Dual color camera microscope and methodology for cell staining and analysis

• US 4,998,284 A
• Filed: 02/24/1989
• Issued: 03/05/1991
• Est. Priority Date: 11/17/1987
• Status: Expired due to Term

• Current Assignee: Cell Analysis Systems, Inc. (Becton, Dickinson & Co)
• Original Assignee: Cell Analysis Systems, Inc. (Becton, Dickinson & Co)
• Inventors: Bacus, James W., Hernicz, Ralph S.
• Primary Examiner(s): Moore, David K.
• Assistant Examiner(s): Razavi, Michael
• Application Number:
  US07/315,443
• Time in Patent Office: 739 Days
• Field of Search: 381/1, 381/6, 358/101, 358/39, 250/573, 250/564, 250/565, 356/39, 356/244, 356/256
• US Class Current: 382/133
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1472 with colour
• CPC Class Codes: G01N 2021/1776 Colour camera
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G06T 7/85 Stereo camera calibration
• CPC Class Codes: G06V 20/69 Microscopic objects, e.g. biological cells or cellular parts

Abstract

A method and apparatus is disclosed for use in performing automated classification of cells and other microscopic specimens. The apparatus provides a compact, adjustable assembly that is operable to provide: an operator-apparatus interactive classification system for the cell analysis; alternative techniques for different cells, cytoplasms and cell populations; and enhanced image or color separation and analysis.

Claims

1. A method of analyzing cells, having at least a first cellular component and a second cellular component, said method comprising:

chemically-optically enhancing the cells with a first and a second spectral stain material,

each of said first and second spectral stain materials combining with one of said first and second cellular components to form a first cell combination and a second cell combination,

said first cell combination having a maximum optical transmittance at a first predetermined wavelength and a lower optical transmittance at a predetermined second wavelength,

said second cell combination having a substantial light absorption at said first predetermined wavelength,

transmitting an image of at least the first and second cell combinations, and communicating said image to a beam splitter;

splitting said image into a first image and a second image;

filtering the first image with a first filter to provide a first substantially monochromatic image at said first wavelength;

filtering the second monochromatic image with the second filter to provide a second substantially monochromatic image at said second wavelength;

sensing the filtered first monochromatic image with a first sensing means to provide a first electrical output representative of the first monochromatic image;

sensing the filtered second monochromatic image with a second sensing means to provide a second electrical output representative of the second monochromatic image; and

,

providing an analysis of characteristics of the cells at a characteristic analyzer based on the electrical outputs from the first and second sensing operations.

2. A method of analyzing cells as claimed in claim 1 and further in which the step of splitting said image in a first and a second images includes providing each said first and second images with different spectral wavelength bandwidths.

3. A method of analyzing cells as claimed in claim 1 and further comprising:

analyzing quantitatively said first monochromatic image for an associated one of said first and second cell components and identifying the other of said first and second cell components utilizing said second monochromatic image.

4. A method of analyzing cells as claimed in claim 3 and further comprising:

measuring the optical density of said associated one of said first and second cell components of said first monochromatic image to provide said quantitative analysis.

5. A method in accordance with claim 1 wherein the step of chemically-optically enhancing the cells with first and second materials comprises treating said cells with a monoclonal antibody stain.

6. A method in accordance with claim 2 further comprising the steps of processing the electrical signals from each sensing means separately to provide cell analysis information and also combining the information from both sensing means to provide a quantitative analysis of the examined cells.

7. A method of analyzing cells as claimed in claim 6 and further comprising:

providing a means for displaying any of said first image said second image and said combined image; and

coupling said display means to said means for analyzing to receive said sensed images.

51. An apparatus as claimed in claim 5 wherein said first and second sensors are monochromatic video cameras.

52. A method as claimed in claim 1 further comprising projecting light through said cell from a means for projecting light and forming a cell image.

53. A method in accordance with claim 1 including the step of transmitting light in wavelengths between 400-700 nm and using marrow band filters of about 20 nm for filtering.

55. A method in accordance with claim 1 including the step of combining a fluorescent spectral stain material with a first cellular component and using a fluorescent microscope for the analysis thereof.

