Method and apparatus for three-dimensional microscopy with enhanced resolution

Abstract

A method and apparatus for three dimensional optical microscopy is disclosed which employs dual opposing objective lenses about a sample and extended incoherent illumination to provide enhanced depth or Z-direction resolution. In a first embodiment, observed light from both objective lenses are brought into coincidence on an image detector and caused to interfere thereon by optical path length adjustment. In a second embodiment, illuminating light from an extended incoherent light source is detected to the sample through both objective lenses and caused to interfere with a section of the sample by adjusting optical path lengths. Observed light from one objective lens is then recorded. In a third embodiment, which combines the first two embodiments, illuminating light from an extended incoherent light source is directed to the sample through both objective lenses and caused to interfere within a section of the sample by adjusting optical path lengths. The observed light from both lenses is caused to interfere on the image detector by the same optical path length adjustment. In a fourth embodiment of the invention, further spatial structure is introduced into the illumination light. Computational processing is used to enhance lateral or XY resolution as well as depth or Z resolution.

What is claimed is:  
     
       1. A three dimensional optical microscopy apparatus, comprising: 
       (a) first and second spaced-apart objective lenses;  
       (b) means for supporting a microscopy sample between said objective lenses;  
       (c) means for beam splitting and recombining light;  
       (d) first and second observation paths, said first observation path extending from said microscopy sample to said beam splitting and recombining means via said first objective lens, said second observation path extending from said microscopy sample to said beam splitting and recombining means via said second objective lens;  
       (e) a plurality of means for directing light, at least one of said light directing means positioned along each of said first and second observation paths to direct observed light from said microscopy sample along said first and second observation paths to said beam splitting and recombining means;  
       (f) optical path length balancing means for adjusting the optical path length of at least one of said first and second observation paths so as to make said optical path lengths of said first and second observation paths be closely equal; and  
       (g) imaging means for detecting and recording images, said imaging means positioned to detect and record all or part of said observed light, said observed light having been combined by said beam splitting and recombining means.  
     
     
       2. An apparatus as recited in  claim 1 , further comprising means for positionally adjusting said microscopy sample relative to said objective lenses. 
     
     
       3. An apparatus as recited in  claim 2 , further comprising means for sensing position of said sample relative to said objective lenses. 
     
     
       4. An apparatus as recited in  claim 1 , further comprising means for positionally adjusting at least one of said objective lenses relative to said other objective lens. 
     
     
       5. An apparatus as recited in  claim 4 , further comprising means for sensing position of at least one of said objective lenses. 
     
     
       6. An apparatus as recited in  claim 5 , wherein said position adjusting means is responsive to said position sensing means. 
     
     
       7. An apparatus as recited in  claim 1 , wherein said optical path length balancing means comprises translation means for positionally adjusting at least one of said light directing means. 
     
     
       8. An apparatus as recited in  claim 1 , further comprising sample illuminating means for providing illuminating light to said sample, said illuminating means positioned to provide said illuminating light to said first objective lens. 
     
     
       9. An apparatus as recited in  claim 8 , further comprising selective transmittance and reflectance means for transmitting said observed light from said objective lenses toward said imaging means and reflecting said illuminating light away from said imaging means. 
     
     
       10. An apparatus as recited in  claim 8 , further comprising filtering means for transmitting said observed light from said objective lenses and filtering said illuminating light from said illuminating means. 
     
     
       11. An apparatus as recited in  claim 8 , further comprising selective transmittance and reflectance means for reflecting said observed light from said objective lenses toward said imaging means and transmitting said illuminating light away from said imaging means. 
     
     
       12. An apparatus as recited in  claim 1 , further comprising phase compensation means for correction of phase differences between different wavelength components of said observed light from said objective lenses. 
     
     
       13. An apparatus as recited in  claim 1 , further comprising means for focusing said light from said beam splitting and recombining means onto said imaging means. 
     
     
       14. An apparatus as recited in  claim 1 , further comprising means for aligning said sample relative to said objective lenses. 
     
     
       15. An apparatus as recited in  claim 1 , further comprising means for determining the amount of adjustment of said path length adjusting means. 
     
