Focusing method, measuring method, principal point detecting method, focusing device, measuring device, and principal point detecting device

• US 10,705,325 B2
• Filed: 08/18/2016
• Issued: 07/07/2020
• Est. Priority Date: 02/24/2014
• Status: Active Grant

• Current Assignee: Evident Corporation
• Original Assignee: Olympus Corporation
• Inventors: Suzuki, Yoshimasa, Kajitani, Kazuo
• Primary Examiner(s): Nguyen, Thong Q
• Application Number:
  US15/240,384
• Publication Number:
  US 20160357002A1
• Time in Patent Office: 1,419 Days
• Field of Search: 359383
• CPC Class Codes: G02B 21/08 Condensers
• CPC Class Codes: G02B 21/24 Base structure
• CPC Class Codes: G02B 21/244 using image analysis techniques
• CPC Class Codes: G02B 21/34 Microscope slides, e.g. mounting specimens on microscope slides
• CPC Class Codes: G02B 7/28 Systems for automatic generation of focusing signals

Abstract

A focusing method includes a step of preparing a microscope, a step of mounting a sample, and a predetermined processing step, the predetermined processing step includes a step of receiving light emitted from the observation optical system, a step of obtaining the quantity of light based on light from a predetermined region of the received light, a step of calculating a difference or a ratio between the quantity of light in the predetermined region and the quantity of light as a reference, a step of comparing a calculation result with a threshold, and a step of changing the distance between the sample and the observation optical system, and in the step of preparing, a partial region of illumination light is shielded or darkened, and when the result of the calculation is equal to or smaller than the threshold, the predetermined processing step is terminated.

Claims

1. A focusing method comprising:

a step of preparing a microscope including an illumination optical system and an observation optical system, in the step of preparing, a partial region of illumination light is shielded or darkened;

a step of mounting a sample having a surface shape that is curved, on the microscope, in the step of mounting, the sample and the observation optical system are opposed to each other in a state in which the surface shape of the sample is not deformed;

a step of specifying a predetermined region, the predetermined region is a partial region of the sample, the step of specifying comprising a step of detecting, the step of detecting comprising;

receiving light from a plurality of regions in a field of view while changing a distance between the sample and the observation optical system;

recording the distance and a quantity of light at the distance for each of the regions;

obtaining a range in which the quantity of light changes monotonously for each of the regions; and

specifying a region in which the range is longest among the regions as the predetermined region; and

a predetermined processing step, the predetermined processing step comprising;

a step of receiving light emitted from the observation optical system,

a step of obtaining a quantity of light based on light from the predetermined region of the received light,

a step of calculating a difference or a ratio between the quantity of light in the predetermined region and a quantity of light as a reference,

a step of comparing a calculation result with a threshold, and

a step of changing a distance between the sample and the observation optical system,

when the calculation result is equal to or smaller than the threshold, the predetermined processing step is terminated.

2. The focusing method according to claim 1, wherein the method comprises a step of setting the quantity of light as a reference before the step of mounting.

3. The focusing method according to claim 2, wherein

the step of setting comprises a step of measuring a quantity of light by directing light emitted from the illumination optical system to the observation optical system, and

the quantity of light as a reference is set based on the measured quantity of light.

4. The focusing method according to claim 1, wherein the method comprises a step of setting the quantity of light as a reference after the step of mounting.

5. The focusing method according to claim 4, wherein

the step of setting comprises a step of measuring a quantity of light in a region where the sample is not present in the field of view by directing light emitted from the illumination optical system to the observation optical system, and

the quantity of light as a reference is set based on the measured quantity of light.

6. The focusing method according to claim 1, wherein a region in which a change in the quantity of light is most monotonous, is specified as the predetermined region.

7. The focusing method according to claim 1, wherein the partial region of illumination light is projected onto a pupil position of the observation optical system.

8. The focusing method according to claim 1, wherein

a projected image is an image of the partial region of illumination light at a pupil position of the observation optical system, and

a size of the projected image is smaller than a size of pupil of the observation optical system.

9. The focusing method according to claim 1, wherein

a projected image is an image of the partial region of illumination light at a pupil position of the observation optical system, and

an area of the projected image is 50% or more of an area of a pupil of the observation optical system.

10. The focusing method according to claim 1, wherein the partial region of illumination light is formed so as to include an optical axis of the illumination optical system.

11. The focusing method according to claim 1, wherein a shape of the partial region of illumination light is a point-symmetric shape.

