Phase distribution measurement method and phase distribution measurement apparatus

• US 9,594,941 B2
• Filed: 02/25/2014
• Issued: 03/14/2017
• Est. Priority Date: 03/22/2013
• Status: Active Grant

• Current Assignee: Evident Corporation
• Original Assignee: Olympus Corporation
• Inventors: Ishiwata, Hiroshi
• Primary Examiner(s): Topgyal, Gelek W
• Application Number:
  US14/189,532
• Publication Number:
  US 20140285650A1
• Time in Patent Office: 1,113 Days
• Field of Search: 250/458.1, 250/208.2, 250/214.1, 250/216, 250/221, 250/358.1, 348 79- 80, 359368-386
• US Class Current: 1/1
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G02B 21/0056 based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
• CPC Class Codes: G02B 21/0068 arrangements using polarisation
• CPC Class Codes: G02B 21/14 affording illumination for phase-contrast observation
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence
• CPC Class Codes: G06V 20/69 Microscopic objects, e.g. biological cells or cellular parts
• CPC Class Codes: G06V 20/695 Preprocessing, e.g. image segmentation

Abstract

A phase distribution measurement method inside a biological sample includes taking in an optical image of the sample formed by a microscope to form a plurality of images with different image contrasts; calculating a component corresponding to phase distribution of the sample and a component corresponding to other than the phase distribution, and dividing the component corresponding to the phase distribution by the component corresponding to other than the phase distribution to forma normalized phase component image; breaking down the phase component image into a plurality of frequency components; performing a deconvolution process to each of the frequency components using an optical response character corresponding to each, and calculating phase distribution of a refraction component and phase distribution of a structure component; and calculating phase distribution of the sample by compounding the phase distribution of the refraction component and the phase distribution of the structure component.

Claims

1. A phase distribution measurement method a for biological sample, comprising:

taking in an optical image of a biological sample formed by a microscope that converts phase distribution into image intensity distribution while changing image contrast to form a plurality of pieces of images with different image contrasts;

calculating a component corresponding to phase distribution of the biological sample and a component corresponding to other than the phase distribution of the biological sample based on the plurality of pieces of images, and dividing the component corresponding to the phase distribution by the component corresponding to other than the phase distribution of the biological sample to form a normalized phase component image;

breaking down the normalized phase component image into a plurality of frequency components based on a spatial frequency of the normalized phase component image;

performing a deconvolution process to each of the frequency components using an optical response character corresponding to each, and calculating phase distribution of a refraction component formed by light refracted inside the biological sample and phase distribution of a structure component formed by light diffracted in a structure inside the biological sample; and

calculating phase distribution of the biological sample by compounding the phase distribution of the refraction component and the phase distribution of the structure component.

2. The phase distribution measurement method according to claim 1, wherein:

the breaking down of the normalized phase component image into a plurality of frequency components comprises performing an averaging process of an image to the normalized phase component image with filters having different kernel sizes, to break down the normalized phase component image, by the spatial frequency of the normalized phase component image, into a background component with a lowest spatial frequency, the refraction component, and the structure component with a highest spatial frequency.

3. The phase distribution measurement method according to claim 1, wherein:

the microscope is a differential interference contrast microscope that performs image capturing with phase distribution of an observed object as image intensity distribution, and

the method further comprises;

switching a shear direction of the microscope; and

compounding two phase distributions of the biological sample calculated in two shear directions before and after switching.

4. The phase distribution measurement method according to claim 3, further comprising:

calculating a position misalignment amount generated when switching the shear direction from correlation of the two phase distributions of the structure component calculated in the two shear directions; and

correcting position misalignment of the two phase distributions of the biological sample calculated in the two shear directions before and after switching using the calculated position misalignment amount.

5. The phase distribution measurement method according to claim 1, further comprising:

performing a deconvolution process to the structure component using an optical response character in a defocus state with respect to an observation plane to calculate a second phase distribution of the structure component;

comparing the second phase distribution of the structure component and phase distribution of the structure component calculated using an optical response character in a focused state with respect to the observation plane; and

removing phase distribution in which blurring has occurred due to the defocus from phase distribution of the structure component based on a comparison result of phase distributions,

wherein the calculating of phase distribution of the biological sample by compounding the phase distribution of the refraction component and the phase distribution of the structure component comprises compounding phase distribution from which the phase distribution in which blurring has occurred due to the defocus from phase distribution of the structure component is removed, and the phase distribution of the refraction component.