     
       16. An apparatus as recited in  claim 1 , further comprising sample illumination means for providing illuminating light to said sample, said illuminating means positioned to direct said illuminating light to said beam splitting and recombining means, said at least one of said light directing means positioned along each of said first and second observation paths to direct said illuminating light along said first and second observation paths to said microscopy sample via said first and second objective lenses. 
     
     
       17. An apparatus as recited in  claim 16 , wherein said optical path length balancing means comprises translation means for positionally adjusting at least one of said light directing means. 
     
     
       18. An apparatus as recited in  claim 16 , further comprising selective transmittance and reflectance means for transmitting said observed light from said objective lenses toward said imaging means and reflecting said illuminating light away from said imaging means. 
     
     
       19. An apparatus as recited in  claim 16 , further comprising phase compensation means for correction of phase differences between different wavelength components of said observed light and said illuminating light. 
     
     
       20. An apparatus as recited in  claim 16 , further comprising filtering means for transmitting said observed light from said objective lenses and filtering said illuminating light from said illuminating means. 
     
     
       21. An apparatus as recited in  claim 16 , further comprising means for focusing said observed light from said beam splitting and recombining means onto said imaging means. 
     
     
       22. An apparatus as recited in  claim 16 , further comprising means for positionally adjusting said microscopy sample relative to said objective lenses. 
     
     
       23. An apparatus as recited in  claim 16 , further comprising second imaging means for detecting and recording images, said second imaging means positioned to record a second beam of combined light from said first beam splitting and recombining means. 
     
     
       24. An apparatus as recited in  claim 16 , wherein said imaging means is positioned to record, as separate images, both beams of combined light from said beam splitting and recombining means. 
     
     
       25. An apparatus as recited in  claim 16 , further comprising selective transmittance and reflectance means for reflecting said observed light from said objective lenses toward said imaging means and transmitting said illuminating light away from said imaging means. 
     
     
       26. An apparatus as recited in  claim 1 , further comprising: 
       (a) sample illuminating means for providing illuminating light to said sample  
       (b) first and second illumination paths; and  
       (c) second means for beam splitting and recombining light;  
       (d) said illuminating means positioned to direct said illuminating light to said second beam splitting and recombining means, said first illumination path extending from said second beam splitting and recombining means to said microscopy sample via said first objective lens, said second illumination path extending from said second beam splitting and recombining means to said microscopy sample via said second objective lens, at least one of said light directing means positioned along each of said first and second illumination paths to direct said illuminating light to said microscopy sample along said first and second illumination paths via said first and second objective lenses.  
     
     
       27. An apparatus as recited in  claim 26 , wherein said optical path length balancing means comprises translation means for positionally adjusting at least one of said light directing means. 
     
     
       28. An apparatus as recited in  claim 26 , further comprising selective transmittance and reflectance means for transmitting said observed light from said objective lenses toward said imaging means and reflecting said illuminating light away from said imaging means. 
     
     
       29. An apparatus as recited in  claim 26 , further comprising phase compensation means for correction of phase differences between different wavelength components of said observed light and said illuminating light. 
     
     
       30. An apparatus as recited in  claim 26 , further comprising filtering means for transmitting said observed light from said objective lenses and filtering said illuminating light from said illuminating means. 
     
     
       31. An apparatus as recited in  claim 26 , further comprising means for positionally adjusting said first objective lens responsive to means for sensing position of said sample. 
     
     
       32. An apparatus as recited in  claim 26 , further comprising means for focusing said observed light from said first beam splitting and recombining means onto said image detection means. 
     
     
       33. An apparatus as recited in  claim 26 , further comprising means for positionally adjusting said microscopy sample relative to said objective lenses. 
     
     
       34. An apparatus as recited in  claim 26 , wherein said imaging means is positioned to record, as separate images, both beams of combined light from said first beam splitting and recombining means. 
     
     
       35. An apparatus as recited in  claim 26 , further comprising second optical path length balancing means, said second optical path length balancing means arranged for adjusting optical path lengths of at least one of said first and second illumination paths so as to make said optical path lengths of said first and second illumination paths be closely equal. 
     
     
       36. An apparatus as recited in  claim 26 , further comprising second imaging means for detecting and recording images, said second imaging means positioned to record a second beam of combined light from said first beam splitting and recombining means. 
     