8. An apparatus for the analysis of cells with at least a first cellular component and a second cellular component, a first spectral material and a second spectral material, one of said first and second cellular components chemically combining with one of the first and second spectral materials to provide a first cell combination and the other of said first and second cellular components chemically combining with the other of said first and second spectral materials to provide a second cell combination, said first cell combination having approximately maximum optical transmittance at a first predetermined wavelength and a lower optical transmittance at a second predetermined wavelength, said second cell combination having substantial light absorption at said first predetermined wavelength,

said apparatus comprising:

a source for projecting light on said cell;

means for forming and magnifying an image of the first and second cell combinations and for communicating the image;

means for splitting the communicated image into a first image and a second image;

first means for filtering the first image to provide a first substantially monochromatic image at said first wavelength;

second means for filtering the second image to provide a second substantially monochromatic image at said second predetermined wavelength;

first means for sensing the first monochromatic image and providing a first electrical output representative thereof;

second means for sensing the second monochromatic image and providing a second electrical output representative thereof; and

,

means for analyzing said cell combinations and thus said cells, which analysis means is operable to receive and store the respective first and second sensing means electrical outputs, to combine said first and second images to provide an analysis therefrom, and to provide an output about at least one cell characteristic.

9. An apparatus for the analysis of cells as claimed in claim 8 wherein said means for analyzing further comprises a means for displaying said first image, said second image, said combined first and second image and said analysis thereof.

31. An apparatus for the analysis of cells as claimed in claim 8 wherein said first spectral material is one of a thionin and a red chromogen activated by alkaline phosphatase, and the second spectral material is the other of said thionin and red chromogen.

32. An apparatus for the analysis of cells as claimed in claim 31 wherein one of said first and second cellular components is a DNA nucleus and said thionin is operable to combine with and distinguish said DNA nucleus in said cell.

33. An apparatus for the analysis of cells as claimed in claim 31 wherein one of said first and second cellular components is selected from among the group comprised of proteins, hormones and lipids, and said red chromogen and alkaline phosphatase is operable to combine with and distinguish said one component for said analysis.

34. An apparatus for the analysis of cells as claimed in claim 8 wherein said first spectral material is one of methyl green and diaminobenzidene with peroxidase, and the second spectral material is the other of said methyl green and diaminobenzidene with peroxidase.

35. An apparatus for the analysis of cells as claimed in claim 34 wherein one of said first and second cellular components is a DNA nucleus and said methyl green is combinable with said nucleus to chemically-optically enhance said DNA nucleus in said cell.

36. An apparatus for the analysis of cells as claimed in claim 35 wherein the other of said first and second components is selected from among the group of proteins, hormones and lipids, and said diaminobenzidene with peroxidase is combinable with said other component for chemical-optical enhancement for analysis.

37. An apparatus for the analysis of cells as claimed in claim 8 wherein said first spectral material is one of methyl green and red chromogen with alkaline phosphatase, and the second spectral material is the other of said methyl green and red chromogen.

38. An apparatus for the analysis of cells as claimed in claim 37 wherein one of said first and second cellular components is a DNA nucleus combinable with said methyl green for chemical-optical enhancement of said DNA nucleus in said cell.

39. An apparatus for the analysis of cells as claimed in claim 37 wherein one of said first and second cellular components is selected from among the group comprised of proteins, hormones and lipids and is combinable with said red chromogen with alkaline phosphatase for chemical-optical enhancement of said one component for analysis.

54. A method as claimed in claim 8 in which the first filter is substantially matched to said first wavelength and the second filter is substantially matched to said second wavelength.

10. An apparatus for the analysis of at least one cell, which cell has at least a first cellular component and a second cellular component, one of said first and second components chemically-optically enhanced by combining with one of a first spectral material and a second spectral material to define a first cell combination, the other of said first and second cellular components chemically-optically enhanced by combining with the other of said first and second spectral materials to define a second cell combination, one of said first and second cell combinations having approximately a maximum transmittance at one of a first and second predetermined wavelength and a lower transmittance at the other of said first and second wavelengths, and the other of said first and second cell combinations having substantial light absorption at said first predetermined wavelength, said apparatus comprising:

means for providing a magnified image of at least the first and second cellular components in a magnified component image light beam with a continuous spectrum;

means for acquiring said magnified image;

said image acquiring means having means for reflecting said magnified component image light beam, means for splitting said magnified component image light beam into at least a first and a second light beam, means for monochromatically filtering said first and second light beams, and, a first sensor and a second sensor operable to receive monochromatic light beams and provide an electrical output signal based on said monochromatic light beams;

said beam splitter means operable to receive said component image light beam and transmit a first light beam and a second light beam;

said first and second light beams communicated to said first and second sensors through said filter means, which filter means are optically matched to said first and second cell combinations and said predetermined first and second wavelengths to filter said first beam to provide one of said first and second sensors approximately a monochromatic fist light beam at approximately one of said first and second predetermined wavelengths, and approximately a monochromatic second light beam at approximately the other of said first and second predetermined wavelengths to the other of said first and second sensors,

each of said first and second sensors operable to provide an electronic signal indicative of said monochromatic images; and

