Reflected fluorescence microscope with multiple laser and excitation light sources

Abstract

Caged reagent release experiments are carried out by introducing laser light of a laser light source for the Caged reagent release of a first reflected illumination optical system light path into a sample side through a first dichroic mirror on a cube turret and also introducing excitation light of a mercury lamp for the excitation light irradiation of a third reflected illumination optical system light path into the sample side through a first dichroic mirror on a slider. Laser trap experiments are carried out by introducing excitation light of a mercury lamp for the excitation light irradiation of a second reflected illumination optical system light path into the sample side through a second dichroic mirror on the cube turrent and also introducing laser light from the laser light source for the laser trap of the third reflected illumination optical system light path into the sample side through a second dichroic mirror on the slider.

1. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, to carry out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
 a first reflected illumination optical system light path provided in a direction which crosses an observation optical system light path to said sample at a point of intersection;  
 a second reflected illumination optical system light path provided in a direction which crosses said observation optical system light path at the same point of intersection as that between said observation optical system light path and said first reflected illumination optical system light path;  
 a first movable member having a cube including a first dichroic mirror and a cube including a second dichroic mirror, for providing a desired one of said cubes to be inserted or detached at said point of intersection between and observation optical system light path and said first or second reflected illumination optical system light path;  
 a third reflected illumination optical system light path provided in a direction which crosses said observation optical system light path at a different point of intersection from that between said first or second reflected illumination optical system light path; and  
 a second movable member having a cube including a third dichroic mirror, for providing said cube with said third dichroic mirror to be inserted or detached at said point of intersection between said observation optical system light path and said third reflected illumination optical system light path,  
 wherein said first dichroic mirror reflects the light from said first reflected illumination optical system light path to a side of said sample, while transmitting fluorescence emitted from said sample.  
 
     
     
       2. A microscope according to  claim 1 , wherein said second dichroic mirror reflects the light from said second reflected illumination optical system light path to a side of said sample, while transmitting fluorescence emitted from said sample. 
     
     
       3. A microscope according to  claim 2 , wherein said third dichroic mirror reflects the light from said third reflected illumination optical system light path to a side of said sample, while transmitting fluorescence emitted from said sample. 
     
     
       4. A microscope according to  claim 1 , wherein said first movable member comprises a cube turret. 
     
     
       5. A microscope according to  claim 1 , wherein said second movable member comprises a slider. 
     
     
       6. A microscope according to  claim 1 , further comprising a laser light source which forms said first reflected illumination optical system light path. 
     
     
       7. A microscope according to  claim 6 , further comprising a mercury light source which forms said second reflected illumination optical system light path. 
     
     
       8. A microscope according to  claim 7 , further comprising at least one of a laser light source and a mercury light source which forms said third reflected illumination optical system light path. 
     
     
       9. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, to carry out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
 a first reflected illumination optical system light path provided in a direction which crosses an observation optical system light path to said sample at a point of intersection;  
 a second reflected illumination optical system light path provided in a direction which crosses said observation optical system light path at the same point of intersection as that between said observation optical system light path and said first reflected illumination optical system light path;  
 a first movable member having a cube including a first dichroic mirror and a cube including a second dichroic mirror, for providing a desired one of said cubes to be inserted or detached at said point of intersection between said observation optical system light path and said first or second reflected illumination optical system light path;  
 a third reflected illumination optical system light path provided in a direction which crosses said observation optical system light path at a different point of intersection from that between said first or second reflected illumination optical system light path;  
 a second movable member having a cube including a third dichroic mirror, for providing said cube with said third dichroic mirror to be inserted or detached at said point of intersection between said observation optical system light path and said third reflected illumination optical system light path; and  
 a fourth reflected illumination optical system light path provided in a direction which crosses said third reflected illumination optical system light path at a point of intersection.  
 
     
     
       10. A microscope according to  claim 9 , further comprising a slider for providing a total reflection mirror to be inserted or detached at a point of intersection between said third and fourth reflected illumination optical system light paths. 
     
     
       11. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, to carry and carrying out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
 an observation optical system light path which passes through an objective lens from the sample to an image forming element for forming an image of the sample:  
 a first reflected illumination optical system light path which crosses said observation optical system light oath path at a point of intersection; and  
 a second reflected illumination optical system light path which crosses said observation optical system light path at the same said point of intersection as that between said observation optical system light path and in a different direction from said first reflected illumination optical system light path,  
 wherein said first reflected illumination optical system light path and said second reflected illumination optical system light path transmit illumination light beams differing in spectral distributions.  
 