     
       37. An apparatus as recited in  claim 26 , further comprising selective transmittance and reflectance means for reflecting said observed light from said objective lenses toward said imaging means and transmitting said illuminating light away from said imaging means. 
     
     
       38. A method for three-dimensional optical microscopy, comprising the steps of: 
       (a) placing a sample between first and second opposing objective lenses;  
       (b) focusing said objective lenses on a section of said sample;  
       (c) directing observed light from said section of said sample along first and second paths to imaging means for detecting and recording images, said first and second paths leading from said section of said sample to said imaging means through said first and second objective lenses respectively, and causing said observed light from said first and second paths to coincide on said imaging means;  
       (d) adjusting optical lengths of at least one of said first and second paths so as to make said first and second optical path lengths be closely equal, thereby causing said observed light from said first and second objective lenses to interfere on said imaging means;  
       (e) recording said interfering observed light on said imaging means;  
       (f) focusing said objective lenses on another section of said sample; and  
       (g) repeating steps (c), (d), (e) and (f) until a plurality of sections of said sample have been observed and recorded, forming a data set of recorded images.  
     
     
       39. A method for three-dimensional optical microscopy according to claimed  claim  38 , further comprising the step of applying means for computational deconvolution to said data set of recorded images to obtain a three-dimensional image of said sample. 
     
     
       40. A method for three-dimensional optical microscopy according to  claim 38 , wherein step (c) is carried out by using a plurality of means for directing light and at least one means for beam splitting and recombining light. 
     
     
       41. A method for three-dimensional optical microscopy according to  claim 38 , further comprising the step of directing illuminating light to said sample through said first objective lens. 
     
     
       42. A method for three-dimensional optical microscopy according to  claim 38 , further comprising the step of preventing said illuminating light from reaching said detection means. 
     
     
       43. A method for three-dimensional optical microscopy according to  claim 38 , further comprising the step of matching the phases of different wavelength components of said observed light. 
     
     
       44. A method for three-dimensional optical microscopy according to  claim 38 , further comprising the step of aligning said sample between said first and second objective lenses. 
     
     
       45. A method for three-dimensional optical microscopy, comprising the steps of: 
       (a) placing a sample between first and second opposing objective lenses;  
       (b) focusing said first and second objective lenses onto a section of said sample;  
       (c) directing illuminating light from an extended, spatially incoherent light source along first and second illumination paths to said section of said sample, said first illumination path extending from said light source to said section of said sample via said first objective lens, said second illumination path extending from said light source to said section of said sample via said second objective lens;  
       (d) directing observed light from said sample along first and second observation paths to imaging means for detecting and recording images, said first and second observation paths extending from said section of said sample to said imaging means via said first and second objective lenses respectively, and causing said observed light from said first and second observation paths to coincide on said imaging means;  
       (e) adjusting optical lengths of at least one of said first and second illumination paths, so as to make said optical lengths of said first and second illumination paths be closely equal thereby causing said illuminating light from said first and second illumination paths to interfere in said section of said sample;  
       (f) recording said observed light on said imaging means;  
       (g) refocusing said first and second objective lenses onto another section of said sample; and  
       (h) repeating steps (c), (d), (e) (f) and (g) until a plurality of sections of said sample have been observed and recorded, forming a data set of recorded images.  
     
     
       46. A method for three-dimensional optical microscopy according to  claim 45 , further comprising the step of applying means for computational deconvolution to said data set of recorded images to obtain a three-dimensional image of said sample. 
     
     
       47. A method for three-dimensional optical microscopy according to  claim 45 , wherein step (c) is carried out by directing said illuminating light from said extended, spatially incoherent light source to means for beam splitting and recombining light and directing said illuminating light from said beam splitting and recombining means along said first and second paths to said sample by a plurality of means for directing light. 
     
     
       48. A method for three-dimensional optical microscopy according to  claim 47 , wherein said first and second observation paths include said beam splitting and recombining means, and wherein segments of said first and second illumination paths that extend between said beam splitting and recombining means and said section of said sample are identical to segments of said first and second observation paths that extend between said section of said sample and said beam splitting and recombining means, respectively. 
     
     
       49. A method for three-dimensional optical microscopy according to  claim 45 , further comprising the step of preventing said illuminating light from reaching said detection means. 
     
