METHOD AND APPARATUS FOR CLASSIFYING AND SEGREGATING PARTICLES WITH ELECTRICAL AND OPTICAL MEANS

• US 3,675,768 A
• Filed: 03/17/1969
• Issued: 07/11/1972
• Est. Priority Date: 03/17/1969
• Status: Expired due to Term

• Current Assignee: Gildardo Legorreta-Sanchez
• Original Assignee: Gildardo Legorreta-Sanchez
• Inventors: Legorreta-Sanchez, Gildardo
• Primary Examiner(s): Kubasiewicz, E. E.
• Application Number:
  US04/809,476
• Time in Patent Office: 1,212 Days
• Field of Search: 324/71PC,61,34E,61 209/1,4,111.7,111.8 210/65
• US Class Current: 209/4
• CPC Class Codes: G01N 1/2813 Producing thin layers of samples on a substrate, e.g. smearing, spinning-on G01N1/30 takes precedence
• CPC Class Codes: G01N 1/312 for samples mounted on planar substrates
• CPC Class Codes: G01N 1/4077 by other techniques involving separation of suspended solids
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1029 Particle size
• CPC Class Codes: G01N 27/74 of fluids G01N24/00 takes precedence
• CPC Class Codes: G06M 1/101 by electro-optical means
• CPC Class Codes: Y10S 209/906 Pneumatic or liquid stream feeding item
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/819 Multifunctional antigen or antibody

Abstract

Apparatus for classifying, counting and segregating microscopic particles such as cells. The particles may be contained in a gas or liquid and may be classified by measuring two characteristics thereof such as the size of the nucleus and the size or volume of the cell. This permits to count and separate red blood corpuscules from leucocytes in the same solution. Normal and atypical cells may be physically separated. The size of the nucleus may be measured by transmitted light and the size of the cell by variations of an electromagnetic field or by scattered light.

Claims

2. Apparatus for classifying microscopic particles suspended in a transparent liquid, comprising:

a. a microscope having a focal plane;

b. means for causing the suspension of particles to flow in a path, said path extending from one side to the other side and once across said focal plane;

c. means for illuminating said focal plane;

d. means for detecting changes of the light absorbed by each particle as it flows through said focal plane; and

e. means disposed adjacent said focal plane for selectively removing a desired particle and collecting it.

3. Apparatus for classifying microscopic cells having a nucleus and suspended in a transparent liquid, comprising:

a. a microscope having a focal plane;

b. means for causing the suspension of cells to flow in a path, said path crossing said focal plane once;

c. means for illuminating said focal plane and for measuring the light absorbed by each cell as it flows through said focal plane, thereby to determine the image of the cell;

d. means for establishing a steady electromagnetic field extending through said focal plane;

e. means for measuring the change of the electromagnetic field caused by the passage of a cell through said focal plane, thereby to trigger the light absorption measuring means; and

f. means for detecting the cells that have predetermined image characteristics.

4. Apparatus for investigating microscopic cells having a nucleus and suspended in a transparent liquid, comprising:

a. a microscope having an objective providing a focal plane, said objective being immersed in the liquid;

b. means for causing the suspension of cells to flow in a path, said path extending from one side across said focal plane and to the other side thereof;

c. means for illuminating said focal plane with light of a wave length aBsorbed by the nucleus of the cells;

d. means for detecting and measuring the light absorbed by the nucleus of each cell as it flows through said focal plane;

e. means for establishing a steady electromagnetic field extending through said focal plane; and

f. means for measuring the change of the electromagnetic field caused by the passage of a cell through said focal plane to determine the volume of the cell.

