Method and apparatus for generating a three-dimensional topographical image of a microscopic specimen

• US 5,831,736 A
• Filed: 08/29/1996
• Issued: 11/03/1998
• Est. Priority Date: 08/29/1996
• Status: Expired due to Term

• Current Assignee: Washington University In St Louis
• Original Assignee: Washington University In St Louis
• Inventors: Conchello, Jose-Angel, Lichtman, Jeff W.
• Primary Examiner(s): Font, Frank G.
• Assistant Examiner(s): Smith, Zandra V.
• Application Number:
  US08/705,412
• Time in Patent Office: 796 Days
• Field of Search: 356/375, 356/376, 250/559.02, 250/559.07, 250/237 G, 359/368, 359/389
• US Class Current: 356/613
• CPC Class Codes: G01B 11/24 for measuring contours or curvatures
• CPC Class Codes: G02B 21/22 Stereoscopic arrangements

Abstract

A method and apparatus for generating an image of a specimen comprises dividing the in-focus plane of an objective of an optical apparatus into a plurality of grid regions. Incident light is directed to the grid regions to illuminate surface portions of the specimen that are within the grid regions. Light reflected from the surface portions is sensed to determine approximate spatial slopes of each surface portion associated with each grid region. The spatial slopes are integrated to generate a topographical map representative of surface portions of the specimen that are within the depth of focus of the objective. Consecutive planes can be obtained to generate topographical maps thicker than the depth of focus of the objective.

Claims

1. A method of generating an image of a specimen comprising:

dividing the in-focus plane of an objective of a confocal microscope into a plurality of discrete regions;

directing incident light to the discrete regions to illuminate surface portions of the specimen that are within the discrete regions;

sensing light reflected from said surface portions;

determining an approximate spatial slope of each surface portion associated with each discrete region; and

integrating said determined spatial slopes to generate a topographical map representative of surface portions of the specimen that are within the depth of focus of the objective.

2. A method as set forth in claim 1 wherein the discrete regions of the in-focus plane of the objective are of a thickness substantially equal to the depth of focus of the objective.

3. A method as set forth in claim 1 wherein the step of directing incident light to the discrete regions comprises directing separate beams of incident light to the regions so that said reflected light comprises separate beams, the beams of reflected light corresponding to the surface portions of the specimen.

4. A method as set forth in claim 1 wherein the step of directing light to the discrete regions comprises serially directing light to each discrete region to separately illuminate each surface portion within a corresponding one of the discrete regions.

5. A method as set forth in claim 1 wherein the approximate spatial slope of each surface portion is determined by:

generating at least one signal representative of an intensity profile associated with light reflected off said each surface portion; and

applying known values to said at least one generated signal.

6. A method as set forth in claim 5 wherein applying known values to said at least one generated signal comprises comparing said at least one generated signal to one of a plurality of known values, each known value being associated with light reflected from a surface of known spatial slope.

7. A method as set forth in claim 1 wherein the step of sensing reflected light comprises sensing an intensity profile associated with the shape and position of a projection of at least a portion of the reflected light onto a particular area of a given plane.

8. A method as set forth in claim 7 wherein said particular area of said given plane comprises a plurality of area portions, and wherein sensing an intensity profile associated with the shape and position of said projection comprises generating a plurality of signals, each of which is representative of an area common to said projection and to one of said plurality of area portions.

9. A method as set forth in claim 8 wherein said plurality of signals are generated by a single light sensor.

10. A method as set forth in claim 7 wherein sensing an intensity profile associated with the shape and position of said projection comprises:

positioning a masking element relative to said given-plane so that a portion of said particular area of said given plane is masked and another portion of said particular area is unmasked, said masking element being configured such that movement of the masking element varies the portion of said particular area which is unmasked;

moving the masking element to a plurality of discrete positions relative to said given plane, a plurality of unmasked area portions of said particular area of said given plane corresponding to the plurality of discrete positions of the masking element; and

generating a plurality of signals, each signal being representative of an area which is common to said projection and to the unmasked area portion associated with each of said positions of the masking element.

11. A method of generating an image of a specimen comprising:

dividing the in-focus plane of an objective of a confocal microscope into a plurality of grid regions;

positioning the specimen a distance from the objective of the optical apparatus so that surface portions of the specimen are within the grid regions of the in-focus plane;

directing incident light to the grid regions to illuminate the surface portions of the specimen that are within the grid regions;

sensing light reflected from said surface portions to determine approximate spatial slopes of each surface portion associated with each grid region;

altering the distance between the specimen and objective so that other surface portions of the specimen, which were previously outside the grid regions of the focal plane, are within previously unoccupied grid regions of the in-focus plane;

directing incident light to the grid regions to illuminate said other surface portions of the specimen that are within said previously unoccupied grid regions;

sensing light reflected from said other surface portions to determine approximate spatial slopes of each of said other surface portions associated with each of said previously unoccupied grid regions; and

integrating said determined spatial slopes to generate a topographical map representative of surface portions of the specimen.