     
       50. A method for three-dimensional optical microscopy according to  claim 45 , further comprising the step of matching the phases of said illuminating light and said observed light. 
     
     
       51. A method for three-dimensional optical microscopy according to  claim 45 , further comprising the step of aligning said sample between said first and second objective lenses. 
     
     
       52. A method for three-dimensional optical microscopy according to  claim 45 , wherein step (d) is carried out by directing said observed light from said section of said sample along said first and second observation paths to means for beam splitting and recombining light and directing said observed light from said beam splitting and recombining means to said imaging means by a plurality of means for directing,  light, said observed light from said first and second observation paths having been combined by said beam splitting and recombining means. 
     
     
       53. A method for three-dimensional optical microscopy, comprising the steps of: 
       (a) placing a sample between first and second opposing objective lenses;  
       (b) focusing said first and second objective lenses onto a section of said sample;  
       (c) directing illuminating light from an extended, spatially incoherent light source along first and second illumination paths to said section of said sample, said first illumination path extending from said light source to said section of said sample via said first objective lens, said second illumination path extending from said light source to said section of said sample via said second objective lens;  
       (d) directing observed light from at least one of said first and second objective lenses to imaging means for detecting and recording images;  
       (e) adjusting optical lengths of at least one of said first and second illumination paths, so as to make said optical lengths of said first and second illumination paths be closely equal, thereby causing said illuminating light from said first and second illumination paths to interfere in said section of said sample;  
       (f) recording said observed light on said imaging means;  
       (g) refocusing said first and second objective lenses onto another section of said sample; and  
       (h) repeating steps (c), (d), (e), (f) and (g) until a plurality of sections of said sample have been observed and recorded, forming a data set of recorded images.  
     
     
       54. A method for three-dimensional optical microscopy according to  claim 53 , further comprising the step of applying means for computational deconvolution to said data set of recorded images to obtain a three-dimensional image of said sample. 
     
     
       55. A method for three-dimensional optical microscopy according to  claim 53 , wherein step (c) is carried out by directing said illuminating light from said extended, spatially incoherent light source to means for beam splitting and recombining light and directing said illuminating light from said beam splitting and recombining means along said first and second illumination paths by a plurality of means for directing light. 
     
     
       56. A method for three-dimensional optical microscopy according to  claim 53 , further comprising the step of preventing said illuminating light from reaching said detection means. 
     
     
       57. A method for three-dimensional optical microscopy according to  claim 53 , further comprising the step of matching the phases of different wavelength components of said illuminating light. 
     
     
       58. A method for three-dimensional optical microscopy according to  claim 53 , further comprising the step of aligning said sample between said first and second objective lenses. 
     
     
       59. A three dimensional optical microscopy apparatus, comprising: 
       (a) a first objective lens and a second objective lens, said objective lenses mounted opposite to each other;  
       (b) means for supporting a microscopy sample between said objective lenses;  
       (c) means for beam splitting light;  
       (d) first and second optical paths, said first optical path extending from said beam splitting means to said microscopy sample via said first objective lens, said second optical path extending from said beam splitting means to said microscopy sample via said second objective lens;  
       (e) illuminating means for producing extended, spatially incoherent light, said illuminating means positioned to provide illuminating light to said beam splitting means;  
       (f) a plurality of means for directing light, at least one of said light directing means positioned along each of said first and second optical paths to direct illuminating light from said beam splitting means along said first and second optical paths to said sample;  
       (g) optical path length balancing means for adjusting optical path lengths of at least one of said first and second optical paths, so as to make said optical path lengths of said first and second optical paths be closely equal; and  
       (h) imaging means for detecting and recording images, said imaging means positioned to record observed light from at least one of said objective lenses.  
     
     
       60. An apparatus as recited in  claim 59 , further comprising means for positionally adjusting said microscopy sample relative to said objective lenses. 
     
     
       61. An apparatus as recited in  claim 60 , further comprising means for sensing position of said sample relative to said objective lenses. 
     
     
       62. An apparatus as recited in  claim 59 , further comprising means for positionally adjusting at least one of said objective lenses relative to said other objective lens. 
     
     
       63. An apparatus as recited in  claim 62 , further comprising means for sensing position of at least one of said objective lenses. 
     
