Method and apparatus for performing high sensitivity fluorescence measurements

• US 5,018,866 A
• Filed: 09/12/1989
• Issued: 05/28/1991
• Est. Priority Date: 09/12/1989
• Status: Expired due to Fees

• Current Assignee: Packard Instrument Co., Inc. (Revvity, Inc.)
• Original Assignee: Packard Instrument Co., Inc. (Revvity, Inc.)
• Inventors: Osten, Donald E.
• Primary Examiner(s): McGraw, Vincent P.
• Application Number:
  US07/405,963
• Time in Patent Office: 623 Days
• Field of Search: 356/73, 356/318, 356/317, 356/417, 356/440, 250/458.1, 250/459.1, 250/461.1
• US Class Current: 356/417
• CPC Class Codes: G01N 2021/516 Multiple excitation of scattering medium, e.g. by retro-reflected or multiply reflected excitation rays
• CPC Class Codes: G01N 2021/6469 Cavity, e.g. ellipsoid
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters

Abstract

An improved method and apparatus is provided for performing high sensitivity fluorescence measurements. A liquid sample contained within a cuvette is exposed to exciting radiation in such a way that the radiation is introduced directly to the sample and is restricted from reaching the container walls. The sample container is in the form of a cylindrical cuvette provided with reflector means on the bottom so that any unabsorbed exciting radiation reaching the cuvette bottom is reflected back through the sample solution. Fluorescent radiation emitted by the sample is collected by an ellipsoidal reflector which substantially surrounds the sample container and focuses radiation incident thereupon onto a small area from which it is efficiently sensed by a photodetetor. The optical arrangement realizes significantly improved signal-to-noise and signal-to-background ratios.

Claims

1. A method of performing fluorescence measurements on a sample capable of being activated by exciting radiation, said method comprising the steps of:

enclosing the sample within a container having

surrounding walls and an open side facing a bottom surface, said bottom surface having reflector means for reflecting any radiation incident thereupon;

introducing a beam of exciting radiation through said open side of container directly onto the sample contained therein in a direction normal to said reflector surface in such a way that said beam does not reach said container walls, thereby avoiding any secondary fluorescence therefrom, any portion of said beam which is unabsorbed by the sample and traverses through the sample to strike said reflector means on si bottom surface being reflected therefrom back through the sample without reaching said container walls, thereby avoiding any secondary fluorescence therefrom;

collecting fluorescent radiation from the sample through collector means surrounding said container walls, said collector means being adapted to focus said fluorescent radiation incident thereupon onto a predefined area; and

detecting and measuring said fluorescent radiation focused onto said predefined area.

2. The method of making fluorescence measurements as set forth in claim 1 wherein said container is in the form of a cuvette formed of glass, quartz, synthetic plastic or like material which is permeable to said fluorescent radiation.

3. The method of making fluorescence measurements as set forth in claim 2 wherein said cuvette is substantially cylindrical, and wherein the walls and bottom surface thereof, in combination, define a cavity for receiving a fluid sample.

4. The method of making fluorescence measurements as set forth in claim 1 wherein said exciting radiation is collimated so as to be focused on the sample through said open side of said sample container without reaching said container walls.

5. The method of making fluorescence measurements as set forth in claim 1 wherein said exciting radiation is filtered to a desired range of wavelength prior to being focused onto the sample.

6. The method of making fluorescence measurements as set forth in claim 1 wherein said collector means is in the form of an ellipsoidal reflector surrounding said container walls, said reflector being disposed with its major axis aligned substantially with said beam of exciting radiation incident on the sample, whereby fluorescent radiation emanating from the sample in all directions is collected and reflected so as to be focused substantially about the focal point of said ellipsoidal reflector.

7. The method of making fluorescence measurements as set forth in claim 6 wherein said focused fluorescent radiation is filtered to a desired wavelength and detected by a photodetector positioned about said focal point of said reflector.

8. Apparatus for performing fluorescence measurements on a sample by detecting fluorescence induced in the sample by focusing exciting radiation thereupon, said apparatus comprising:

a sample container having surrounding walls and an open side facing a bottom surface, said walls and bottom surface defining a sample-containing cavity, said bottom surface being provided with reflector means for reflecting any radiation incident thereupon;

means for introducing a beam of exciting radiation through said open side of container directly onto the sample contained therein in a direction normal to said reflector surface in such a way that said beam does not reach said container walls, thereby avoiding any secondary fluorescence therefrom, any portion of said beam which is unabsorbed by the sample and traverses through the sample to strike said reflector means on said bottom surface being reflected therefrom back through the sample without reaching said container walls, thereby avoiding any secondary fluorescence therefrom;

collector means for collecting fluorescent radiation emitted from the sample, said collector means substantially surrounding said container walls and being adapted to focus said fluorescent radiation incident thereupon onto a predefined area; and

means for detecting said focused fluorescent radiation.

9. The apparatus for making fluorescence measurements as set forth in claim 8 wherein said sample container is in the form of a cuvette formed of glass, quartz, synthetic plastic or like material which is permeable to said fluorescent radiation.

10. The apparatus for making fluorescence measurements as set forth in claim 9 wherein said cuvette is substantially cylindrical, and wherein the walls and bottom surface thereof, in combination, define a cylindrical cavity for receiving a fluid sample.

11. The method of making fluorescence measurements as set forth in claim 8 further including means for collimating said exciting radiation so as to be focused on the sample substantially about the central section of said open side of said sample container without reaching said container walls.

12. The apparatus for making fluorescence measurements as set forth in claim 8 further including means for filtering said exciting radiation to a desired wavelength prior to being focused onto the sample.

13. The apparatus for making fluorescence measurements as set forth in claim 8 wherein said collector means is in the form of an ellipsoidal reflector surrounding said container walls, said reflector being disposed with its major axis aligned substantially with s id beam of exciting radiation incident on the sample, whereby fluorescent radiation emanating from the sample in all directions is collected and reflected so as to be focused substantially about the focal point of said ellipsoidal reflector.

14. The apparatus for making fluorescence measurements as set forth in claim 13 further including means for filtering said focused fluorescent radiation to a desired range of wavelength.

15. The apparatus for making fluorescence measurements as set forth in claim 14 wherein said means for detecting said focused fluorescent radiation comprises a photodetector positioned about said focal point of said reflector.