12. The focusing method according to claim 1, wherein the illumination light is light of a single wavelength or a light in a narrow band.

13. The focusing method according to claim 1, wherein an area of light flux passing through a pupil of the observation optical system is changed.

14. The focusing method according to claim 1, wherein a size of the partial region of illumination light is changed.

15. A measuring method comprising:

holding a sample with a holding container having a bottom surface provided with a marker;

performing focusing on a surface of the sample using the focusing method of claim 1 and acquiring first focus information when focus is achieved;

performing focusing on the marker using a focusing method for performing focusing based on a change in contrast, and acquiring second focus information when focus is achieved; and

acquiring a difference between the first focus information and the second focus information.

16. The measuring method according to claim 15, wherein the first focus information and the second focus information are any one of a position of the sample, a position of the observation optical system, and a distance between the sample and the observation optical system.

17. The measuring method according to claim 15, wherein a distance in a plane orthogonal to an optical axis of the observation optical system is measured.

18. A measuring method comprising:

performing focusing on a first position on a surface of the sample using the focusing method of claim 1, and acquiring first focus information when focus is achieved;

performing focusing on a second position on the surface of the sample using the focusing method of claim 1, and acquiring second focus information when focus is achieved; and

acquiring a difference between the first focus information and the second focus information.

19. A principal point detecting method comprising detecting a principal point of a sample having a surface shape that is curved, using the focusing method of claim 1.

20. A focusing method comprising:

a step of preparing a microscope including an illumination optical system and an observation optical system, the illumination optical system includes a condenser lens, the observation optical system includes an objective lens, in the step of preparing, a partial region of illumination light is shielded or darkened by an aperture, wherein the aperture is between the condenser lens and a light source;

a step of mounting a sample having a surface shape that is curved, on the microscope, in the step of mounting, the sample and the observation optical system are opposed to each other in a state in which the surface shape of the sample is not deformed; and

a predetermined processing step, the predetermined processing step comprising;

a step of receiving light emitted from the observation optical system;

a step of obtaining a quantity of light based on light from a predetermined region of the received light, the predetermined region is a partial region of the sample;

a step of calculating a difference or a ratio between the quantity of light in the predetermined region and a quantity of light as a reference;

a step of comparing a calculation result with a threshold; and

a step of changing a distance between the sample and the observation optical system,

when the calculation result is equal to or smaller than the threshold, the predetermined processing step is terminated,

following conditional expression is satisfied;

0.6≤

(R₀×

β

)/R_ob<

0.995

where

R₀ is a length from an optical axis of the illumination optical system to an outer edge of the partial region of illumination light;

R_ob is a radius of a pupil of the objective lens; and

β

is a value obtained by dividing a focal length of the objective lens by a focal length of the condenser lens.

21. A focusing method comprising:

a step of preparing a microscope including an illumination optical system and an observation optical system, in the step of preparing, a partial region of illumination light is shielded or darkened by an aperture, wherein a first region is the partial region of illumination light, a second region is formed outside the first region with a region interposed therebetween to allow the illumination light to pass through, and shields or darkens part of the illumination light, an image of an outer edge of the first region is formed inside an outer edge of a pupil of the observation optical system, and an image of an inner edge of the second region is formed outside the outer edge of the pupil of the observation optical system, wherein the aperture is between a light source of the illumination light and a sample;

a step of mounting a sample having a surface shape that is curved, on the microscope, in the step of mounting, the sample and the observation optical system are opposed to each other in a state in which the surface shape of the sample is not deformed; and

a predetermined processing step, the predetermined processing step comprising;

a step of receiving light emitted from the observation optical system,

a step of obtaining a quantity of light based on light from a predetermined region of the received light, the predetermined region is a partial region of the sample,

a step of calculating a difference or a ratio between the quantity of light in the predetermined region and a quantity of light as a reference,

a step of comparing a calculation result with a threshold, and

a step of changing a distance between the sample and the observation optical system,

when the calculation result is equal to or smaller than the threshold, the predetermined processing step is terminated.

22. The focusing method according to claim 21, wherein

the illumination optical system includes a condenser lens,

the observation optical system includes an objective lens, and

following conditional expression is satisfied:

1.01<

(R₀×

β

)/R_ob≤

2

where

R₁ is a length from an optical axis of the illumination optical system to the inner edge of the second region;

R_ob is a radius of a pupil of the objective lens; and

β

is a value obtained by dividing a focal length of the objective lens by a focal length of the condenser lens.