5. Apparatus for classifying microscopic cells having a nucleus and suspended in a transparent liquid, comprising:

a. a microscope having an objective providing a focal plane, said objective being immersed in the liquid;

b. means for causing the suspension of cells to flow in a path, said path extending once across said focal plane;

c. means for illuminating said focal plane with light of a wave length absorbed by the nucleus of the cells;

d. means for detecting and measuring the light absorbed by the nucleus of each cell as it flows through said focal plane;

e. means for establishing a steady electromagnetic field extending through said focal plane;

f. means for measuring the change of the electromagnetic field caused by the passage of a cell through said focal plane to determine the volume of the cell; and

g. means disposed adjacent said focal plane for selectively removing a desired cell and for collecting it.

6. Apparatus for counting simultaneously both red and white blood corpuscles suspended in a transparent liquid, the white blood corpuscles having each a nucleus, said apparatus comprising:

a. a microscope having a focal plane;

b. means for causing the blood corpuscles to flow in a path, said path passing once through said focal plane;

c. means for illuminating said focal plane both with direct and indirect light;

d. means for measuring the light absorbed by the nucleus of each white blood corpuscle as it flows through said focal plane, thus enabling the detection of each white blood corpuscle;

e. means for establishing a steady electromagnetic field extending through said focal plane;

f. means for measuring the change of the electromagnetic field caused by the passage of a blood corpuscle through said focal plane, thus enabling the detection of the total number of blood corpuscles; and

g. means for counting the total blood corpuscles and including the white blood corpuscles.

7. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a. a compound microscope having an objective immersed in the liquid and an ocular and providing a focal plane adjacent said objective;

b. means for causing the suspension of cells to flow across said focal plane;

c. means for illuminating said focal plane with light having a wave length range absorbed by the nucleus of the cells of said suspension;

d. means including a light sensitive means disposed adjacent the ocular of said microscope and responsive to the variation of light caused by the nucleus of each cell as it moves across said focal plane;

e. means for establishing a steady electromagnetic field within a volume including said focal plane;

f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell;

g. hydraulic pressure means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

h. means responsive to a predetermined range of values of the light absorbed by the nucleus and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined size from other cells.

8. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a. a compound microscope having an objective immersed in the liquid and an ocular and providing a focal plane adjacent said objective;

b. means for causing the suspension of cells to flow across said focal plane;

c. means for illuminating said focal plane with light having a wave length range absorbed by the nucleus of the cells of said suspension;

d. means including a light sensitive device disposed adjacent the ocular of said microscope and responsive to the variation of light caused by the absorption of light by each cell as it moves across said focal plane;

e. means for establishing a steady electromagnetic field within a volume including said focal plane;

f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell;

g. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

h. means responsive to a predetermined range of values of the variation of the light caused by at least a portion of the cell and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined size from other cells.

9. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a. a compound microscope having an objective, said objective being immersed in the liquid and an ocular and providing a focal plane adjacent said objective;

b. means for causing the suspension of cells to flow across said focal plane;

c. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

d. an opaque diaphragm having a variable slit disposed in said focal plane;

e. means including a light sensitive device disposed adjacent the ocular of said microscope and responsive to the variation of the light absorption caused by the nucleus of each cell as it moves across said focal plane;

f. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

g. means responsive to a predetermined range of values of the variation of light caused by a nucleus for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.

10. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a. a compound microscope having an objective, said objective being immersed in the liquid and an ocular and providing a focal plane adjacent said objective;

b. means for causing the suspension of cells to flow across said focal plane;

c. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

d. means including a light sensitive means disposed adjacent the ocular of said microscope and responsive to the variation of the light absorption caused by at least a portion of each cell as it moves across said focal plane;

e. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

f. means responsive to a predetermined range of values of the variation of light caused by a cell for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.

11. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a. a compound microscope having an objective, said objective being immersed in the liquid, and an ocular and providing a focal plane adjacent said objective;

b. said microscope having a central annular light condenser and an outer annular light condenser spaced therefrom, both being immersed in the liquid for illuminating said focal plane by direct and tangential light;

c. means for causing the suspension of cells to flow across said focal plane;

d. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

e. means including a light sensitive device disposed adjacent the ocular of said microscope and responsive to the variation of the light absorption caused by the nucleus of each cell as it moves across said focal plane;

f. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

g. means responsive to a predetermined range of values of the variation of light caused by a nucleus for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.