,

means for digitizing and storing an electrical signal for each of said first and second light beams, which digitizing means is operable to receive said electrical output signals from said first and second sensors to digitize, combine, store and project said first and second light beam signals for review and analysis of said cell.

11. An apparatus as claimed in claim 10 further comprising means for reflecting;

said means for beam splitting having a first beam splitter providing a split path for the magnified component light beam and a second beam splitter, which first beam splitter and reflecting means cooperate to communicate said magnified image light beam to said second beam splitter;

said second beam splitter having a predetermined splitting wavelength operable to split said magnified light beam image into a first light beam below said predetermined splitting wavelength and a second light beam above said predetermined splitting wavelength for communication to said first and second sensors;

said filter means operable to filter said first light beam and second light beam communicated to said first and second sensors at said predetermined first and second wavelengths, one of said first and second wavelengths approximately at the maximum optical transmittance of the first and second light beams.

12. An apparatus as claimed in claim 11 wherein said filter means are a first monochromatic optical filter operable to filter and first light beam at approximately said first predetermined wavelength and a second monochromatic optical filter operable to filter and said second light beam at approximately said second predetermined wavelength, which filtered first and second light beams are communicated to said first and second sensors, respectively.

13. An apparatus as claimed in claim 12 wherein said first and second sensors comprise a first video camera and a second video camera;

said fist camera positioned and operable to receive said filtered first light beam, and said second camera positioned and operable to receive said filtered second light beam, which first and second cameras are operable to provide an electrical output representative of said image at said first and second wavelengths, respectively.

14. An apparatus as claimed in claim 13 wherein said reflecting means comprises a first mirror, a second mirror and a third mirror;

said image acquisition apparatus further comprising a focusing lens, which lens is interposed between said first mirror and said first beam splitter to provide a focused and magnified image cell component light beam to said first mirror; and

, said second mirror is operable to receive and communicate said focused and reflected first light beam image from said first mirror to said second beam splitter.

15. An apparatus as claimed in claim 14 wherein said third mirror is positioned and operable to reflect said first beam from said second beam splitter to said first sensor.

16. An apparatus as claimed in claim 15 wherein said second beam splitter, said third mirror and first sensor cooperate to define a first communication path distance therebetween for said first light beam;

said second beam splitter and said second sensor cooperate to define a second communication path distance therebetween for said second light beam,

which first and second communication path distances are approximately equal.

17. An apparatus as claimed in claim 13 wherein said first sensor has a first longitudinal axis, which first sensor is movable along said first axis to focus said first sensor and one of said filtered first and second light beam image in said digitizing means;

and

said second sensor has a second longitudinal axis, which second sensor is movable along and rotatable about said second axis to focus said second sensor and the other of said first and second light beam image in said digitizing means.

18. An apparatus as claimed in claim 17 wherein said first sensor and second sensor further comprise a first electronic gain adjust and a second electronic gain adjust, respectively, to focus said first and second light beam image in said digitizing means.

19. An apparatus as claimed in claim 17 further comprising a housing;

said housing having a top panel, a front panel, rear panel, a bottom panel, and a first and a second sidewall, which cooperate to define a chamber for said image acquisition means;

said first beam splitter including a focus lens mounted in said housing; and

said reflecting means, said first and second monochromatic optical filters, said second beam splitter, and said first and second sensors are adjustably mounted in said housing.

20. An apparatus as claimed in claim 19 further comprising a first mirror mounting base and a second mirror mounting base, each base having a generally cylindrical first pintle and second pintle for said first and second mounting base, respectively;

said first mirror and said second mirror mounted and operable on said first mounting base and second mounting base, respectively;

said bottom panel defining a horizontal reference plane and, a first pivot socket and a second pivot socket to receive said first and second mounting base pintles, and, a first threaded bore and a second threaded bore open to said first and second pivot sockets, respectively;

said first pintle positioned and rotatable in said first pivot socket and said second pintle positioned and rotatable in said second pivot socket to rotatably adjust said first mirror and second mirror, respectively, for reflecting said magnified component beam from said first beam splitter to said second beam splitter; and

,

a first threaded locking screw and a second threaded locking screw matable with said first threaded bore and second threaded bore, respectively, to contact said pintles and secure said pintles and mirrors in position.