     
       64. An apparatus as recited in  claim 59 , wherein said optical path length balancing means comprises translation means for positionally adjusting at least one of said light directing means. 
     
     
       65. An apparatus as recited in  claim 59 , further comprising selective transmittance and reflectance means for transmitting said observed light from said objective lenses toward said imaging means and reflecting said illuminating light away from said imaging means. 
     
     
       66. An apparatus as recited in  claim 59 , further comprising filtering means for transmitting observed light from said objective lenses and filtering said illuminating light from said illuminating means. 
     
     
       67. An apparatus as recited in  claim 59 , further comprising phase compensation means for correction of phase differences between different wavelength components of said illuminating light from said illuminating means. 
     
     
       68. An apparatus as recited in  claim 59 , further comprising means for focusing said observed light from said objective lenses onto said imaging means. 
     
     
       69. An apparatus as recited in  claim 59 , further comprising means for determining the amount of adjustment of said path length adjusting means. 
     
     
       70. An apparatus as recited in  claim 59 , further comprising selective transmittance and reflectance means for reflecting said observed light from said objective lenses toward said imaging means and transmitting said illuminating light away from said imaging means. 
     
     
       71. An apparatus as recited in  claim 1 , further comprising means for providing spatially structured illuminating light to said sample, said means for providing spatially structured illuminating light comprising means for providing at least two mutually coherent beams of light to said sample, said at least two mutually coherent beams of light arranged so as to interfere with each other at said sample. 
     
     
       72. An apparatus as recited in  claim 71 , wherein said spatially structured illuminating light comprises a standing wave. 
     
     
       73. An apparatus as recited in  claim 72 , wherein said standing wave has a direction, wherein said first and second objective lenses have a common optic axis, and wherein said direction of said standing wave is parallel to said optic axis. 
     
     
       74. An apparatus as recited in  claim 72 , wherein said standing wave has a direction, wherein said first and second objective lenses have a common optic axis, and wherein said direction of said standing wave is perpendicular to said optic axis. 
     
     
       75. An apparatus as recited in  claim 72 , wherein said standing wave has a direction, wherein said first and second objective lenses have a common optic axis, and wherein said direction of said standing wave is neither parallel nor perpendicular to said optic axis. 
     
     
       76. An apparatus as recited in  claim 1 , further comprising means for providing spatially structured illuminating light to said sample, said means for providing spatially structured illuminating light comprising means for providing two mutually coherent beams of light to said sample, said two mutually coherent beams of light arranged so as to interfere with each other at said sample, said two beams of light directed to said sample through said first objective lens. 
     
     
       77. An apparatus as recited in  claim 71 , wherein at least one of said at least two beams of light is directed to said sample through said first objective lens, and wherein at least one of said at least two beams of light is directed to said sample through said second objective lens. 
     
     
       78. An apparatus as recited in  claim 59 , wherein said illuminating means comprises means for producing spatially structured illuminating light, and wherein said means for producing spatially structured illuminating light comprises light source means for providing light, an illumination path from said light source means to said beam splitting means, and at least one mask located along said illumination path. 
     
     
       79. An apparatus as recited in  claim 71 , further comprising structure altering means for altering spatial structure of said spatially structured illuminating light. 
     
     
       80. An apparatus as recited in  claim 79 , wherein said structure altering means comprises means for altering a phase of spatial structure of said spatially structured illuminating light. 
     
     
       81. An apparatus as recited in  claim 80 , wherein said structure altering means further comprises means for altering an orientation of spatial structure of said spatially structured illuminating light. 
     
     
       82. An apparatus as recited in  claim 79 , further comprising computational processing means for processing a plurality of images from said imaging means to produce a reconstruction of said sample with improved resolution. 
     
     
       83. An apparatus as recited in  claim 78 , wherein at least one of said at least one masks is located at a position that is conjugate to an image plane of at least one of said first and second objective lenses. 
     
     
       84. An apparatus as recited in  claim 78 , further comprising structure altering means for altering spatial structure of said spatially structured illuminating light. 
     
     
       85. An apparatus as recited in  claim 78 , further comprising computational processing means for processing a plurality of images from said imaging means to produce a reconstruction of said sample with improved resolution. 
     