12. An attachment for a compound microscope of the type having an objective and ocular and providing a focal plane adjacent said objective, said attachment comprising:

a. means for causing a suspension of cells to flow across said focal plane;

b. means for illuminating the microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

c. means including a light sensitive device disposed adjacent the ocular of the microscope and responsive to the variation of light absorbed by the nucleus of each cell as it moves across said focal plane;

d. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

e. means responsive to a predetermined range of values of the light absorbed by a nucleus for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption from other cells.

13. Apparatus for classifying microscopic particles suspended in a fluid to provide a suspension of particles, said apparatus comprising:

a. means for establishing a steady electromagnetic field within a predetermined volume of a liquid;

b. means for measuring the change of the field caused by the passage of each individual particle through said volume and indicative of the volume of the particle;

c. means disposed in the vicinity of said volume for selectively removing a desired particle and collecting it; and

d. means responsive to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a particle, thereby to segregate particles having a predetermined volume from other particles.

14. Apparatus for classifying microscopic particles suspended in a fluid to provide a suspension of particles, said apparatus comprising:

a. means for establishing a steady electromagnetic field within a predetermined volume of a liquid;

b. means for measuring the change of the field caused by the passage of each individual particle through said volume and indicative of the volume of the particle;

c. means disposed in the vicinity of said volume for selectively removing a desired particle and collecting it;

d. means responsive to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a particle, thereby to segregate particles having a predetermined volume from other particles; and

e. means for counting the collected particles.

15. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a. a compound microscope having a reflective objective and a reflective ocular immersed in the liquid and providing a focal plane adjacent said objective;

b. means including a pressure differential for causing the suspension of cells to flow across said focal plane;

c. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

d. means including a photomultiplier disposed adjacent the ocular of said Microscope and responsive to the amount of light absorbed by the nucleus of each cell as it moves across said focal plane;

e. means for establishing a steady electromagnetic field within a volume including said focal plane;

f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell;

g. means disposed adjacent said focal plane for developing a pressure differential to selectively remove a desired cell and collect it; and

h. means responsive to a predetermined range of values of the light absorbed by a nucleus and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined volume from other cells.

16. Apparatus for classifying microscopic cells suspended in a transparent liquid to provide a suspension of cells, said apparatus comprising:

a. a compound microscope having an objective and an ocular and providing a focal plane adjacent said objective;

b. means for causing the suspension of cells to flow across said focal plane;

c. means for illuminating said microscope with light having a wave length range absorbed by the nucleus of the cells of said suspension;

d. means including a photocathode disposed adjacent the ocular of said microscope and responsive to the amount of light absorbed by the nucleus of each cell as it moves across said focal plane;

e. means including a capacitor for establishing a steady electromagnetic field within a volume including said focal plane;

f. means for measuring the change of the field caused by the passage of a cell across said focal plane and indicative of the volume of the cell;

g. means disposed adjacent said focal plane for selectively removing a desired cell and collecting it; and

h. means responsive to a predetermined range of values of the light absorbed by a nucleus and to a predetermined range of values of the change of the electromagnetic field for actuating said means for removing a cell, thereby to segregate cells having predetermined light absorption and a predetermined volume from other cells.

17. A method of investigating microscopic particles which comprises the steps of:

a. suspending the particles to be classified in a liquid;

b. providing a restricted testing passage;

c. adjusting the size of said testing passage depending upon the size of the particles to be measured and to assure an unclogged condition;

d. causing the suspended particles to flow past said adjusted testing passage;

e. providing a steady electromagnetic field within said testing passage; and

f. measuring the change of said electromagnetic field caused by the passage of each individual particle through said testing passage, thereby to determine the volume of the particle.