     
     
       12. A microscope according to  claim 11 , further comprising:
 a light path splitting member provided at a point on said observation optical system light path between said objective lens and said image forming element, and which splits light into split light paths; and  
 an eyepiece provided at a point on a light path which is one of the split light paths split by said light path splitting member.  
 
     
     
       13. A microscope according to  claim 11 , further comprising a third related illumination optical system light path which crosses said observation optical system light path at a different point of intersection from that of said first or second reflected illumination optical system light path. 
     
     
       14. A microscope according to  claim 13 , further comprising wavelength discriminating means provided to be inserted or detected at said point of intersection between said observation optical system light path and said third reflected illumination optical system light path. 
     
     
       15. A microscope according to  claim 13 , further comprising a fourth reflected illumination optical system light path which crosses said observation optical system light path at a different point of intersection from that of said first, second or third reflected illumination optical system light path. 
     
     
       16. A microscope according to  claim 13 , further comprising a slider having a cube including a dichroic mirror, for providing said cube to be inserted or detached at said point of intersection between said observation optical system light path and said third reflected illumination optical system light path. 
     
     
       17. A microscope according to  claim 13 , further comprising at least one of a laser light source and a mercury light source which forms said third reflected illumination optical system light path. 
     
     
       18. A microscope according to  claim 11 , further comprising a cube turret having a cube including a first dichroic mirror and a cube including a second dichroic mirror, for providing a desired one of said cubes to be inserted or detached of said point of intersection between said observation optical system light path and said first or second reflected illumination optical system light path. 
     
     
       19. A microscope according to  claim 11 , further comprising a laser light source which forms said first reflected illumination optical system light path. 
     
     
       20. A microscope according to  claim 19 , further comprising a mercury light source which forms said second reflected illumination optical system light path. 
     
     
       21. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, and carrying out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
   an observation optical system light path which passes through an objective lens from the sample to an image forming element for forming an image of the sample;        a first reflected illumination optical system light path which is formed by a first light source and which crosses said observation optical system light path at a first point of intersection;        a second reflected illumination optical system light path which is formed by a second light source and which crosses said observation optical system light path at a second point of intersection different from the first point of intersection, the second point of intersection being located between said objective lens and the first point of intersection; and        means for inhibiting and allowing transmission of a light beam from the second light source of said second reflected illumination to said observation optical system light path;        wherein a wavelength of a light beam from the first light source of said first reflected illumination optical system light path is longer than a wavelength of a light beam from the second light source of said second reflected illumination optical system.     
     
     
       22. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, and carrying out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
   an observation optical system light path which passes through an objective lens from the sample to an image forming element for forming an image of the sample;        a first reflected illumination optical system light path which is formed by a first light source and which crosses said observation optical system light path at a first point of intersection;        a second reflected illumination optical system light path which is formed by a second light source and which crosses said observation optical system light path at a second point of intersection different from the first point of intersection, the second point of intersection being located between said objective lens and the first point of intersection; and        means for inhibiting and allowing transmission of a light beam from the second light source of said second reflected illumination to said observation optical system light path;        wherein said means for inhibiting and allowing transmission of a light beam from the second light source comprises a cube which includes a dichroic mirror and which is capable of being inserted to and detached from said observation optical system light path at the second point of intersection.     
     
     
       23. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, and carrying out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
   an observation optical system light path which passes through an objective lens from the sample to an image forming element for forming an image of the sample;        a first reflected illumination optical sample light path which is formed by a first light source and which crosses said observation optical system light path at a first point of intersection;        a second reflected illumination optical system light path which is formed by a second light source and which crosses said observation optical system light path at a second point of intersection different from the first point of intersection, the second point of intersection being located between said objective lens and the first point of intersection; and        means for inhibiting and allowing transmission of a light beam from the second light source of said second reflected illumination to said observation optical system light path;        wherein said means for inhibiting and allowing transmission of a light beam from the second light source comprises a plurality of cubes, each of which is capable of being inserted to and detached from said observation optical system light path at the second point of intersection.     
     