6. The phase distribution measurement method according to claim 5, wherein the calculating of a second phase distribution of the structure component comprises performing a deconvolution process to the structure component using an optical response character calculated in view of a phase transfer function generated due to the defocus.

7. The phase distribution measurement method according to claim 5, wherein the removing of phase distribution in which blurring has occurred due to the defocus from phase distribution of the structure component comprises forming a subtraction image by subtracting the phase distribution calculated using an optical response character in the defocus state from the phase distribution of the structure component, to separate phase distribution of the structure component within a certain depth of focus from the observation plane.

8. The phase distribution measurement method according to claim 5, wherein the removing of phase distribution in which blurring has occurred due to the defocus from phase distribution of the structure component comprises:

extracting a phase distribution in part out of a depth of focus of the microscope from the second phase distribution of the structure component, and performing convolution of the extracted phase distribution by the optical response character in the defocus state to calculate phase distribution in which blurring has occurred; and

subtracting the calculated phase distribution in which blurring has occurred from the phase distribution of the structure component.

9. The phase distribution measurement method according to claim 1, further comprising:

changing a focal plane with respect to an observation plane in an optical axis direction;

comparing phase distributions of the structure component calculated at respective focal planes to identify phase distribution leaking into the observation plane from a structure of the biological sample existing above and below the observation plane; and

removing the identified phase distribution from the phase distribution of the structure component.

10. The phase distribution measurement method according to claim 9, wherein comparing of the phase distributions comprises:

at respective focal planes, extracting an area in an XY plane that is orthogonal to an optical axis in which a phase amount of the structure component at the focal plane becomes larger than a phase amount of the structure component at adjacent focal planes above and below the focal plane; and

performing a convolution process to the phase distribution of the structure component in the extracted area, using an optical response character calculated in view of a phase transfer function generated by defocus with the adjacent focal planes above and below the focal plane.

11. The phase distribution measurement method according to claim 1, further comprising:

changing a focal plane with respect to an observation plane in an optical axis direction;

comparing phase distributions of the structure component calculated at respective focal planes to identify phase distributions of a structure of the biological sample existing continuously from front and back of the observation plane to the observation plane; and

connecting the identified phase distributions of the structure continuously.

12. A phase distribution measurement apparatus comprising:

a microscope configured to convert phase distribution of a biological sample into image intensity distribution, comprising an image contrast changing unit configured to change image contrast of the image intensity distribution;

a control unit configured to control the image contrast changing unit so as to obtain a plurality of pieces of images with different image contrasts; and

a calculating unit configured to calculate phase distribution of the biological sample from the plurality of pieces of images obtained by control the control unit performs,

wherein the calculating unit is configured to;

take in an optical image of the biological sample formed by the microscope while changing image contrast to form the plurality of pieces of images with different image contrasts,

calculate a component corresponding to phase distribution of the biological sample and a component corresponding to other than the phase distribution of the biological sample based on the plurality of pieces of images, and divide the component corresponding to the phase distribution by the component corresponding to other than the phase distribution of the biological sample to form a normalized phase component image,

break down the normalized phase component image into a plurality of frequency components based on a spatial frequency of the normalized phase component image,

perform a deconvolution process to each of the frequency components using an optical response character corresponding to each, and calculate phase distribution of a refraction component formed by light refracted inside the biological sample and phase distribution of a structure component formed by light diffracted in a structure inside the biological sample, and

calculate phase distribution of the biological sample by compounding the phase distribution of the refraction component and the phase distribution of the structure component.

13. The phase distribution measurement apparatus according to claim 12, wherein the phase distribution measurement apparatus is configured to measure an inside structure of an cultured ES cell or iPS cell being the biological sample.

14. The phase distribution measurement apparatus according to claim 13, wherein the phase distribution measurement apparatus is configured to distinguish a normal cell and a mutated cell inside a colony of the ES cell or the iPS cell.

15. The phase distribution measurement apparatus according to claim 13, wherein the phase distribution measurement apparatus is configured to identify an undifferentiated state of the ES cell or the iPS cell.

