Fluoresecence imaging system

• US 5,672,880 A
• Filed: 03/15/1996
• Issued: 09/30/1997
• Est. Priority Date: 12/08/1994
• Status: Expired due to Term

• Current Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Dynamics Inc. (General Electric Company)
• Inventors: Kain, Robert C.
• Primary Examiner(s): Hannaher, Constantine
• Application Number:
  US08/616,174
• Time in Patent Office: 564 Days
• Field of Search: 250/458.1
• US Class Current: 250/458.1
• CPC Class Codes: G02B 21/002 Scanning microscopes scanning near field optical microscopes G01Q60/18
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence

Abstract

A fluorescence imaging system that includes an objective that is both achromatic and has an external entrance pupil. The objective also serves as a condenser for the system which substantially reduces the system'"'"'s cost and footprint. With the objective positioned above a sample so that they are in close proximity to one another, a laser directs a collimated beam of light to a scan device located at the objective entrance pupil. The scan device reflects, refracts, or diffracts the light through the lens to illuminate a spot on the sample'"'"'s surface. The scan device illuminates a line or an area on the sample surface by varying the angle of laser light, in one or two dimensions, into the objective. The sample emits fluorescent light in response to the illumination. The fluorescence light is collected by the objective and passes through the system along the path of the illumination light. A wavelength-discriminating dichroic filter is placed along the optical axis between the laser and the objective to direct the fluorescent light onto a photo-detector to produce a signal representing the sample surface emitting the fluorescent light. A display device is provided that displays the digitized data in a raster format.

Claims

1. An optical scanning system for microscopy of a sample comprising,

a photodetector,

a source for emitting an incident beam of light,

a telecentric objective disposed proximate to said sample, defining an optical axis, a high numerical aperture and a single pupil, with said single pupil located external to said objective in said optical axis, said objective being positioned to receive said incident beam therethrough to illuminate a region of said sample, said objective being afocal at said pupil and adapted to collect light emitted from said region forming a retro-beam,

means, positioned at said pupil, for scanning said region across said sample, with substantially all light associated with said retro-beam impinging upon said scanning means,

means, positioned in said optical axis between said source and said objective, for separating said incident 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 photo-detector, said photo-detector producing signals representing light impinging thereon, and

means, connected to receive said signals, for producing a visual display of said sample.

2. The optical system of claim 1 further including a wavelength selective filter disposed between said photodetector and said separating means to transmit light having a predetermined wavelength while attenuating all other light.

3. The optical scanning system of claim 1 wherein said objective is achromatic.

4. The optical scanning system of claim 1 wherein said objective has first and second ends, with said first end facing said scanning means and spaced apart therefrom a first distance, with said second end opposing said first end and positioned proximate to said sample, defining a working distance therebetween, with said working distance being substantially less than said first distance.

5. The optical system of claim 1 further including a back aperture positioned between said separating means and said photodetector, and focusing optics, positioned between said back aperture and said separating means in the path of said retro-beam, with said back aperture adapted to be optically conjugate to said region.

6. The optical scanning system of claim 1 further including means, positioned between said beam source and said separating means, for changing the diameter of said incident beam.

7. The optical scanning system of claim 1 wherein said beam source is a non-coherent source of light optically coupled to collimating optics.

8. The optical scanning system of claim 1 wherein said separating means is 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.

9. The optical scanning system of claim 1 wherein said separating means is a dichroic filter.

10. The optical scanning system of claim 1 wherein said separating means is a prism.

11. The optical scanning system of claim 1 wherein said separating means is a grating.

12. The optical scanning system of claim 1 wherein said separating means is a fresnel reflector.

13. The optical scanning system of claim 1 wherein said scanning means includes a reflective surface.

14. The optical scanning system of claim 1 wherein said scanning means includes a refractive scanning deflector.

15. The optical scanning system of claim 1 wherein said scanning means includes a diffractive scanning deflector.

16. The optical scanning system of claim 1 further including means, disposed in the path of said incident beam between said source and said separating means, for illuminating said region as a line.