     
       24. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, and carrying out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
   an observation optical system light path which passes through an objective lens from the sample to an image forming element for forming an image of the sample;        a first reflected illumination optical system light path which is formed by a first light source and which crosses said observation optical system light path at a first point of intersection;        a section reflected illumination optical system light path which is formed by a second light source and which crosses said observation optical system light path at a second point of intersection different from the first point of intersection, the second point of intersection being located between said objective lens and the first point of intersection;        means for inhibiting and allowing transmission of a light beam from the second light source of said second reflected illumination to said observation optical system light path; and        a slider having a cube including a dichroic mirror, and wherein said slider is adapted to insert and remove said cube from said observation optical system light path at the first point of intersection.     
     
     
       25. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, and carrying out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
   an observation optical system light path which passes through an objective lens from the sample to an image forming element for forming an image of the sample;        a first reflected illumination optical system light path which is formed by a first light source and which crosses said observation optical system light path at a first point of intersection;        a second reflected illumination optical system light path which is formed by a second light source and which crosses said observation optical system light path at a second point of intersection different from the first point of intersection, the second point of intersection being located between said objective lens and the first point of intersection; and        means for inhibiting and allowing transmission of a light beam from the second light source of said second reflected illumination to said observation optical system light path;        wherein said second light source includes at least one of a laser light source and a mercury light source which forms said first reflected illumination optical system light path; and        wherein said second light source produces an N   2    laser having a wavelength at  337  nm.     
     
     
       26. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, and carrying out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
   an observation optical system light path which passes through an objective lens from the sample to an image forming element for forming an image of the sample;        a first reflected illumination optical system light path which is formed by a first light source and which crosses said observation optical system light path at a first point of intersection;        a second reflected illumination optical system light path which is formed by a second light source and which crosses said observation optical system light path at a second point of intersection different from the first point of intersection, the second point of intersection being located between said objective lens and the first point of intersection; and        means for inhibiting and allowing transmission of a light beam from the second light source of said second reflected illumination to said observation optical system light path;        wherein a wavelength of a light beam from the first light source of said first reflected illumination optical system light path is different from a wavelength of a light beam from the second light source of said second reflected illumination optical system.     
     
     
       27. A reflected fluorescence microscope for allowing a plurality of reflected illumination light beams to be selectively irradiated onto a sample, and carrying out optical operations of said sample and its fluorescence observation simultaneously, said microscope comprising:
   an observation optical system light path which passes through an objective lens from the sample to an image forming element for forming an image of the sample;        a first reflected illumination optical system light path which is formed by a first light source and which crosses said observation optical system light path at a first point of intersection;        a second reflected illumination optical system light path which is formed by a second light source and which crosses said observation optical system light path at a second point of intersection different from the first point of intersection, the second point of intersection being located between said objective lens and the first point of intersection; and        means for inhibiting and allowing transmission of a light beam from the second light source of said second reflected illumination to said observation optical system light path;        wherein said means for inhibiting and allowing transmission of a light beam from the second light source comprises a cube which includes a dichroic mirror and which is capable of being inserted to and detached from said observation optical system light path at the second point of intersection.     
     
     
       28. A microscope according to  claim 27 , wherein a wavelength of a light beam from the first light source of said first reflected illumination optical system light path is different from a wavelength of a light beam from the second light source of said second reflected illumination optical system. 
     
     
       29. A microscope according to  claim 27 , wherein a wavelength of a light beam from the first light source of said first reflected illumination optical system light path is longer than a wavelength of a light beam from the second light source of said second reflected illumination optical system. 
     
     
       30. A microscope according to  claim 27 , wherein said means for inhibiting and allowing transmission of a light beam from the second light source comprises a plurality of cubes, each of which is capable of being inserted to and detached from said observation optical system light path at the second point of intersection. 
     
     
       31. A microscope according to  claim 27 , further comprising a light path splitting member which is provided at a point on said observation optical system light path between said objective lens and said image forming element, and which splits light into two split light paths. 
     
     
       32. A microscope according to  claim 27 , further comprising a slider having a cube including a dichroic mirror, and wherein said slider is adapted to insert and remove said cube from said observation optical system light path at the first point of intersection. 
     
     
       33. A microscope according to  claim 27 , wherein said second light source includes at least one of a laser light source and a mercury light source which forms said first reflected illumination optical system light path. 
     
     
       34. A microscope according to  claim 33 , wherein said second light source produces an N 2    laser having a wavelength of  337  nm.