Methods devices and systems for sorting and separating particles

• US 20020198928A1
• Filed: 03/26/2002
• Published: 12/26/2002
• Est. Priority Date: 03/29/2001
• Status: Active Grant

• Current Assignee: Cellect Technologies, LLC
• Original Assignee: Cellect Technologies, LLC
• Inventors: Bukshpan, Shmuel, Zilberstein, Gleb
• Granted Patent: US 7,354,733 B2
• Time in Patent Office: Days
• US Class Current: 709/200
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: G01N 1/4044 by chemical techniques; Digestion; Chemical decomposition
• 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 2001/2826 Collecting by adsorption or absorption
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 2474/20 : Immunohistochemistry assay
• CPC Class Codes: Y10S 435/968 : High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/801 : Electron dense compounds, e.g. ferritin
• CPC Class Codes: Y10S 436/905 : Photochemical activation of reactions

Abstract

Methods, devices, and systems for sorting and/or separating and/or purifying particles based on selective adherence or attachment of particles to metal grains formed in a light sensitive layer or substrate containing a photosensitizable metal compound by irradiation of the layer or substrate with light and development of the photosensitized substrate or layer.

Claims

1. A method for sorting or separating different particles, the method comprising the steps of:

providing a fluid containing a mixture of different particles suspended therein, said mixture of particles includes one or more target particle;

selectively labeling at least one target particle of said one or more target particles with a photophoric probe to form at least one labeled target particle in said fluid, said photophoric probe is capable of being controllably activated to produce localized emission of light in the vicinity of said at least one labeled target particle;

providing a light sensitive substrate comprising at least one photosensitizable metal compound;

applying said fluid to said light sensitive substrate such that at least one labeled target particle is adjacent to said light sensitive substrate;

activating said photophoric probe to produce localized emission of light in the vicinity of said at least one labeled target particle, said light photosensitizes at least portions of said light sensitive substrate to form photosensitized portions of said substrate;

developing said photosensitized portions to form metal grains in said photosensitized portions;

allowing said at least one labeled target particle to adhere to said metal grains; and

removing the particles of said mixture of different particles which do not adhere to said metal grains.

2. The method according to claim 1 wherein said step of activating comprises activating said photophoric probe to produce light or to induce the production of light in the vicinity of said at least one labeled target particle using an activation method selected from a photoactivation method, a chemical activation method, a thermal activation method, an electrical activation method.

3. The method according to claim 1 wherein said step of activating comprises inducing said photophoric probe to produce light or to induce the production of light in the vicinity of said at least one labeled target particle by participating in a light producing chemical reaction.

4. The method according to claim 1 further including the step of detaching from said substrate at least one of the particles attached to said substrate.

5. The method according to claim 4 further including the step of collecting the particles detached from said substrate in said step of detaching.

6. The method according to claim 4 wherein said step of detaching comprises applying to said substrate a detaching agent.

7. The method according to claim 6 wherein said detaching agent is selected from, n-hexane, and a solution comprising an enzyme capable of detaching the particles attached to said substrate.

8. The method according to claim 7 wherein said enzyme is a proteolytic enzyme.

9. The method according to claim 8 wherein said proteolytic enzyme is selected from the group consisting of pepsin, trypsin, and papaine, and combinations thereof.

10. The method according to claim 6 wherein said substrate comprises a polymerized calcium alginate matrix and said detaching agent comprises a solution containing a calcium sequestering agent.

11. The method according to claim 10 wherein said calcium sequestering agent comprises sodium citrate, or EDTA, or EDTA salts.

12. The method according to claim 1 wherein said photosensitizable metal compound comprises a photosensitizable metal salt.

13. The method according to claim 1 wherein said photosensitizable metal salt comprises a silver halide.

14. The method according to claim 1 wherein said step of developing comprises applying a developer solution to said substrate.

15. The method according to claim 14 wherein said particles are living cells and wherein said step of developing comprises applying to said substrate a developing solution formulated to allow a substantial portion of said living cells to remain viable and to proliferate after exposure to said developer solution.

16. The method according to claim 4 further including the step of collecting the particles detached from said substrate in said step of detaching.

17. The method according to claim 16 further comprising the step of treating the particles collected in said step of collecting with a metal dissolving solution for dissolving metal grains attached to said particles.

