Fluorescence imaging system having reduced background fluorescence

A fluorescence microscopy system for stimulating a sample to fluoresce, said system comprising:

• a photodetector;
• a source for emitting an excitation beam of light along an optical path, said excitation beam having optical properties to cause said sample to fluoresce;
• an air-spaced objective disposed in said optical path to receive said excitation beam therethrough to illuminate a region of said sample and collect fluorescent light emitted from said region, forming a retro-beam, said objective including a plurality of lens elements, with each of said plurality of lens elements being spaced-apart from an adjacent lens element of said air-spaced objective, thereby avoiding the presence of adhesives in the portion of the optical path so as to reduce auto-fluorescence produced by said air-spaced objective;
• means, positioned in said optical path between said source and said objective, for separating said excitation beam from said retro-beam, wherein said objective directs said retro-beam onto said separating means, with said separating means directing said retro-beam toward said photodetector, said photodetector producing signals representing light impinging thereon;
• and
• beam shaping optics disposed in said optical path, said beam shaping optics having optical properties to produce auto-fluorescence that radiates spherically in response to said excitation beam impinging thereon and a filtering means, disposed in said optical path, for attenuating auto-fluorescence propagating over a solid angle of sufficient size to prevent said auto-fluorescence from impinging upon said photodetector.

A fluorescence microscopy system for stimulating a sample to fluoresce, said system comprising:

• a photodetector;
• a source for emitting an excitation beam of light along an optical path, said excitation beam having optical properties to cause said sample to fluoresce;
• an air-spaced objective disposed in said optical path to receive said excitation beam therethrough to illuminate a region of said sample and collect fluorescent light emitted from said region, forming a retro-beam, said objective including a plurality of lens elements, with each of said plurality of lens elements being spaced-apart from an adjacent lens element of said air-spaced objective, thereby avoiding the presence of adhesives in the portion of the optical path so as to reduce auto-fluorescence produced by said air-spaced objective;
• means, positioned in said optical path between said source and said objective, for separating said excitation beam from said retro-beam, wherein said objective directs said retro-beam onto said separating means, with said separating means directing said retro-beam toward said photodetector, said photodetector producing signals representing light impinging thereon;
• and
• beam shaping optics disposed in said optical path, said beam shaping optics having optical properties to produce auto-fluorescence in response to said excitation beam impinging thereon and a filtering means, disposed to in said optical path, for attenuating light associated with said excitation beam, thereby preventing the same from impinging upon said beam shaping optics.

A fluorescence microscopy system for stimulating a sample to fluoresce, said system comprising:

• a photodetector;
• a source for emitting an excitation beam of light alone an optical path, said excitation beam having optical properties to cause said sample to fluoresce;
• an air-spaced objective disposed in said optical path to receive said excitation beam therethrough to illuminate a region of said sample and collect fluorescent light emitted from said region, forming a retro-beam, said objective including a plurality of lens elements, with each of said plurality of lens elements being spaced-apart from an adjacent lens element of said air-spaced objective, thereby avoiding the presence of adhesives in the portion of the optical path so as to reduce auto-fluorescence produced by said air-spaced objective;
• means, positioned in said optical path between said source and said objective, for separating said excitation beam from said retro-beam, wherein said objective directs said retro-beam onto said separating means, with said separating means directing said retro-beam toward said photodetector, said photodetector producing signals representing light impinging thereon;
• and
• wherein said objective has optical properties to provide a substantially uniform detection sensitivity across said field of view by introducing, into said system, a specified amount of axial chromatic aberrations while reducing lateral chromatic aberrations and a focusing lens means, disposed in the path of said retro-beam, for directing said retro-beam onto said photodetector and further including a focusing lens means, disposed between said objective and said photodetector, for reducing axial chromatic aberrations introduced by said objective.

A fluorescence microscopy system for stimulating a sample to fluoresce, said system comprising:

• a photodetector;
• a source for emitting an excitation beam of light along an optical path, said excitation beam having optical properties to cause said sample to fluoresce;
• an air-spaced objective disposed in said optical path to receive said excitation beam therethrough to illuminate a region of said sample and collect fluorescent light emitted from said region, forming a retro-beam, said objective including a plurality of lens elements, with each of said plurality of lens elements being spaced-apart from an adjacent lens element of said air-spaced objective, thereby avoiding the presence of adhesives in the portion of the optical path so as to reduce auto-fluorescence produced by said air-spaced objective;
• means, positioned in said optical path between said source and said objective, for separating said excitation beam from said retro-beam, wherein said objective directs said retro-beam onto said separating means, with said separating means directing said retro-beam toward said photodetector, said photodetector producing signals representing light impinging thereon;
• and
• wherein said separating means includes a mirror having a diameter greater than the diameter of said incident beam and smaller than the diameter of said retro-beam, with the diameter of the retro-beam being substantially larger than the diameter of the incident beam.

A fluorescence microscopy system for stimulating a sample to fluoresce, said system comprising:

• a photodetector;
• a source for emitting an excitation beam of light along an optical path, said excitation beam having optical properties to cause said sample to fluoresce;
• an objective disposed in said optical path to receive said excitation beam therethrough to illuminate a region of said sample and collect fluorescent light emitted from said region, forming a retro-beam;
• a spatial filter, positioned proximate to said photodetector, having a substantially transmissive aperture, restricting light scattered rearwardly to increase signal response;
• a focusing lens means, disposed in the path of said retro-beam, for directing said retro-beam onto said aperture, and onto said photodetector, with said photodetector producing signals representing light impinging thereon, said focusing lens means having optical properties to produce auto-fluorescence in response to said excitation beam passing therethrough;
• and
• first filtering means, disposed in said optical path between said focusing lens means and said separating means, for reducing the auto-fluorescence sensed by said detector.

A fluorescence microscopy system for stimulating a sample to fluoresce, said system comprising:

• a photodetector;
• a source for emitting an excitation beam of light along an optical path, said excitation beam having optical properties to cause said sample to fluoresce;
• an air-spaced objective disposed in said optical path to receive said excitation beam therethrough to illuminate a region of said sample and collect fluorescent light emitted from said region, forming a retro-beam;
• said objective including a plurality of lens elements, with each of said plurality of lens elements being spaced-apart from an adjacent lens element of said objective;
• a spatial filter, positioned proximate to said photodetector, having a substantially transmissive aperture, restricting light scattered rearwardly to increase signal response;
• a focusing lens means, disposed in the path of said retro-beam, for directing said retro-beam onto said aperture, and onto said photodetector, with said photodetector producing signals representing light impinging thereon, said focusing lens means having optical properties to produce auto-fluorescence in response to said excitation beam passing therethrough;
• means, positioned in said optical path between said beam source and said objective, for separating said incident beam from said retro-beam, said separating means directing said retro-beam toward said photodetector, with said photodetector producing signals representing light impinging thereon;
• and
• first filtering means, disposed in said optical path between said focusing lens means and said separating means, for reducing the auto-fluorescence sensed by said detector.