16. The phase distribution measurement apparatus according to claim 12, wherein the phase distribution measurement apparatus is configured to identify a mutated cell inside a tissue formed by differentiation of an ES cell or an iPS cell.

17. The phase distribution measurement apparatus according to claim 12, wherein the phase distribution measurement apparatus is configured to identify a structure in a tissue and a mutated cell formed by differentiation of a stem cell.

18. The phase distribution measurement apparatus according to claim 12, wherein the phase distribution measurement apparatus is configured to identify a mutated cell in a living tissue without labeling.

19. A phase distribution measurement apparatus comprising:

a microscope configured to convert phase distribution of a biological sample into image intensity distribution, comprising an image contrast changing unit configured to change image contrast of the image intensity distribution;

a control unit configured to control the image contrast changing unit so as to obtain a plurality of pieces of images with different image contrasts; and

a calculating unit configured to calculate phase distribution of the biological sample from the plurality of pieces of images obtained by control the control unit performs,

wherein the calculating unit is configured to;

take in an optical image of the biological sample formed by the microscope while changing image contrast to form the plurality of pieces of images with different image contrasts,

calculate a component corresponding to phase distribution of the biological sample and a component corresponding to other than the phase distribution of the biological sample based on the plurality of pieces of images, and divide the component corresponding to the phase distribution by the component corresponding to other than the phase distribution of the biological sample to form a normalized phase component image,

break down the normalized phase component image into a plurality of frequency components based on a spatial frequency of the normalized phase component image,

perform a deconvolution process to each of the frequency components using an optical response character corresponding to each, and calculate phase distribution of a refraction component formed by light refracted inside the biological sample and phase distribution of a structure component formed by light diffracted in a structure inside the biological sample,

perform a deconvolution process to the structure component using an optical response character in a defocus state with respect to an observation plane to calculate a second phase distribution of the structure component,

compare the second phase distribution of the structure component and phase distribution of the structure component calculated using an optical response character in a focused state with respect to the observation plane,

remove phase distribution in which blurring has occurred due to the defocus from phase distribution of the structure component based on a comparison result of phase distributions, and

calculate phase distribution of the biological sample by compounding the phase distribution of the refraction component and the phase distribution of the structure component,

wherein the calculating of phase distribution of the biological sample by compounding the phase distribution of the refraction component and the phase distribution of the structure component comprises compounding phase distribution from which the phase distribution in which blurring has occurred due to the defocus from phase distribution of the structure component is removed, and the phase distribution of the refraction component.

20. A phase distribution measurement apparatus comprising:

a microscope configured to convert phase distribution of a biological sample into image intensity distribution, comprising an image contrast changing unit configured to change image contrast of the image intensity distribution;

a focus position control unit configured to change a focal plane with respect to an observation plane in an optical axis direction;

a control unit configured to control the image contrast changing unit so as to obtain a plurality of pieces of images with different image contrasts; and

a calculating unit configure to calculate phase distribution of the biological sample from which phase distribution leaking from a structure of the biological sample existing above and below the observation plane is removed, from the plurality of pieces of images obtained by control the control unit performs,

wherein the calculating unit is configured to;

take in an optical image of the biological sample formed by the microscope while changing image contrast to form the plurality of pieces of images with different image contrasts,

calculate a component corresponding to phase distribution of the biological sample and a component corresponding to other than the phase distribution of the biological sample based on the plurality of pieces of images, and divide the component corresponding to the phase distribution by the component corresponding to other than the phase distribution of the biological sample to form a normalized phase component image,

break down the normalized phase component image into a plurality of frequency components based on a spatial frequency of the normalized phase component image,

perform a deconvolution process to each of the frequency components using an optical response character corresponding to each, and calculate phase distribution of a refraction component formed by light refracted inside the biological sample and phase distribution of a structure component formed by light diffracted in a structure inside the biological sample,

change a focal plane with respect to an observation plane in an optical axis direction,

compare phase distribution of the structure component calculated at respective focal planes to identify phase distribution leaking into the observation plane from a structure of the biological sample existing above and below the observation plane,

remove the identified phase distribution from the phase distribution of the structure component, and

calculate phase distribution of the biological sample by compounding the phase distribution of the refraction component and the phase distribution of the structure component.