18. The method according to claim 17 wherein said photosensitizable metal compound comprises a photosensitizable silver compound, said metal grains are silver metal grains, and wherein said metal dissolving solution is a solution comprising K₃(Fe(CN)₆ and NH₄SCN.

19. The method according to claim 1 wherein said particles are selected from inorganic particles, macromolecules, cellular aggregates, eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, dead cells, fixed cells, subcellular organelles, sub-cellular particles, cell membranes or fragments thereof, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, prions, nanobacteria, unicellular organisms, multicellular organisms, isolated genes or fragments thereof, chromosomes, parts or fragments of chromosomes, single subunit or multi-subunit protein molecules, modified protein molecules, proteoglycans, glycoproteins, DNA, RNA, and olygonucleotides.

20. A system for sorting particles, the system comprising:

optical means configured for identifying a selected particle type based on at least one property of said particle type;

light generating means configured for applying light to at least a portion of a light sensitive substrate, said portion is adjacent to or in contact with a target particle of said selected particle type, said light is adapted to photosensitive a metal compound included within said substrate, said photosensitized metal compound is capable of being developed to form metal grains within said portion of said substrate, said metal grains are capable of binding to or adhering to said target particle to attach the target particle to said substrate;

removing means for removing particles which are not attached to said substrate.

21. The particle sorting system according to claim 20 wherein said light generating means is selected from a coherent light source, and an incoherent light source.

22. The particle sorting system according to claim 20 wherein said coherent light source is a laser.

23. The particle sorting system according to claim 20 wherein said light generating means comprises a light source configured for producing light having a wavelength range suitable for photosensitizing said metal compound.

24. The particle sorting system according to claim 20 wherein said light generating means are optically coupled to said optical means.

25. The particle sorting system according to claim 20 wherein said optical means comprise a microscope.

26. The particle sorting system according to claim 25 wherein said microscope comprises a second light source configured for illuminating said particles during said identifying of said selected particle type.

27. The particle sorting system according to claim 26 wherein said second light source is configured for producing light having a wavelength range and a light intensity value adapted for not photosensitizing said metal compound within the time period required for said identifying of particles of said selected type within a field of view of said microscope.

28. The particle sorting system according to claim 20 wherein said optical means are configured for visually identifying said selected particle type by a human operator, and wherein said light generating means is configured for being controlled by said operator to apply light to at least a portion of said light sensitive substrate.

29. The particle sorting system according to claim 20 further comprising an image acquisition system operatively coupled to said optical means for acquiring an image of a field of view of said optical means.

30. The particle sorting system according to claim 29 further comprising a processing unit coupled to said image acquisition system for processing said image to identify said selected particle type based on said at least one property of said particle type.

31. The particle sorting system according to claim 30 wherein said processing unit is operatively coupled to said light generating means for automatically operating said light generating means to perform said applying of said light to said at least a portion of said light sensitive substrate upon detecting a particle of said selected particle type based on at least one property of said particle.

32. The particle sorting system according to claim 31 wherein said removing means comprise an automatic fluidics system operatively coupled to said processing unit and configured for controllably applying fluids to said substrate and for controllably removing fluids from said substrate in response to control signals received from said processing unit.

33. The particle sorting system according to claim 20 wherein said removing means comprise a fluidics system configured for applying fluids to said substrate and for removing fluids from said substrate.

34. The particle sorting system according to claim 20 wherein said particles are selected from inorganic particles, macromolecules, cellular aggregates, eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, dead cells, fixed cells, subcellular organelles, sub-cellular particles, cell membranes or fragments thereof, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, prions, nanobacteria, unicellular organisms, multicellular organisms, isolated genes or fragments thereof, chromosomes, parts or fragments of chromosomes, single subunit or multi-subunit protein molecules, modified protein molecules, proteoglycans, glycoproteins, DNA, RNA, and olygonucleotides.

36. The method according to claim 35 wherein said step of developing comprises applying a developer solution to said substrate.

37. The method according to claim 35 wherein said particles are selected from inorganic particles, macromolecules, cellular aggregates, eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, dead cells, fixed cells, subcellular organelles, sub-cellular particles, cell membranes or fragments thereof, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, prions, nanobacteria, unicellular organisms, multicellular organisms, isolated genes or fragments thereof, chromosomes, parts or fragments of chromosomes, single subunit or multi-subunit protein molecules, modified protein molecules, proteoglycans, glycoproteins, DNA, RNA, and olygonucleotides.

