Sample observation apparatus
View Patent ↗A sample observation apparatus includes excitation light irradiation unit to irradiate sample with excitation light; excitation light modulator to modulate spatial intensity distribution of the excitation light on the sample; excitation light modulation control unit to control the excitation light modulator according to modulation control signal; photo detection unit to detect light emission from the sample and to generate a detection signal; image generation unit to generate image data of the sample according to the modulation control signal and the detection signal; modulation control signal generation unit to generate the modulation control signal such that Nyquist frequency of the image data will be larger than cut-off frequency in the spatial intensity distribution of the excitation light on the sample; and image processing unit to emphasize high-frequency component that exceeds the cut-off frequency included in the image data.
1. A sample observation apparatus comprising:
an excitation light irradiation unit to irradiate a sample with an excitation light;
an excitation light modulation unit to modulate a spatial intensity distribution of the excitation light on the sample;
an excitation light modulation control unit to control the excitation light modulation unit according to a modulation control signal;
a photo detection unit to detect light emission from the sample caused by irradiation with the excitation light to generate a detection signal;
an image generation unit to generate image data of the sample according to the modulation control signal and the detection signal;
a modulation control signal generation unit to generate the modulation control signal such that a Nyquist frequency of the image data will be larger than a cut-off frequency in the spatial intensity distribution of the excitation light on the sample; and
an image processing unit to emphasize a high-frequency component that exceeds the cut-off frequency included in the image data.
2. The sample observation apparatus according to claim 1 , wherein
the excitation light irradiation unit includes a light condensing unit to condense at least a portion of the excitation light onto the sample, and
the excitation light modulation unit is a scanning unit that scans the sample by moving a light-condensing position to which the light condensing unit condenses the excitation light.
3. The sample observation apparatus according to claim 2 , wherein
the modulation control signal generation unit generates the modulation control signal such that the Nyquist frequency of the image data will be equal to or larger than 1.5 times the cut-off frequency in the spatial intensity distribution of the excitation light on the sample.
4. The sample observation apparatus according to claim 2 , wherein
the modulation control signal generation unit generates the modulation control signal such that the Nyquist frequency of the image data will be equal to or smaller than four times the cut-off frequency in the spatial intensity distribution of the excitation light on the sample.
5. The sample observation apparatus according to claim 2 , wherein
the cut-off frequency in the spatial intensity distribution of the excitation light is determined by a diffraction limit that is calculated from a wavelength of the excitation light and a numerical aperture on an emission side of the light condensing unit.
6. The sample observation apparatus according to claim 2 , wherein
the Nyquist frequency of the image data is determined by sampling intervals at which the scanning unit scans the sample.
7. The sample observation apparatus according to claim 2 , further comprising
a confocal stop that configures an aperture at an optically conjugate position with a light-condensing position of the light condensing unit.
8. The sample observation apparatus according to claim 7 , wherein
an aperture diameter of the confocal stop is equal to or smaller than a Rayleigh diameter.
9. The sample observation apparatus according to claim 2 , wherein
the image processing unit emphasizes the high-frequency component by using a digital convolution filter.
10. The sample observation apparatus according to claim 9 , wherein
a coefficient of the digital convolution filter is adjusted in accordance with a wavelength of the excitation light.
11. The sample observation apparatus according to claim 9 , wherein
a coefficient of the digital convolution filter is adjusted in accordance with a wavelength of the light emission.
12. The sample observation apparatus according to claim 9 , wherein
a coefficient of the digital convolution filter is adjusted in accordance with a numerical aperture on an emission side of the light condensing unit.
13. The sample observation apparatus according to claim 9 , wherein
a coefficient of the digital convolution filter is adjusted in accordance with a magnification at which an optical image of the sample is projected onto a light-receptive plane of the photo detection unit.
14. The sample observation apparatus according to claim 9 , wherein
a coefficient of the digital convolution filter is adjusted in accordance with a Nyquist frequency of the image data.
15. The sample observation apparatus according to claim 1 , wherein
the light emission non-linearly depends on irradiation strength of the excitation light on the sample.
16. The sample observation apparatus according to claim 15 , wherein
the light emission is a multi-photon fluorescent light generated at the sample.
17. The sample observation apparatus according to claim 15 , wherein
the light emission is a higher-order harmonics from the sample.
18. The sample observation apparatus according to claim 15 , wherein
the excitation light includes two different wavelength components, and
the light emission includes anti-Stokes components included in a Raman scattering light from the sample.
19. The sample observation apparatus according to claim 15 , wherein
the excitation light includes two or more different wavelength components, and
the light emission is caused by Four-Wave Mixing at the sample.
20. The sample observation apparatus according to claim 1 , wherein
the image processing unit processes the image data in real time in accordance with the image data generated by the image generation unit.