12. A method as set forth in claim 11 wherein the approximate spatial slope of each surface portion is determined by:

generating at least one signal representative of a characteristic associated with light reflected off said each surface portion; and

applying known values to said at least one generated signal.

13. A method as set forth in claim 12 wherein applying known values to said at least one generated signal comprises comparing said at least one generated signal to one of a plurality of known values, each known value being associated with light reflected from a surface of known spatial slope.

14. A method of approximating a spatial slope of a surface portion of a specimen comprising:

directing incident light along an incident light path of a confocal microscope to said surface portion of the specimen to reflect light from said surface portion;

sensing a characteristic associated with said reflected light; and

applying the sensed characteristic to values relating to spatial slope.

15. A method as set forth in claim 14 wherein sensing a characteristic associated with said reflected light comprises sensing an intensity profile associated with said reflected light.

16. A method as set forth in claim 15 wherein said values comprise a plurality of known characteristics, each of said known characteristics being associated with reflection of light from a surface of known spatial slope, and wherein the step of applying the sensed characteristic comprises matching the sensed intensity profile to one of said known characteristics.

17. A method as set forth in claim 15 wherein the step of directing light comprises directing incident light of a particular shape and direction to said surface portion so that said reflected light is of a particular shape.

18. A method as set forth in claim 17 wherein the step of sensing an intensity profile associated with said reflected light comprises sensing an intensity profile associated with the shape and position of a projection of at least a portion of the reflected light onto a particular area of a given plane.

19. A method as set forth in claim 18 wherein said particular area of said given plane comprises a plurality of area portions, and wherein sensing an intensity profile associated with the shape and position of said projection comprises generating a plurality of signals, each of which is representative of an area common to said projection and to one of said plurality of area portions.

20. A method as set forth in claim 19 wherein said plurality of signals are generated by a single sensor.

21. A method as set forth in claim 18 wherein sensing an intensity profile associated with the shape and position of said projection comprises:

positioning a masking element relative to said given plane so that a portion of said particular area of said given plane is masked and another portion of said particular area is unmasked, said masking element being configured such that movement of the masking element varies the portion of said particular area which is unmasked;

moving the masking element to a plurality of discrete positions relative to said given plane, a plurality of unmasked area portions of said particular area of said given plane corresponding to the plurality of discrete positions of the masking element; and

generating a plurality of signals, each signal being representative of an area which is common to said projection and to the unmasked area portion associated with each of said positions of the masking element.

22. A method as set forth in claim 20 wherein the step of applying the sensed characteristic to values relating to spatial slope comprises applying the generated signals to values relating to spatial slope.

23. A method as set forth in claim 22 wherein said values comprise a plurality of known values, each known value being associated with reflection of light from a surface of known spatial slope, and wherein the step of applying the generated signals comprises matching the generated signals to one of said known values.

24. A method of generating a topographical image of a specimen, said method comprising the steps of:

directing incident light through an objective of a confocal microscope onto said specimen to thereby illuminate a plurality of surface portions thereof,

sensing light reflected from only those surface portions thereof that are within said objective'"'"'s depth of field and numerical aperture,

determining from said sensed reflected light a spatial slope for said surface portions, and

integrating said determined spatial slopes to thereby generate a topographical map representative of said light reflecting surface portions, said topographical map thereby comprising said topographical image of said specimen.

25. A method as set forth in claim 24 further comprising the step of re-positioning said specimen with respect to said objective, as required, to bring more of the surface portions of said specimen within the objective'"'"'s depth of field.

26. The method as set forth in claim 25 wherein the step of directing incident light includes the step of scanning said specimen with the incident light to thereby sequentially illuminate separate surface portions of the specimen.

27. The method as set forth in claim 26 wherein the step of sensing the reflected light includes the step of sensing a profile of said reflected light.

28. The method as set forth in claim 27 wherein the step of sensing a profile includes the step of marking said reflected light from each of said surface portions with an aperture, and illuminating again each of said surface portions as said aperture is re-positioned.

29. The method as set forth in claim 28 wherein the integrating step includes the step of normalizing the values of height of adjacent determined spatial slopes.

30. The method as set forth in claim 27 wherein the step of determining the spatial slope includes the step of calculating a spatial slope from the sensed profile of each surface portion.

31. The method as set forth in claim 30 wherein the step of calculating a spatial slope includes the step of referring to a look up table.