38. The method according to claim 35 wherein said step of selectively exposing comprises illuminating portions of said substrate in the vicinity of or in contact with said one or more target particles with a beam of electromagnetic radiation capable of photosensitizing said portions.

39. The method according to claim 35 wherein said at least one photosensitizable metal compound comprises a silver halide.

40. The method according to claim 39 wherein said silver halide is selected from the group consisting of silver chloride, silver bromide, silver iodide, and combinations thereof.

35. A method for attaching particles to a substrate, the method comprising the steps of:

providing a light sensitive substrate comprises at least one photosensitizable metal compound;

contacting said substrate with a fluid having particles suspended therein such that at least some of said particles are in contact with said substrate;

selectively exposing to light portions of said light sensitive substrate, said portions are in the vicinity of or in contact with one or more of said particles, to form photosensitized portions of said substrate;

developing said photosensitized portions to form metal grains in said photosensitized portions; and

allowing at least one of said particles to adhere to said metal grains.

41. A method for sorting or separating different particles, the method comprising the steps of:

providing a light sensitive substrate comprising at least one photosensitizable metal compound;

contacting said substrate with a mixture of different particles suspended in a fluid such that at least some of said particles are in contact with said substrate;

selectively exposing to light portions of said substrate, said portions are in the vicinity of or in contact with one or more target particles included in said mixture of different particles, to form photosensitized portions of said substrate;

developing said photosensitized portions to form metal grains in said photosensitized portions;

allowing at least one of said target particles to adhere to said metal grains; and

removing particles which do not adhere to said metal grains.

42. The method according to claim 41 wherein said step of developing comprises applying a developer solution to said substrate.

43. The method according to claim 42 wherein said particles are living cells and wherein said step of developing comprises applying to said substrate a developer solution formulated to allow a substantial portion of said living cells to remain viable and to proliferate after exposure to said developer solution.

44. The method according to claim 41 wherein said particles are selected from inorganic particles, macromolecules, cellular aggregates, eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, dead cells, fixed cells, subcellular organelles, sub-cellular particles, cell membranes or fragments thereof, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, prions, nanobacteria, unicellular organisms, multicellular organisms, isolated genes or fragments thereof, chromosomes, parts or fragments of chromosomes, single subunit or multi-subunit protein molecules, modified protein molecules, proteoglycans, glycoproteins, DNA, RNA, and olygonucleotides.

45. The method according to claim 41 wherein said step of selectively exposing comprises illuminating portions of said substrate in the vicinity of or in contact with said one or more target particles with a beam of electromagnetic radiation capable of photosensitizing said portions.

46. The method according to claim 41 wherein said at least one photosensitizable metal compound comprises a silver halide.

47. The method according to claim 46 wherein said silver halide is selected from the group consisting of silver chloride, silver bromide, silver iodide, and combinations thereof.

49. The method according to claim 48 wherein said step of developing comprises applying a developer solution to said substrate.

50. The method according to claim 49 wherein said particles are living cells and wherein said step of developing comprises applying to said substrate a developer solution formulated to allow a substantial portion of said living cells to remain viable and to proliferate after exposure to said developer solution.

51. The method according to claim 50 further including the step of washing the cells collected in said step of collecting to remove most of said developer solution.

52. The method according to claim 48 wherein said particles are selected from inorganic particles, macromolecules, cellular aggregates, eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, dead cells, fixed cells, subcellular organelles, sub-cellular particles, cell membranes or fragments thereof, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, prions, nanobacteria, unicellular organisms, multicellular organisms, isolated genes or fragments thereof, chromosomes, parts or fragments of chromosomes, single subunit or multi-subunit protein molecules, modified protein molecules, proteoglycans, glycoproteins, DNA, RNA, and olygonucleotides.

53. The method according to claim 48 wherein said at least one photosensitizable metal compound comprises a silver halide.

54. The method according to claim 53 wherein said silver halide is selected from the group consisting of silver chloride, silver bromide, silver iodide, and combinations thereof.