18. A method of investigating microscopic particles which comprises the steps of:

a. suspending the particles to be classified in a liquid having a dielectric constant different from that of the particles;

b. causing the suspended particles to flow past a predetermined testing volume;

c. establishing a steady electromagnetic field within the testing volume;

d. measuring the change of the electromagnetic field caused by the passage of each individual particle through the testing volume, thereby to determine the volume of the particle; and

e. separating the particles in accordance with their volumes.

19. A method of investigating microscopic particles which comprises the steps of:

a. suspending the particles to be classified in a liquid;

b. causing the suspended particles to flow past a predetermined testing volume;

c. establishing an electromagnetic field within the testing volume;

d. measuring the change of the electromagnetic field caused by the passage of each individual particle through the testing volume, therEby to determine one characteristic of the particle;

e. simultaneously projecting light through the particle; and

f. detecting at least another characteristic of the particle from the absorption of light passing through the particle.

20. A method of classifying cells which comprises the steps of:

a. suspending the cells to be classified in a liquid;

b. causing the suspended cells to flow past a predetermined testing volume;

c. establishing an electromagnetic field within the testing volume;

d. measuring the change of the electromagnetic field caused by the passage of a cell through the testing volume, thereby to determine the volume of the cell;

e. simultaneously illuminating the testing volume; and

f. measuring the light absorbed by the cell within the testing volume, thereby to detect at least one characteristic of the cell from the light absorbed by the cell.

21. A method of classifying microscopic particles which comprises the steps of:

a. suspending the particles to be classified in a liquid;

b. causing the suspended particles to flow past a predetermined testing volume;

c. projecting light through the particle in the same direction of the particle flow;

d. detecting at least one characteristic of the particle from the light absorbed by passing through the particle; and

e. separating the particle in accordance with said characteristic.

22. A method of classifying microscopic particles which comprises the steps of:

a. suspending the particles to be classified in a liquid;

b. causing the suspended particles to flow past a predetermined testing volume;

c. establishing an electromagnetic field within the testing volume;

d. measuring the change of the electromagnetic field caused by the passage of a particle through the testing volume, thereby to determine one characteristic of the particle;

e. projecting light through the particle;

f. detecting at least another characteristic of the particle from the absorption of the light passing through the particle; and

g. separating the particles in accordance with both of said characteristics.

23. A method of automatically counting simultaneously red and white blood cells suspended in a transparent liquid comprising the steps of:

a. staining the white blood cells;

b. causing the blood cells to flow through a predetermined focal plane;

c. projecting light through said focal plane having a wave length which is absorbed by the stained white blood cells;

d. measuring the light absorbed by each white cell passing through said focal plane;

e. establishing an electromagnetic field in a volume including said focal plane; and

f. measuring the change of the electromagnetic field caused by the movement of a blood cell through said focal plane thereby to count the total number of blood cells and the number of white blood cells.

24. A method of automatically classifying white blood cells suspended in a transparent liquid comprising the steps of:

a. staining the nuclei and the cytoplasm of the white blood cells;

b. causing the white blood cells to flow through a predetermined focal plane;

c. projecting light through said focal plane;

d. measuring the light absorbed by the nucleus of each white cell passing through said focal plane;

e. detecting the color of the cytoplasm;

f. establishing an electromagnetic field in a volume including said focal plane; and

g. measuring the change of the electromagnetic field caused by the movement of a blood cell through said focal plane, thereby to determine characteristics of the white blood cells.

25. A method of automatically classifying biological cells comprising the steps of:

a. digesting the intercellular substance;

b. suspending the cells in a transparent liquid;

c. staining the nucleus and the cytoplasm of the cells;

d. causing the cells to flow once across a focal plane;

e. projecting liGht on said focal plane;

f. detecting the change in the intensity of light caused by a cell;

g. detecting the color of the image;

h. establishing an electromagnetic field in a volume including said focal plane;

i. detecting the change of the electromagnetic field caused by the particle when crossing the focal plane, thereby to determine one characteristic of the particle; and

j. separating and counting the particles in accordance with said characteristics.

