatial distribution or polarization states of the illumination light beams so that a high-frequency component is accentuated in a spatial frequency distribution of the point spread function.
29. An image acquisition method comprising:
the measurement method according to claim 17 ; and
generating a sample image by using a point spread function measured in accordance with the measurement method.
30. The image acquisition method according to claim 29 ,
wherein the generating of the sample image includes generating the sample image acquired as a result of scanning the illumination light beams from the light source onto the sample and detecting the signal light generated at the irradiation positions in the sample, and reconfiguring the sample image generated by using the point spread function measured in accordance with the measurement method.
31. The image acquisition method according to claim 30 ,
wherein the generating of the sample image includes reconfiguring the sample image by performing deconvolution on the sample image by using the point spread function.
32. The point-spread-function measurement method according to claim 17 ,
wherein the calculating of the point spread function includes integrating the detected signal light to obtain the light intensity distribution.