48. A method for sorting or separating different particles, the method comprising the steps of:

providing a light sensitive substrate comprising at least one photosensitizable metal compound;

contacting said substrate with a mixture of different particles suspended in a fluid such that most of said particles are in contact with said substrate, said particles include target particles and non-target particles;

selectively exposing to light portions of said substrate, said portions are in the vicinity of or in contact with one or more of said non-target particles included in said mixture of different particles, to form photosensitized portions of said substrate;

developing said photosensitized portions to form metal grains in said photosensitized portions;

allowing at least some of said non-target particles to adhere to said metal grains; and

collecting the target particles which do not adhere to said metal grains.

55. A method for sorting or separating different particles, the method comprising the steps of:

providing a fluid containing a mixture of different particles suspended therein, said mixture of particles includes one or more target particles;

selectively labeling at least one target particle of said one or more target particles with a photophoric probe to form at least one labeled target particle in said fluid, said photophoric probe is capable of being controllably activated to produce localized emission of light in the vicinity of said at least one labeled target particle;

providing a light sensitive substrate, said substrate comprises at least one photosensitizable metal compound;

applying said fluid to said light sensitive substrate such that at least one labeled target particle is adjacent to or in contact with the surface of said light sensitive substrate;

activating said photophoric probe to produce localized emission of light in the vicinity of said at least one labeled target particle, said light photosensitizes at least portions of said light sensitive substrate to form photosensitized portions of said substrate;

developing said photosensitized portions to form metal grains in said photosensitized portions;

allowing said at least one labeled target particle to adhere to said metal grains; and

removing the particles which do not adhere to said metal grains.

56. The method according to claim 55 wherein said step of activating comprises activating said photophoric probe to produce light or to induce the production of light in the vicinity of said at least one labeled target particle using an activation method selected from a photoactivation method, a chemical activation method, a thermal activation method, an electrical activation method.

57. The method according to claim 55 wherein said step of activating comprises inducing said photophoric probe to produce light or to induce the production of light in the vicinity of said at least one labeled target particle by participating in a light producing chemical reaction.

58. The method according to claim 55 wherein said particles are selected from inorganic particles, macromolecules, cellular aggregates, eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, dead cells, fixed cells, subcellular organelles, sub-cellular particles, cell membranes or fragments thereof, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, prions, nanobacteria, unicellular organisms, multicellular organisms, isolated genes or fragments thereof, chromosomes, parts or fragments of chromosomes, single subunit or multi-subunit protein molecules, modified protein molecules, proteoglycans, glycoproteins, DNA, RNA, and olygonucleotides.

59. The method according to claim 55 wherein said photophoric probe comprises:

a first affinity probe capable of specifically and selectively binding to said target particles or to at least a second affinity probe bound to said target particles; and

a second portion linked to said first affinity probe and capable of being controllably induced to emit light or to cause the emission of light in the vicinity of a target particle to which said photophoric probe is bound.

60. The method according to claim 59 wherein said first affinity probe is selected from an antibody or a fragment thereof, a toxin having an affinity for at least a portion of a target particle of said target particles, an oligonucleotyde probe, a protein based affinity probe, a glycoprotein based affinity probe, and a hapten or molecule having an affinity for at least a portion of a target particle of said target particles.

61. The method according to claim 59 wherein said second portion of said photophoric probe is selected from, a chemiluminescent moiety or agent, a fluorescent moiety or agent, an upconverting moeity or particle or agent, an inorganic two photon upconverting anti-stokes phosphor particle, a two photon upconverting dye, a bioluminescent protein, a bioluminescent or a chemiluminescent molecule, a thermoluminescent moiety or agent or particle, and an electroluminescent moiety or agent or particle.

62. The method according to claim 59 wherein said second portion of said photophoric probe comprises an enzyme capable of participating in a chemiluminescent chemical reaction, or capable of activating or catalyzing of a chemiluminescent chemical reaction resulting with the production of light, or capable of catalyzing a chemical reaction for producing a reaction product capable of reacting with at least one chemical in a chemiluminescent reaction resulting in the production of light.

63. The method according to claim 59 wherein said second portion of said photophoric probe comprises aequorin or obelin.

64. The method according to claim 59 wherein said second portion of said photophoric probe comprises an enzyme selected from the group consisting of a peroxidase, a phosphatase, an alkaline-phosphatase, a galactosidase, and a β

-glucuronidase.