21. An apparatus as claimed in claim 20 further comprising a means for adjusting said second beam splitter, which adjusting means has a mounting frame and a gimballed base;

said second beam splitter mounted and operable in said frame, which frame defines at least a first, a second and a third through-hole;

said bottom panel defining a washer well having a first threaded and generally centered bore and, a recessed seat with a sidewall and a second threaded bore, said recessed seat operable to receive said gimballed base which is adjustable therein, and a threaded locking passage;

said gimballed base defining a generally centrally located cavity and, a first, a second and a third threaded base bore, and a pivot passage alignable with said recessed seat second threaded bore;

a spherical bearing mounted in said centrally located cavity;

a first, a second and a third threaded means for securing, and a threaded pivot means for securing;

a first, second and third vertical biasing means positioned between said frame and gimballed base and aligned with said first, second and third base threaded bores, said mounting frame and second beam splitter positioned on said gimballed base with said first, second and third through-holes aligned with said first, second and third threaded gimballed base bores and vertical bias means to receive said first, second and third threaded securing means to mate, respectively, with said first, second and third threaded base bores to secure said frame and second beam splitter against the bias of said vertical biasing means;

said threaded pivot means for securing extending through said gimballed base pivot passage and threadably engaging said recessed seat second threaded bore;

a lateral bias means positioned and operable between said recessed seat sidewall and gimballed base to pivotably bias said base in said recessed seat about said threaded pivot securing means;

a third locking screw to control the rotation of said gimballed base positioned and operable in said threaded locking passage to contact said gimballed base positioned in said recess opposite said lateral biasing means;

a washer with a washer through-bore;

a threaded means for securing said washer;

said washer positioned in said washer well with said washer through-bore aligned with said washer-well threaded bore to receive said washer securing means;

said washer contacting said laterally biased gimballed base, and cooperating with said threaded pivot securing means and said third locking screw to secure said base at a fixed position with said washer and washer securing means against rotational movement by said lateral biasing means.

22. An apparatus as claimed in claim 21 further comprising a generally vertical member and a second gimballed mounting having a lower section with at least a first and a second mounting bolt port;

said third reflecting mirror mounted on said vertical member;

a pair of bolts for securing said second gimbal;

said bottom panel defining at least a first threaded gimbal port and a second threaded gimbal port;

said gimballed mounting lower section first and second bolt ports aligned with said bottom panel first and second threaded gimbal ports, to receive one of said pair of second gimbal bolts, respectively, which bolts secure said second gimbal mounting assembly to said bottom panel;

said gimbal vertical member defining a first and a second threaded adjustment port, which adjustment ports are both vertically and horizontally offset from each other;

a first threaded adjustment member and a second threaded adjustment member, which first and second threaded adjustment members are positioned in said first and second adjustment ports, respectively, and operable to contact and adjust said third mirror both vertically and horizontally, to reflect and communicate said first beam from said second beam splitter to said first sensor.

23. An apparatus as claimed in claim 22 further comprising a first monochromatic optical filter mounting frame and a second monochromatic optical filter mounting frame;

each of said frames further defining means for receiving and retaining said optical filters therein;

each of said frames defining a first mounting bolt bore and a second mounting bolt bore;

a pair of threaded bolts for each of said first and second frames;

said bottom panel defining a pair of first filter threaded bolt holes, and a pair of second filter threaded bolt holes, which bolt holes are alignable with said first and second filter frame bores, respectively, to receive said threaded bolts to secure said frames to said bottom panel.

24. An apparatus as claimed in claim 23 wherein each of said frame means for retaining and securing said optical filters is a slotted frame.

26. An apparatus as set forth in claim 12 wherein said first optical filter is an optical bandpass filter operable to filter a light beam at a wavelength of about 620 nanometers.

27. An apparatus as set forth in claim 26 wherein said first optical filter is an optical bandpass filter operable to filter a light beam at a wavelength between about 610 nanometers and 630 nanometers.

28. An apparatus as set forth in claim 12 wherein said second optical filter is an optical bandpass filter operable to filter a light beam at a wavelength of about 500 nanometers.

