High-speed multiple wavelength illumination source, apparatus containing the same, and applications thereof to methods of irradiating luminescent samples and of quantitative luminescence ratio microscopy

• US 5,491,343 A
• Filed: 03/25/1994
• Issued: 02/13/1996
• Est. Priority Date: 03/25/1994
• Status: Expired due to Term

• Current Assignee: Atto Instruments, LLC
• Original Assignee: Gary Brooker
• Inventors: Brooker, Gary
• Primary Examiner(s): Hannaher, Constantine
• Application Number:
  US08/217,883
• Time in Patent Office: 690 Days
• Field of Search: 250/459.1, 250/458.1
• US Class Current: 250/458.1
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16

Abstract

A high-speed, non-mechanical multiple wavelength illumination source is provided, which contains a first light source, a second light source providing a different wavelength of light, and a controlling means for controlling electric current to the first and second light sources. The illumination source is particularly useful in an improved apparatus for quantitative luminescence ratio photometry and/or luminescence ratio imaging. Methods of luminescence emission ratio photometry and luminescence emission ratio imaging using the illumination source and apparatus are also provided. The present invention offers particular advantages in photomicroscopy and in microscopic imaging.

Claims

1. A vibration-free method of irradiating a sample, comprising the steps of:

(A) irradiating said sample with light having a first wavelength, a first intensity or both a first wavelength and a first intensity, by passing an electric current having a first value through a first arc and passing an electric current having a second value through a second arc, said first value being greater than said second value; and

within a period of time of from 1 nanosecond (1 ns) to 1 second (1 s) of said irradiating step (A),

(B) irradiating said sample with light having a second wavelength, a second intensity or both a second wavelength and a second intensity by reducing the value of said electric current passed through said first light source to a third value and increasing said electric current through said second light source to a fourth value, said fourth value being greater than said third value,

wherein each of said arcs has a wattage of from 10 to 2000 W.

2. The method of claim 1, further comprising, within a period of time of from 1 ns to 1 s after said irradiating step (B), repeating said irradiating step (A).

3. The method of claim 1, wherein said first current is greater than 90% of the total of said first and second currents.

4. The method of claim 1, wherein said irradiating step (A) irradiates said sample with light having a first wavelength and a first intensity, and said irradiating step (B) irradiates said sample with light having a second wavelength and a second intensity, wherein said second wavelength differs from said first wavelength.

5. The method of claim 1, wherein said irradiating step (B) is conducted within a period of time of from 1 ns to 2.5 milliseconds.

6. An apparatus for sequentially irradiating a sample with two or more wavelengths, intensities, or wavelengths and intensities of light, comprising:

(A) a first arc which generates a first light beam having a first wavelength, a first intensity or both a first wavelength and a first intensity;

(B) a second arc which generates a second light beam having a second wavelength, a second intensity or both a second wavelength and a second intensity, wherein said second wavelength differs from said first wavelength, said second intensity differs from said first intensity, or said second wavelength and said second intensity differ from said first wavelength and said first intensity; and

(C) a controlling means for controlling electric current to each of said first and second arcs, said controlling means being electrically connected to each of said first and second arcs,

wherein each of said first and second arcs has a wattage of from 10 to 2000 W.

7. The apparatus of claim 6, further comprising:

(D) a first vibration-free means for selecting a band of light which intercepts said first light beam, and

(E) a second vibration-free means for selecting a band of light which intercepts said second light beam.

8. The apparatus of claim 7, wherein said apparatus sequentially irradiates a sample with two or more wavelengths of light, said first wavelength is a mean or average wavelength of a first band of wavelengths, and said second wavelength is a mean or average wavelength of a second band of wavelengths, each of said first and second bands of wavelengths either (a) having a band width of 40 nm or less, or (b) being greater than or equal to a desired or pre-selected wavelength.

9. The apparatus of claim 6, further comprising a beam combiner.

10. The apparatus of claim 6, further comprising at least one additional light source.

11. The apparatus of claim 6, wherein said controlling means provides a minimum current to one of said first and second arcs and a larger current to the other of said first and second arcs, wherein said minimum current is one sufficient to support a small arc, but one which does not cause interference with measurement of an emission resulting from irradiating said sample with light from said other of said first and second arcs.

12. The apparatus of claim 6, wherein at least one of said first and second light sources is a multiple arc lamp, comprising a pilot arc and a running arc.

13. The apparatus of claim 12, wherein said pilot arc and said running arc are electrically connected to one another.

14. The apparatus of claim 12, wherein the beam of light generated by said multiple arc lamp has an optical axis, said running arc is aligned with said optical axis, and said pilot arc is not aligned with said optical axis.

15. An apparatus for luminescence emission photometry, comprising:

(a) a first arc which generates a first light beam having sufficient energy to electronically excite at least one luminescent compound in a sample,

(b) a second arc which generates a second light beam having sufficient energy to electronically excite at least one luminescent compound in said sample,

(c) a controlling means for controlling electric current to each of said first and second arcs, said controlling means being electrically connected to each of said first and second arcs,

(d) a first vibration-free means for selecting a wavelength of light, positioned in the path of said first light beam to provide said first light beam with a first wavelength,

(e) a second vibration-free means for selecting a wavelength of light, positioned in the path of said second light beam to provide said second light beam with a second wavelength, said second wavelength differing from said first wavelength,

(f) a mounting platform for said sample, positioned such that each of said first and second light beams strikes said sample so that said luminescent compound produces a luminescent emission, and

(g) a detector positioned to receive said luminescent emission,

wherein each of said first and second arcs has a wattage of from 10 to 2000 W.

16. The apparatus of claim 15, further comprising:

(h) a dichroic mirror positioned in the path of each of said first and second light beams, to reflect each of said first and second light beams to a location on said mounting platform where said sample may be located.

17. The apparatus of claim 15, wherein said detector has an electronic switching device for adjusting the sensitivity of said detector at least two times within a period of time of from about 2 ns to 1 min.

18. The apparatus of claim 15, further comprising a beam combiner.

19. The apparatus of claim 15, wherein said detector detects an emission from said sample in response to excitation by said first light beam, and said apparatus further comprises a second detector which detects an emission from said sample in response to excitation by said second light beam.

20. A vibration-free method of determining the concentration of a luminescence-affecting chemical species in a sample capable of emitting luminescent radiation, comprising the steps of:

(A) irradiating said sample with light having a first wavelength and having sufficient energy to cause said sample to emit a first luminescent emission by passing an electric current having a first value through a first arc and passing an electric current having a second value through a second arc, said first value being greater than said second value;

then, within a period of time of from 1 ns to 1 s,

(B) irradiating said sample with light having a second wavelength and having sufficient energy to cause said sample to emit a second luminescent emission by reducing the value of said electric current passed through said first light source to a third value and increasing said electric current through said second light source to a fourth value, said fourth value being greater than said third value,

(C) measuring the intensity of each of said first and second luminescent emissions,

(D) determining a ratio of one of said first and second luminescent emission intensities to the remaining one of said first and second luminescent emission intensities, and

(E) correlating said ratio to said concentration of said luminescence-affecting chemical species in said sample,

wherein each of said arcs has a wattage of from 10 to 2000 W.

21. The method of claim 20, wherein said luminescence is fluorescence.