17. The optical scanning system of claim 16 wherein said illuminating means is a binary optic.

18. The optical scanning system of claim 16 wherein said illuminating means is a holographic lens.

19. The optical scanning system of claim 16 wherein said illuminating means is a cylindrical lens.

20. The optical scanning system of claim 1 wherein said retro-beam comprises fluorescent light.

21. The optical scanning system of claim 1 wherein said retro-beam comprises specularly reflected light.

22. The optical scanning system of claim 1 wherein said retro-beam comprises dispersive light emitted from said region.

23. An optical scanning system for fluorescence microscopy of a sample, said system comprising,

a photodetector,

a beam source for emitting a collimated beam of coherent light,

a telecentric objective lens means for directing said collimated beam onto said sample, forming an illuminated region thereon, and collecting light emitted from said region, forming a fluorescent retro-beam, said objective lens means defining a single pupil, a high numerical aperture and a working distance, with said pupil located external to said objective lens means, said objective lens means being afocal at said pupil, said working distance measured between said sample and said objective lens means, with said objective lens means adapted to provide different numerical apertures with said numerical apertures being inversely proportional to said working distance so as to maintain said objective lens means proximate to said sample,

means, in said optical axis, for scanning said region across said sample, said scanning means including a reflective element positioned at said pupil or said objective lens means,

means, positioned in said optical axis between said beam source and said objective lens means, for separating said incident beam from said retro-beam, wherein said objective lens means directs said retro-beam onto said separating means, with said separating means directing said retro-beam toward said photo-detector, said photo-detector producing signals representing light impinging thereon, and

means, connected to receive said signals, for producing a visual display or said sample.

24. The optical scanning system of claim 23 wherein said separating means is a mirror having a diameter greater than a diameter of said collimated beam and smaller than a diameter of said retro-beam, with the diameter of the retro-beam being substantially larger than the diameter of the collimated beam.

25. The optical scanning system of claim 23 wherein said objective lens means includes a plurality of objective lenses each of which is adapted to be selectively coupled to said collimated beam.

26. The optical scanning system of claim 23 wherein said objective lens means comprises of a zoom lens.

27. An optical scanning system for fluorescence microscopy of a sample, said system comprising,

a beam source for emitting a collimated beam of coherent light,

a photo-detector,

a telecentric objective defining an optical axis, a high numerical aperture and a single pupil, with said pupil located external to said objective, said objective positioned to receive said collimated beam therethrough to illuminate a spot on the surface of said sample, said objective being achromatic and afocal at said pupil and directing said incident beam onto said surface at a right angle with respect to the surface of said sample, said objective collecting light emitted from said spot, and forming a fluorescent retro-beam,

means, in said optical axis, for raster scanning said spot, on said sample, said scanning means including a reflective element positioned at said pupil of said objective, with said telecentric objective adapted to form said retro-beam with a cross-sectional area of sufficient size relative to said reflective element to ensure substantially all light associated with said retro-beam impinges upon said reflective element,

a dichroic filter positioned in said optical axis between said beam source and said objective, wherein said objective directs said retro-beam onto said dichroic filter, with said dichroic filter separating said retro-beam from said collimated beam and directing said retro-beam toward said photo-detector, with said photo-detector producing signals representing said substantially all light emitted from said spot, and

means, connected to receive said signals, for producing a visual display of said sample.

28. The optical scanning system of claim 27 wherein said objective has first and second ends, with said first end facing said scanning means, and spaced apart therefrom a first distances, with said second end opposing said first end and positioned proximate to said sample, defining a working distance therebetween, said working distance being substantially less than said first distance.

29. The optical scanning system of claim 28 wherein said scanning means further includes means for rotating said reflective element about a pivot axis.

30. The optical scanning system of claim 28 wherein said working distance is in the range between 3 and 3.5 mm inclusive.