65. The method according to claim 55 wherein said step of developing comprises applying a developer solution to said substrate.

66. The method according to claim 55 further including the step of detaching from said substrate at least one of the particles attached to said substrate.

67. The method according to claim 66 further comprising the step of treating the particles detached in said step of detaching with a metal dissolving solution for dissolving metal grains attached to said particles.

68. The method according to claim 67 wherein said photosensitizable metal compound comprises a photosensitizable silver compound, said metal grains are silver metal grains, and wherein said metal dissolving solution is a solution comprising K₃(Fe(CN)₆ and NH₄SCN.

69. The method according to claim 66 further including the step of collecting the particles detached from said substrate in said step of detaching.

70. The method according to claim 66 wherein said step of detaching comprises applying to said substrate a detaching agent.

71. The method according to claim 66 wherein said detaching agent is selected from, n-hexane, and a solution comprising an enzyme capable of detaching the particles attached to said substrate.

72. The method according to claim 71 wherein said enzyme is a proteolytic enzyme.

73. The method according to claim 72 wherein said proteolytic enzyme is selected from the group consisting of pepsin, trypsin, and papaine, and combinations thereof.

74. The method according to claim 70 wherein said detaching agent is formulated for dissolving at least portions of said metal grains to detach particles attached to said metal grains.

75. The method according to claim 65 wherein said particles are living cells and wherein said step of developing comprises applying to said substrate a developing solution formulated to allow a substantial portion of said living cells to remain viable and to proliferate after exposure to said developing solution.

76. The method according to claim 75 further including the step of detaching from said substrate at least one of the living cells attached to said substrate.

77. The method according to claim 76 further including the step of collecting the cells detached from said substrate in said step of detaching.

78. The method according to claim 77 further including the step of washing the cells collected in said step of collecting to remove most of said developing solution.

79. The method according to claim 55 wherein said at least one photosensitizable metal compound comprises a silver halide.

80. The method according to claim 79 wherein said silver halide is selected from the group consisting of silver chloride, silver bromide, silver iodide, and combinations thereof.

81. A method for separating particles, the method comprising the steps of:

providing a fluid containing a mixture of different particles suspended therein, said mixture of particles includes one or more target particles and one or more non-target particles;

selectively labeling most of said non-target particles of said fluid with a photophoric probe to form labeled non-target particles in said fluid, said photophoric probe is capable of being controllably activated to produce localized emission of light in the vicinity of said labeled non-target particles;

providing a light sensitive substrate, said substrate comprises at least one photosensitizable metal compound;

applying said fluid to said light sensitive substrate such that most labeled non-target particles are adjacent to or in contact with the surface of said light sensitive substrate;

activating said photophoric probe to produce localized emission of light in the vicinity of said labeled non-target particles, said light photosensitizes at least portions of said light sensitive substrate to form photosensitized portions of said substrate;

developing said photosensitized portions to form metal grains in said photosensitized portions;

allowing said labeled non-target particle to adhere to said metal grains; and

collecting the particles which do not adhere to said metal grains to obtain an enriched particle population having a higher ratio of said target particles to said non-target particles.

82. The method according to claim 81 wherein said step of activating comprises activating said photophoric probe to produce light or to induce the production of light in the vicinity of said at labeled non-target particles using an activation method selected from a photoactivation method, a chemical activation method, a thermal activation method, an electrical activation method.

83. The method according to claim 81 wherein said step of activating comprises inducing said photophoric probe to produce light or to induce the production of light in the vicinity of said labeled non-target particles by participating in a light producing chemical reaction.

84. The method according to claim 81 wherein said step of developing comprises applying a developing solution to said substrate.

85. The method according to claim 81 wherein said particles are selected from inorganic particles, macromolecules, cellular aggregates, eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, dead cells, fixed cells, subcellular organelles, sub-cellular particles, cell membranes or fragments thereof, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, prions, nanobacteria, unicellular organisms, multicellular organisms, isolated genes or fragments thereof, chromosomes, parts or fragments of chromosomes, single subunit or multi-subunit protein molecules, modified protein molecules, proteoglycans, glycoproteins, DNA, RNA, and olygonucleotides.