26. A method of automatically classifying biological cells comprising the steps of:

a. digesting the intercellular substance;

b. suspending the cells in a liquid;

c. eliminating the clusters of cells;

d. differentially staining the nucleus;

e. suspending the cells in a transparent liquid;

f. causing the cells to flow across a focal plane so that each cell is momentarily exactly in said focal plane;

g. projecting light on said focal plane;

h. detecting and measuring the change in the light caused by the nucleus;

i. establishing an electromagnetic field in a volume including said focal plane;

j. detecting and measuring the change in the electromagnetic field caused by the passage of a cell through said focal plane; and

k. separating the cells in accordance with the light change and the change in the electromagnetic field.

27. A method of separating microscopical particles in a microscope having a focal plane, comprising the steps of:

a. suspending the particles in a transparent liquid;

b. causing a flow of liquid through the focal plane;

c. illuminating the focal plane;

d. measuring the variation in the light produced by the particle; and

e. producing a reduction of pressure in the suspending liquid to aspirate the said particle.

28. A method of separating and classifying particles by their size comprising the steps of:

a. suspending the particles in a liquid;

b. causing a flow of the liquid;

c. producing an electromagnetic field in the liquid;

d. sizing the particles by the change in the electromagnetic field, and e. producing a reduction of ambient liquid pressure to cause a selected particle to flow in a desired direction for collecting it.

29. Apparatus for classifying leucocytes which have previously been stained and which are suspended in a transparent liquid, said apparatus comprising:

a. a compound microscope having an objective, said objective being immersed in the liquid and a plurality of oculars, each receiving light from said objective, said microscope providing a focal plane adjacent said objective;

b. means for causing the suspended leucocytes to flow across said focal plane;

c. means for illuminating said focal plane with light having a wave length range absorbed by stained leucocytes;

d. means including a color filter and light sensitive means disposed adjacent each ocular of said microscope and responsive to the variation of light caused by light absorption by at least a portion of a leucocyte as it moves across said focal plane;

e. means disposed adjacent said focal plane for selectively removing a desired leucocyte and collecting it;

f. means responsive to a predetermined range of values of the variation of light absorption caused by a leucocyte for actuating said means for removing a leucocyte, thereby to segregate leucocytes having predetermined color absorption from other leucocytes; and

g. means for counting the leucocytes in each predetermined range of values.

30. A method of investigating particles by their size, comprising the steps of:

a. suspending the particles in a liquid;

b. moving the particles through a testing passage;

c. adjusting the size of said testing passage in accordance with the size of the particles to be measured and to assure an unclogged condition;

d. sizing the particles by subjecting to an applied field; and

e. triggering one or more actions when the size of a particle is in a range of predetermined values.

31. An apparatus for classifying particles suspended in a fluid, to provide a suspension of particles, said apparatus comprising:

a. a testing passage of a restricted size;

b. means for adjustably increasing and decreasing the size of said testing passage in proportion to the size of the particle to be measured, and relieve any existing clogging;

c. means for causing the suspension of particles to cross through said testing passage;

d. means for providing an applied field within said testing passage; and

e. means for detecting the change of the field caused by the passage of each particle through said testing passage.

32. A method of investigating particles suspended in a liquid which comprises the steps of:

a. causing the suspended particles to cross past a predetermined testing volume;

b. establishing a steady electric field within the testing volume;

c. detecting the change of the field caused by the passage of a particle through the testing volume; and

d. separating the desired particles from the particle suspension, according to predetermined values of said change of the field.

33. An apparatus for classifying particles suspended in a fluid to provide a suspension of particles, said apparatus comprising:

a. a testing passage of a restricted size;

b. means for adjustably increasing and decreasing the size of said testing passage in proportion to the size of the particle to be detected;

c. means for causing the suspension of particles to cross through said testing passage;

d. means for providing a magnetic field within said testing passage;

e. means for detecting the change of the field caused by the passage of each particle through said testing passage; and

f. means for classifying the particles in response to a predetermined range of values of the change of the magnetic field.