     
       86. An apparatus as recited in  claim 1 , further comprising means for providing spatially structured illuminating light to said sample, said means for providing spatially structured illuminating light comprising light source means for providing light, an illumination path from said light source means to said sample, and at least one mask located along said illumination path. 
     
     
       87. An apparatus as recited in  claim 86 , wherein at least one of said at least one masks is positioned at a plane that is conjugate to an image plane of at least one of said first and second objective lenses. 
     
     
       88. An apparatus as recited in  claim 86 , wherein said light source means comprises an extended, spatially incoherent light source. 
     
     
       89. An apparatus as recited in  claim 16 , wherein said illuminating means comprises means for providing spatially structured illuminating light to said sample, said means for providing spatially structured illuminating light comprising light source means for providing light, an illumination path from said light source means to said beam splitting and recombining means, and at least one mask located along said illumination path. 
     
     
       90. An apparatus as recited in  claim 86 , further comprising means for processing a plurality of images from said imaging means to produce a reconstruction of said sample with improved resolution. 
     
     
       91. An apparatus as recited in  claim 16 , wherein said illuminating means comprises means for providing spatially structured illuminating light to said sample, said means for providing spatially structured illuminating light comprising means for providing at least two mutually coherent beams of light to said beam splitting and recombining means, said at least two mutually coherent beams of light arranged so as to interfere with each other at said sample. 
     
     
       92. An apparatus as recited in  claim 89 , further comprising structure altering means for altering spatial structure of said spatially structured illuminating light. 
     
     
       93. An apparatus as recited in  claim 91 , further comprising means for altering a phase of spatial structure of said spatially structured illuminating light. 
     
     
       94. An apparatus as recited in  claim 91 , further comprising means for altering a direction of spatial structure of said spatially structured illuminating light. 
     
     
       95. An apparatus as recited in  claim 26 , wherein said illuminating means comprises means for providing spatially structured illuminating light to said sample, said means for providing spatially structured illuminating light comprising means for providing at least two mutually coherent beams of light to said beam splitting and recombining means, said at least two mutually coherent beams of light arranged so as to interfere with each other at said sample. 
     
     
       96. An apparatus as recited in  claim 95 , further comprising structure altering means for altering spatial structure of said spatially structured illuminating light. 
     
     
       97. An apparatus as recited in  claim 95 , further comprising means for altering a phase of spatial structure of said spatially structured illuminating light. 
     
     
       98. An apparatus as recited in  claim 95 , further comprising means for altering a direction of spatial structure of said spatially structured illuminating light. 
     
     
       99. An apparatus as recited in  claim 89 , wherein at least one of said least one masks is disposed at a position that is conjugate to an image plane of at least one of said first and second objective lenses. 
     
     
       100. An apparatus as recited in  claim 89 , wherein said light source means comprises an extended, spatially incoherent light source. 
     
     
       101. An apparatus as recited in  claim 91 , wherein said at least two mutually coherent beams of light emanate from at least two mutually coherent point sources of light located at positions approximately conjugate to a back focal plane of at least one of said first and second objective lenses. 
     
     
       102. An apparatus as recited in  claim 91 , further comprising structure altering means for altering spatial structure of said spatially structured illuminating light. 
     
     
       103. An apparatus as recited in  claim 26 , wherein said illuminating means comprises means for providing spatially structured illuminating light to said sample, said means for providing spatially structured illuminating light comprising light source means for providing light, an illumination path from said light source means to said second beam splitting and recombining means, and at least one mask located along said illumination path. 
     
     
       104. An apparatus as recited in  claim 103 , wherein at least one of said at least one masks is disposed at a position that is conjugate to an image plane of at least one of said first and second objective lenses. 
     
     
       105. An apparatus as recited in  claim 103 , further comprising structure altering means for altering spatial structure of said spatially structured illuminating light. 
     
     
       106. An apparatus as recited in  claim 103 , wherein said light source means comprises an extended, spatially incoherent light source. 
     
     
       107. An apparatus as recited in  claim 95 , wherein said at least two mutually coherent beams of light emanate from at least two mutually coherent point sources of light located at positions approximately conjugate to a back focal plane of at least one of said first and second objective lenses. 
     
     
       108. A method for three- dimensional microscopy as recited in    claim 45   , further comprising the step of introducing lateral structure into said illuminating light, and further comprising the step of computationally processing said data set of recorded images to obtain a reconstruction of said sample with improved resolution.   
     