29. An apparatus as set forth in claim 28 wherein said second optical filter is operable to filter a light beam at a wavelength between about 490 and 510 nanometers wide.

30. An apparatus as claimed in claim 12 wherein said second beam splitter is a dichroic beam splitter.

25. An apparatus for the analysis of at least one cell, each of said cells having at least one nuclear DNA with a nucleus, that is chemically-optically enhanced by a first spectral stain material, and at least one protein and associated protein site, that is chemically-optically enhanced by an immunohistochemical spectral stain material,

said stained nucleus having approximately maximum optical transmittance at a first predetermined wavelength and a lower optical transmittance at a second predetermined wavelength,

said stained proteins at said protein sites having a substantial light absorption at said first predetermined wavelength, said apparatus comprising:

means for providing a magnified objective image of said at least one cell in a magnified cell component;

means for acquiring said magnified image;

said image acquiring means having means for beam splitting said magnified image, means for reflecting said image, means for monochromatically filtering light beams and at least a first sensor and a second sensor each operable to receive a monochromatic light beam and provide an analog electrical output signal representative of said sensed monochromatic light beam;

said image from said magnifying means comprising a plurality of wavelengths encompassing a broadband spectrum from one of the visible and infrared light ranges;

said beam splitter means operable to receive said magnified cell component light beam and transmit a first light beam below a predetermined splitting wavelength and a second light beam above said splitting wavelength to said first and second sensors, respectively, through said monochromatic filter means, which monochromatic filter means are operable to filter said first and second light beams at said first and second predetermined wavelengths to provide said first and second sensor a monochromatic first light beam at approximately said first predetermined wavelength and a monochromatic second light beam at approximately said second predetermined wavelength to the other of said first and second sensors; and

,

means for digitizing and storing an analog signal for each first and second light beams, which digitizing means is operable to receive said analog electrical output signals from said first and second sensors to digitize, combine, store and project said first and second light beam signals for review.

43. An apparatus as claimed in claim 25 wherein said immunohistochemical staining material contains a specific antigen.

44. An apparatus as claimed in claim 43 wherein said immunohistochemical staining material has a monoclonal antibody against said specific antigen.

45. An apparatus as claimed in claim 44 wherein said immunohistochemical staining material includes staining with an alkaline phosphatase compound.

46. An apparatus as claimed in claim 25 wherein said means for providing a magnified image further comprises a mounting stage, which mounting stage is operable to receive a specimen holder.

47. An apparatus as claimed in claim 46 wherein said specimen holder is operable to retain an alignment grid for said first and second cameras, which grid has a predetermined matrix.

48. An apparatus as claimed in claim 47 wherein said grid is a matrix of a plurality of horizontally and vertically aligned squares approximately 0.01 millimeters on a side.

49. An apparatus as claimed in claim 46 wherein said first and second sensors each have a means for adjusting said sensors, which adjusting means is operable to adjust the focus and magnification of said sensor output.

50. An apparatus as claimed in claim 49 wherein said adjusting means includes a rotational adjustment and a translational adjustment for aligning said patterns on said first and second sensors.

40. A method of calibration for an apparatus utilizing two solid state sensors in the image analysis of cells, said method comprising:

providing means for analyzing cells, which analyzing means has a microscope with a cell examination location, at least one beam splitter, a first optical filter, a second optical filter, means for projecting light through said cell, and a means for providing a cell characteristic;

providing a calibration pattern to be viewed by a microscope at the examination location of the cells;

projecting a light beam through said pattern to provide an image;

acquiring an image of the pattern and communicating the image to a beam splitter;

splitting the image into first and second images of the pattern;

sensing the first image by the first sensor and providing a first electrical output representative of the first image of the pattern;

sensing the image of the pattern by a second sensor and providing a second electrical output representative of the second image pattern, each of said first and second electrical outputs communicated to said means for providing a cell characteristic;

combining and displaying the first and second pattern images overlying one another; and

adjusting the sensor positions to align the overlying patterns to provide a substantially pixel-by-pixel alignment of the image patterns being sensed by the first and second sensors.

41. A method of calibration in accordance with claim 40 further comprising providing a grid pattern for said pixel-by-pixel alignment and including the step of overlying the grid image from one of said first and second sensors over the second grid image from the other of said first and second sensors.

42. A method of calibration in accordance with claim 41 further comprising adjusting at least one of said first and second sensors until the first and second grid images are substantially aligned one over the other.