34. A method of investigating particles which comprises the steps of:

a. suspending the particles to be investigated in a fluid;

b. causing the suspended particles to cross through a testing passage;

c. adjusting the size of said testing passage in accordance with the size of the particles to be investigated and to assure an unclogged condition;

d. providing a magnetic field within said testing passage; and

e. measuring the size of the particles by detecting the change in the field caused by the passage of a particle through said testing passage.

35. An apparatus for investigating particles suspended in a fluid, to provide a suspension of particles, said apparatus comprising:

a. a testing volume restricted as to size according to the size of the particles to be investigated;

b. means for adjusting the size of said testing volume;

c. means for causing the suspension of particles to cross through said testing volume;

d. means for establishing a steady electric field within the testing volume;

e. means for measuring the change of the capacitance of said electric field caused by the passage of each particle through said testing volume;

f. means for establishing a steady magnetic field within the testing volume; and

g. means for measuring the change of the permeability of said magnetic field caused by the passage of each particle through said testing volume.

36. A combination as defined in claim 35 including means for classifying the particles according to predetermined values of changes of the electric and the magnetic fields.

37. A method of investigating particles which comprises the steps of:

a. suspending the particles to be investigated in a fluid;

b. causing the suspended particles to cross past a testing volume;

c. changing the size of the testing volume according to the size of the particles to be investigated and to provide an unclogged condition;

d. establishing a steady electric field within the testing volume;

e. measuring the change of the capacitance of said electRic field caused by the passage of each particle through said testing volume;

f. establishing a steady magnetic field within the testing volume; and

g. measuring the change of the permeability of said magnetic field caused by the passage of each particle through said testing volume.

38. An apparatus for studying the dielectric properties of fluids comprising:

a. a testing passage;

b. means for adjusting in position the size of said testing passage;

c. means to cause a sample of the fluid to flow through said testing passage;

d. means to produce a steady electric field within said testing passage;

e. means to detect the change in capacitance of said field by said fluid; and

f. means for diverting a portion of the fluid according to predetermined values of the capacitance change.

39. Apparatus for investigating particles suspended in a liquid comprising:

a. a testing volume;

b. means for causing the suspension of particles to cross through said testing volume;

c. means for establishing a magnetic field within the testing volume to induce an electrical field in the flowing liquid;

d. means, including a pair of pick-up electrodes, for detecting the change of the induced electrical field caused by the passage of each particle through said testing volume and e. means for classifying the particles according to predetermined values of the change detected by said pick-up electrodes.

40. An apparatus for investigating particles, such as cells, suspended in a transparent liquid, comprising:

a. a microscope having a focal plane;

b. means, including an opaque diaphragm with an aperture, which coincide with said focal plane, for causing the suspension of cells to flow through said focal plane;

c. means for changing the size of the focal plane;

d. means for illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane;

e. means for detecting the image produced by each particle;

f. means, including a light-proof shield, for preventing stray light reaching the image detecting means;

g. means for producing an electrical current through said focal plane; and

h. means for measuring the variations produced in the electrical current by each particle, as it flows through said focal plane, and thereby determining the size of the particle.

41. A combination as defined in claim 40 including means for classifying the particles according to predetermined values of the measured characteristics.

42. A combination as defined in claim 41 including means responsive to a predetermined range of variations of the light caused by at least a portion of the cell, and to a predetermined range of values of the change of the electrical current for actuating said means for classifying said particles.

43. A combination as defined in claim 41 including means disposed in the vicinity of said testing volume for removing the desired particles from the particle suspension.