     
       109. A method for three- dimensional microscopy as recited in    claim 108   , wherein said reconstruction of said sample possesses improved lateral resolution.   
     
     
       110. A method for three- dimensional microscopy as recited in    claim 108   , wherein the step of introducing lateral structure into said illuminating light is carried out by directing said illuminating light past at least one mask.   
     
     
       111. A method for three- dimensional microscopy as recited in    claim 53   , further comprising the step of introducing lateral structure into said illuminating light, and further comprising the step of computationally processing said data set of recorded images to obtain a reconstruction of said sample with improved resolution.   
     
     
       112. A method for three- dimensional microscopy as recited in    claim 111   , wherein said reconstruction of said sample possesses improved lateral resolution.   
     
     
       113. A method for three- dimensional microscopy as recited in    claim 112   , wherein the step of introducing lateral structure into said illuminating light is carried out by directing said illuminating light past at least one mask.   
     
     
       114. An apparatus for optical microscopy, comprising: 
       ( a )  means for supporting a sample;    
       ( b )  means for providing spatially structured illuminating light to said sample, said spatially structured illuminating light containing lateral structure, said means for providing spatially structured illuminating light comprising light source means for producing light, an illuminating path from said light source means to said sample, and at least one mask located along said illuminating path;    
       ( c )  optical magnification means for producing magnified images of said sample illuminated by said spatially structured illumination light;    
       ( d )  imaging means for detecting and recording said magnified images of said sample; and    
       ( e )  processing means for processing said recorded images from said imaging means to obtain a reconstruction of said sample with improved resolution including improved lateral resolution, said processing means arranged to cause information components from said recorded images to assume new positions in Fourier space.   
     
     
       115. An apparatus as recited in  claim 114 , further comprising structure altering means for altering spatial structure of said spatially structured illuminating light. 
     
     
       116. An apparatus as recited in  claim 114 , further comprising means for altering a phase of spatial structure of said spatially structured illuminating light. 
     
     
       117. An apparatus as recited in  claim 114 , further comprising means for altering a direction of spatial structure of said spatially structured illuminating light. 
     
     
       118. An apparatus as recited in  claim 114 , wherein said means for providing spatially structured illuminating light further comprises polarizing means located along said illuminating path. 
     
     
       119. An apparatus as recited in  claim 114 , further comprising focusing means for refocusing said optical magnification means relative to said sample to allow said optical magnification means to produce refocused images of said sample, said imaging means arranged to detect and record a multiplicity of said refocused images, said processing means arranged to process said multiplicity of recorded images from said imaging means to obtain a three- dimensional reconstruction of said sample.   
     
     
       120. An apparatus as recited in  claim 114 , wherein at least one of said at least one mask is positioned at a plane that is conjugate to an image plane of said optical magnification means. 
     
     
       121. An apparatus as recited in  claim 114 , wherein said light source means comprises an extended, spatially incoherent light source. 
     
     
       122. An apparatus for optical microscopy, comprising: 
       ( a )  means for supporting a sample;    
       ( b )  means for providing spatially structured illuminating light to said sample, said spatially structured illuminating light containing lateral structure, said means for providing spatially structured illuminating light comprising means for providing at least two mutually coherent beams of light to said sample, said at least two mutually coherent beams of light arranged so as to interfere with each other at said sample;    
       ( c )  optical magnification means for producing magnified images of said sample illuminated by said spatially structured illumination light;    
       ( d )  imaging means for detecting and recording said magnified images of said sample; and    
       ( e )  processing means for processing said recorded images from said imaging means to obtain a reconstruction of said sample with improved resolution, including improved lateral resolution.   
     
     
       123. An apparatus as recited in  claim 122 , further comprising structure altering means for altering spatial structure of said spatially structured illuminating light. 
     
     
       124. An apparatus as recited in  claim 122 , further comprising means for altering a phase of spatial structure of said spatially structured illuminating light. 
     
     
       125. An apparatus as recited in  claim 122 , further comprising means for altering a direction of spatial structure of said spatially structured illuminating light. 
     
     
       126. An apparatus as recited in  claim 122 , wherein said spatially structured illuminating light has at least one characteristic wave vector, said apparatus further comprising means for altering at least one of said at least one characteristic wave vector. 
     