86. The method according to claim 81 wherein said photophoric probe comprises:

a first affinity probe capable of specifically and selectively binding to said non-target particles or to at least a second affinity probe bound to said non-target particles; and

a second portion linked to said first affinity probe and capable of being controllably induced to emit light or to cause the emission of light in the vicinity of a non-target particle to which said photophoric probe is bound.

87. The method according to claim 86 wherein said first affinity probe is selected from an antibody or a fragment thereof, a toxin having an affinity for at least a portion of a target particle of said target particles, an oligonucleotyde probe, a protein based affinity probe, a glycoprotein based affinity probe, and a hapten or molecule having an affinity for at least a portion of a target particle of said target particles.

88. The method according to claim 86 wherein said second portion of said photophoric probe is selected from, a chemiluminescent moiety or agent, a fluorescent moiety or agent, an upconverting moeity or particle or agent, an inorganic two photon upconverting anti-stokes phosphor particle, a two photon upconverting dye, a bioluminescent protein, a bioluminescent or a chemiluminescent molecule, a thermoluminescent moiety or agent or particle, and an electroluminescent moiety or agent or particle.

89. The method according to claim 86 wherein said second portion of said photophoric probe comprises an enzyme capable of participating in a chemiluminescent chemical reaction, or capable of activating or catalyzing of a chemiluminescent chemical reaction resulting with the production of light, or capable of catalyzing a chemical reaction for producing a reaction product capable of reacting with at least one chemical in a chemiluminescent reaction resulting in the production of light.

90. The method according to claim 86 wherein said second portion of said photophoric probe comprises aequorin or obelin.

91. The method according to claim 86 wherein said second portion of said photophoric probe comprises a an enzyme selected from the group consisting of a peroxidase, a phosphatase, an alkaline-phosphatase, a galactosidase, and a β

-glucuronidase.

92. The method according to claim 81 wherein said step of developing comprises applying a developer solution to said substrate.

93. The method according to claim 81 wherein said at least one photosensitizable metal compound comprises a silver halide.

94. The method according to claim 93 wherein said silver halide is selected from the group consisting of silver chloride, silver bromide, silver iodide, and combinations thereof.

95. A method for attaching particles to a substrate, the method comprising the steps of:

providing a light sensitive substrate comprises at least one photosensitizable metal compound;

contacting said substrate with a fluid having particles suspended therein such that at least some of said particles are in contact with said substrate;

exposing said light sensitive substrate to light, to photosensitize said substrate;

developing the photosensitized substrate to form metal grains in said substrate; and

allowing at least one of said particles to adhere to said metal grains.

96. The method according to claim 95 wherein said step of developing comprises applying a developer solution to said substrate.

97. The method according to claim 95 wherein said particles are selected from inorganic particles, macromolecules, cellular aggregates, eukaryotic cells, prokaryotic cells, mammalian cells, non-mammalian cells, viable cells, dead cells, fixed cells, subcellular organelles, sub-cellular particles, cell membranes or fragments thereof, pathogenic organisms, non-pathogenic organisms, bacterial cells, viruses, prions, nanobacteria, unicellular organisms, multicellular organisms, isolated genes or fragments thereof, chromosomes, parts or fragments of chromosomes, single subunit or multi-subunit protein molecules, modified protein molecules, proteoglycans, glycoproteins, DNA, RNA, and olygonucleotides.

98. The method according to claim 95 wherein said step of exposing comprises exposing said substrate to electromagnetic radiation having a wavelength range capable of photosensitizing said at least one photosensitizable metal compound.

99. The method according to claim 95 wherein said at least one photosensitizable metal compound comprises a photosensitizable metal salt.

100. The method according to claim 99 wherein said photosensitizable metal salt comprises a silver halide.

101. The method according to claim 100 wherein said photosensitizable metal salt is selected from the group consisting of silver chloride, silver bromide, silver iodide, and combinations thereof.

102. A method for attaching particles to a substrate, the method comprising the steps of:

providing a light sensitive substrate comprises at least one photosensitizable metal compound;

contacting said substrate with a fluid having at least one particle suspended therein such that said at least one particle is in contact with said substrate;

exposing said light sensitive substrate to light, to photosensitive said substrate;

developing the photosensitized substrate to form metal grains in said substrate; and

allowing said at least one particle to adhere to said metal grains.