44. A method of investigating particles which comprises the steps of:

a. suspending the particles to be investigated in a transparent liquid;

b. causing the suspended particles to flow through the focal plane of a microscope, said focal plane having an opaque diaphragm with an aperture;

c. changing the size of said aperture in accordance with the size of the particles to be investigated and to assure an unclogged condition;

d. illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane;

e. detecting the image produced by each particle;

f. shielding the image detection against stray-light;

g. producing an electrical current through said focal plane; and

h. measuring the variations in the electrical current produced by each particle as it flows through said focal plane, and thereby determining the size of the particle.

45. A method of studying biological cells suspended in a liquid, comprising the steps of:

a. measuring the size of the nucleus by the ultraviolet light absorbed by the cell, and at the same time producing an image of the cell;

b. measuring the size of the cytoplasm by the disturbance produced in different applied fields by the same cell;

c. determining the ratio of nucleus size to cytoplasm size in each cell; and

d. multiplying the number of cell images when the nucleus-cytoplasm ratio is within a predetermined range of values, and measuring the amount of light absorbed by a number of areas in which the cell is divided.

46. An apparatus for investigating particles suspended in a transparent liquid, comprising:

a. a microscope having a focal plane;

b. means for causing the suspension of particles to flow through said focal plane;

c. means for illuminating said focal plane with light absorbed by the particles to thereby produce an image;

d. means for measuring the image density, therefore determining the absorption of light produced by a particle;

e. means for sizing the particles as they cross said focal plane, thereby triggering the image density-measuring means when the size of a particle is between predetermined values, and f. means responsive to predetermined image characteristics and to a predetermined range of cell-size for removing a cell from the suspension.

47. An apparatus for investigating particles, such as cells, suspended in a transparent liquid, comprising:

a. a microscope having a focal plane;

b. means, including an opaque diaphragm with an aperture, which coincide with said focal plane, for causing the suspension of cells to flow through said focal plane;

c. means for changing the size of the focal plane;

d. means for illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane;

e. means for detecting the image produced by each particle;

f. means, including a light-proof shield, for preventing stray light reaching the image detecting means;

g. means for producing a magnetic field through said focal plane to induce an electrical field in the liquid flowing through said focal plane; and

h. means, including a pair of pick-up electrodes, for measuring the variations produced in the electrical field by each particle, as it flows through said focal plane, and thereby determining the size of the particle.

48. A method of investigating particles which comprises the steps of:

a. suspending the particles to be investigated in a transparent liquid;

b. causing the suspended particles to flow through the focal plane of a microscope, said focal plane having an opaque diaphragm with an aperture;

c. changing the size of said aperture in accordance with the size of the particles to be investigated and to assure an unclogged condition;

d. illuminating said focal plane with light that is absorbed by the particles, therefore to produce an image of a particle as it flows through said focal plane;

e. detecting the image produced by each particle;

f. shielding the image detection against stray-light;

g. producing an electrical field through said focal plane; and

h. measuring the variations in the electrical field produced by each particle as it flows through said focal plane, and thereby determining the size of the particle.

49. An improvement for an apparatus for investigating particles suspended in a liquid, said apparatus having means for producing an electrical current through a testing volume, said improvement comprising:

a. means for measuring the variation produced in the electrical current, by each particle, as it flows through the testing volume, and thereby determining its size;

b. a microscope having a focal plane which coincides with said testing volume;

c. means for changing the size of thE testing volume;

d. means for illuminating said focal plane with light that is absorbed by a particle as it flows through said focal plane, and thereby produce an image of the particle; and

e. means for detecting the image produced by each particle.

50. A method of studying biological cells suspended in a liquid comprising the steps of:

a. producing an image of a cell through a microscope;

b. dividing the image of the cell in a number of areas;

c. measuring the light in each one of said areas;

d. measuring the size of the nucleus by the number of areas in which the amount of light reaches a predetermined threshold;

e. measuring the size of the cytoplasm by the disturbance produced in different applied mediums by the same cell;

f. determining the ratio of nucleus size to cytoplasm size; and

g. triggering one or more actions when the ratio of nucleus-cytoplasm is within predetermined range of values.