     
       127. An apparatus as recited in  claim 122 , wherein said at least two mutually coherent beams of light emanate from at least two mutually coherent sources of light, said at least two mutually coherent sources of light located at positions approximately conjugate to a back focal plane of said optical magnification means. 
     
     
       128. An apparatus as recited in  claim 122 , wherein said spatially structured illuminating light further contains axial structure. 
     
     
       129. An apparatus as recited in  claim 122 , wherein said reconstruction further possesses improved axial resolution. 
     
     
       130. An apparatus as recited in  claim 122 , further comprising focusing means for refocusing said optical magnification means relative to said sample to allow said optical magnification means to produce refocused images of said sample, said imaging means arranged to detect and record a multiplicity of said refocused images, said processing means arranged to process said multiplicity of recorded images from said imaging means to obtain a three- dimensional reconstruction of said sample.   
     
     
       131. A method of optical microscopy comprising the steps of: 
       ( a )  placing a luminescent sample in a microscope containing image detecting and recording means;    
       ( b )  illuminating said sample with an illumination pattern that contains lateral structure;    
       ( c )  recording at least one image of said sample using said image detecting and recording means;    
       ( d )  altering said illumination pattern at least one time, each time recording at least one image of said sample illuminated with said altered illumination pattern;    
       ( e )  collecting said images into a data set; and    
       ( f )  computationally processing said data set to obtain a reconstruction of said sample with improved resolution, including improved lateral resolution, said step of computationally processing said data comprising the steps of separating a plurality of information components, causing said information components to assume new positions in Fourier space, and recombining said information components.   
     
     
       132. A method for optical microscopy as recited in  claim 131 , wherein said reconstruction of said sample possesses improved lateral resolution. 
     
     
       133. A method for optical microscopy as recited in  claim 131 , wherein step (b) is carried out by directing illuminating light past at least one mask to said sample. 
     
     
       134. A method for optical microscopy as recited in  claim 131 , wherein step (b) is carried out by causing at least two mutually coherent beams of light to interfere at said sample. 
     
     
       135. A method for optical microscopy as recited in  claim 131 , wherein step ( d )  comprises the step of altering a phase of said illumination pattern at least one time.   
     
     
       136. A method for optical microscopy as recited in  claim 135 , further comprising the step of applying computational deconvolution means to said data set. 
     
     
       137. A method for optical microscopy as recited in  claim 131 , wherein step ( d )  comprises the step of altering a direction of said illumination pattern at least one time.   
     
     
       138. A method for optical microscopy as recited in  claim 131 , further comprising the step of illuminating said sample with laterally uniform illumination and recording at least one image of said sample illuminated by said laterally uniform illumination. 
     
     
       139. A method for optical microscopy as recited in  claim 138 , further comprising the steps of 
       
         refocusing said microscope relative to said sample at least one time;  
       
       
         each time said microscope is thus refocused  
       
       ( i )  repeating steps  ( b ) ,  ( c ) ,  ( d )  and  ( e )  and    
       ( ii )  illuminating said sample with laterally uniform illumination and recording at least one image of said sample illuminated by said laterally uniform illumination;    
         collecting said data sets into a three - dimensional data set; and    
         processing said three - dimensional data set to generate a three - dimensional reconstruction of said sample.   
     
     
       140. A method for optical microscopy as recited in  claim 131 , further comprising the steps of refocusing said microscope relative to said sample at least one time, repeating steps ( b ) ,  ( c ) ,  ( d )  and  ( e )  each time the microscope is thus refocused, collecting said data sets into a three - dimensional data set, and processing said three - dimensional data set to generate a three - dimensional reconstruction of said sample.   
     
     
       141. A method for optical microscopy as recited in  claim 131 , wherein step ( a )  is carried out by placing said sample in a microscope having first and second opposing objective lenses, and wherein step  ( b )  is carried out by providing illuminating light that contains lateral structure, splitting said illuminating light into first and second beams of structured illuminating light, directing said first beam of structured illuminating light to said sample along a first illuminating path through said first obiective lens, directing said second beam of structured illuminating light to said sample along a second illuminating path through said second objective lens, to allow said first and second beams of structured illuminating light to interfere at said sample.