39. A microscope as set forth in claim 30 wherein said detecting means further comprises means for generating a plurality of signals, each signal being representative of an area which is common to said projection and to the unmasked area portion associated with each of a plurality of discrete positions of the masking element relative to said particular area.

32. A confocal microscope for generating a topographical image of a specimen, said device comprising:

an incident light source for directing incident light through an objective onto said specimen to thereby illuminate a plurality of surface portions thereof,

a detector in a reflected light path for detecting light reflected from only those surface portions that are within the objective'"'"'s depth of field a numerical aperture, and

an electronic processor configured for determining a spatial slope for said surface portions, and integrating said determined spatial slopes into a topographical map to thereby generate a topographical image of the specimen.

33. A confocal microscope for approximating a spatial slope of a surface portion of a specimen, said microscope having:

optical elements configured for directing incident light along an incident light path from a light source to the surface portion of the specimen and for directing light reflected from the surface portion along a return light path, one of the optical elements including an exit pupil in the return light path, the optical elements being positioned and configured so that the exit pupil is illuminated by the reflected light in a manner dependent upon the spatial slope of the surface portion of the specimen;

a sensor in the return light path for generating at least one signal representative of the manner of illumination of the exit pupil; and

a processor for matching the generated signal to a signal associated with light reflected from a surface of a known spatial slope.

34. A confocal microscope for approximating a spatial slope of a surface portion of a specimen, said microscope having:

optical elements configured for directing incident light along an incident light path from a light source to the surface portion of the specimen and for directing light reflected from the surface portion along a return light path, one of the optical elements including an exit pupil in the return light path, one of the optical elements including an objective having a numerical aperture angle of at least approximately 50°

, the optical elements being positioned and configured so that the exit pupil is illuminated by the reflected light in a manner dependent upon the spatial slope of the surface portion of the specimen; and

a sensor in the return light path for generating at least one signal representative of the manner of illumination of the exit pupil.

35. A confocal microscope for approximating a slope and orientation of a surface portion of a specimen, said microscope having:

optical elements configured for directing incident light along an incident light path from a light source to the surface portion of the specimen and for directing light reflected from the surface portion along a return light path; and

means for detecting characteristics of the reflected light sufficient to approximate the spatial slope of the surface portion of the specimen.

36. A microscope as set forth in claim 35 wherein said optical elements are configured for directing incident light of a particular shape and direction to said surface portion so that the reflected light is of a particular shape.

37. A microscope as set forth in claim 36 wherein said detecting means comprises means for sensing a characteristic associated with the shape and position of a projection of at least a portion of the reflected light onto a particular area of a given plane.

38. A microscope as set forth in claim 37 wherein said sensing means includes a masking element positioned relative to said given plane and configured so that a portion of said particular area is masked by the masking element and another portion of said particular area is unmasked by the masking element, the masking element being moveable relative to said particular area for varying the portion of said particular area which is unmasked.

40. A confocal microscope for generating an image of a specimen, said microscope having:

an objective configured for being spaced a distance from the specimen at which at least part of the specimen is within the in-focus plane of the objective;

optical elements configured for directing incident light to discrete regions of the in-focus plane of the objective to illuminate surface portions of the specimen that are within the discrete regions;

a sensor for sensing light reflected from the surface portions and for generating signals representative of the sensed light; and

a processor configured for approximating, in response to the signals generated by the sensor, spatial slopes of each surface portion of the specimen within the discrete regions of the in-focus plane.

41. A microscope as set forth in claim 40 wherein the processor is further configured for integrating the determined spatial slopes to generate a topographical map representative of surface portions of the specimen.

42. A microscope as set forth in claim 40 wherein said optical elements are configured for directing incident light of a particular shape and direction to said discrete regions so that the reflected light is of a particular shape.

43. A microscope as set forth in claim 42 wherein said optical elements are configured for projecting at least a portion of the reflected light onto a particular area of a given plane.

44. A microscope as set forth in claim 43 further comprising a masking element positioned relative to said given plane and configured so that a portion of said particular area is masked by the masking element and another portion of said particular area is unmasked by the masking element, the masking element being moveable relative to said particular area for varying the portion of said particular area which is unmasked.

45. A microscope as set forth in claim 44 wherein the sensor is configured for generating a plurality of signals, each signal being representative of an area which is common to the projection and to the unmasked area portion associated with each of a plurality of discrete positions of the masking element relative to said particular area.

46. A microscope as set forth in claim 40 wherein the objective has a numerical aperture angle of at least approximately 50°

.

47. A microscope as set forth in claim 40 wherein the objective has a numerical aperture angle of at least approximately 60°

.