23. A focusing device comprising:

an illumination optical system;

an observation optical system;

a holding member;

a drive device;

a light-receiving device; and

a processing device, wherein

the illumination optical system includes a light source, a condenser lens, and an aperture member,

the observation optical system includes an objective lens and an imaging lens,

the holding member is configured to hold a sample and is disposed between the illumination optical system and the observation optical system,

the drive device changes a distance between the holding member and the observation optical system,

the light-receiving device is disposed at a position where light emitted from the observation optical system is received,

the processing device comprises

a unit configured to obtain a quantity of light from a predetermined region of the received light,

a unit configured to calculate a difference or a ratio between the quantity of light in the predetermined region and a quantity of light as a reference,

a unit configured to compare a result of the calculation with a threshold, and

a unit configured to control a change in the distance,

the aperture member has a light-shielding part or a darkening part, and a transmission part,

at least part of the transmission part is located outside the light-shielding part or the darkening part,

the predetermined region is a partial region of the sample,

when the calculation result is equal to or smaller than the threshold, processing in the processing device is terminated,

the focusing device further comprises a unit configured to detect the predetermined region and a unit configured to store the predetermined region,

the unit configured to detect includes

a unit configured to record the distance and a quantity of light at the distance, for each of the regions,

a unit configured to obtain a range in which the quantity of light changes monotonously, for each of the regions, and

a unit configured to specify a region in which the range is longest among the regions.

24. The focusing device according to claim 23, wherein

the focusing device further comprises a unit configured to store the quantity of light as a reference, and

light emitted from the illumination optical system is emitted to the observation optical system in a state in which the sample is placed on the holding member, and the quantity of light as a reference is obtained based on light from a region where the sample is not present in a field of view.

25. The focusing device according to claim 23, wherein a region in which a change in the quantity of light is most monotonous is the predetermined region.

26. The focusing device according to claim 23, wherein the aperture member is projected onto a pupil position of the objective lens.

27. The focusing device according to claim 23, wherein an image of the light-shielding part or the darkening part is smaller in size than a pupil of the objective lens.

28. The focusing device according to claim 23, wherein an area of an image of the light-shielding part or the darkening part is 50% or more of an area of a pupil of the objective lens.

29. The focusing device according to claim 23, wherein the aperture member is disposed such that the light-shielding part or the darkening part includes an optical axis of the illumination optical system.

30. The focusing device according to claim 23, wherein a shape of the light-shielding part or a shape of the darkening part is a point-symmetric shape.

31. The focusing device according to claim 23, wherein a permissible range of displacement between the light-shielding part or the darkening part and a pupil position of the condenser lens is within 20% of a focal length of the condenser lens.

32. The focusing device according to claim 23, wherein the light source is a monochromatic light source, or the illumination optical system includes wavelength selecting means.

33. The focusing device according to claim 23, wherein

the observation optical system includes an aperture member, and

the aperture member is disposed at a pupil position of the objective lens or at a position conjugate with the pupil position of the objective lens.

34. The focusing device according to claim 23, further comprising:

another aperture member that is different from the aperture member; and

a moving mechanism configured to move the aperture member and the other aperture member.

35. A measuring device configured to hold a sample with a holding container having a bottom surface provided with a marker, the measuring device comprising:

a unit configured to perform focusing on a surface of the sample using the focusing device according to claim 23 and to acquire first focus information when focus is achieved; and

a unit configured to perform focusing on the marker using a focusing device that performs focusing based on a change in contrast and to acquire second focus information when focus is achieved,

wherein a difference between the first focus information and the second focus information is acquired.

36. The measuring device according to claim 35, wherein the first focus information and the second focus information is any one of a position of the sample, a position of the observation optical system, and a distance between the sample and the observation optical system.

37. The measuring device according to claim 35, further comprising a unit configured to measure a distance in a plane orthogonal to an optical axis of the observation optical system.

38. A measuring device comprising:

a unit configured to perform focusing on a first position on a surface of the sample using the focusing device according to claim 23 and to acquire first focus information when focus is achieved; and

a unit configured to perform focusing on a second position on the surface of the sample using the focusing device according to claim 23 and to acquire second focus information when focus is achieved,

wherein a difference between the first focus information and the second focus information is acquired.

39. A principal point detecting device comprising:

a focusing device according to claim 23, wherein

a principal point of a sample having a surface shape that is curved is detected using the focusing device.