High sensitivity fluorescent single particle and single molecule detection apparatus and method

• US 4,979,824 A
• Filed: 05/26/1989
• Issued: 12/25/1990
• Est. Priority Date: 05/26/1989
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Stryer, Lubert, Peck, Konan, Mathies, Richard A.
• Primary Examiner(s): Evans, F. L.
• Application Number:
  US07/358,782
• Time in Patent Office: 578 Days
• Field of Search: 356/72, 356/73, 356/317, 356/318, 356/417, 250/458.1, 250/459.1, 250/461.1, 250/461.2
• US Class Current: 356/318
• CPC Class Codes: G01N 15/1429 Signal processing
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/0222 from dynamic light scattering, e.g. photon correlation spectroscopy
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging

Abstract

Apparatus is described for ultrasensitive detection of single fluorescent particles down to the single fluorescent molecule limit in a fluid or on a substrate comprising means for illuminating a predetermined volume of the fluid or area of the substrate whereby to emit light including background light from the fluid and burst of photons from particles residing in the area. The photon burst is detected in real time to generate output representative signal. The signal is received and the burst of energy from the fluorescent particles is distinguished from the background energy to provide an indication of the number, location or concentration of the particles or molecules.

Claims

1. An apparatus for detection of single fluorescent particles and/or molecules in a fluid which comprises:

means for illuminating a predetermined volume of the fluid;

means for causing particles to transit through said illuminated volume whereby fluorescent energy emitted from said volume responsive to the illumination includes background energy and bursts of energy from particles or molecules as they transit through said volume;

means for detecting said energy and providing an output signal; and

means for receiving said output signal and distinguishing bursts of energy from said particles from background energy or electronic noise and providing an indication of the detection of a particle or molecule transiting through said volume.

2. Apparatus as in claim 1 wherein said means for receiving and distinguishing particle bursts of energy comprises means for detecting the amplitude and duration of said bursts.

3. Apparatus as in claim 2 in which said means for detecting the amplitude and duration of said burst comprises a frequency to voltage converter which produces an analog signal, a pulse height discriminator for receiving said analog signal and providing output pulses having a pulse width equal to the time which the signal exceeds a predetermined value and a pulse width discriminator providing an output when the pulse width exceeds a predetermined value indicative of the transit time of a particle.

4. Apparatus as in claim 3 in which said pulse width discriminator includes a pulse width to voltage converter for receiving said output pulses and providing an output voltage having an amplitude dependent upon pulse width and a pulse height discriminator for detecting when the output voltage exceeds a predetermined amplitude or lies within a predetermined window.

5. Apparatus as in claim 2 in which said means for detecting the amplitude and duration of said bursts comprises a current to voltage converter for receiving the output of said detecting means and providing an analog output signal, a pulse height discriminator for receiving said analog signal and providing output pulses having a pulse width equal to the time said signal exceeds a predetermined value and a pulse width discriminator providing an output signal when the output pulse width exceeds a predetermined value or lies in a predetermined window or range indicative of the transit time of a particle through said volume.

6. Apparatus as in claim 2 in which said means for detecting the amplitude and duration of said bursts comprises means for counting photons which are emitted over predetermined periods and providing an output signal indicative of a particle when the count exceeds a predetermined count.

7. An apparatus according to claim 2 wherein said means for illuminating a predetermined volume of the fluid is composed of a laser.

8. Apparatus as in claim 2 wherein said means for detecting and identifying particle bursts of energy operates in real time.

9. An apparatus for detecting single fluorescent particles in a fluid comprising means for illuminating an area of said fluid to cause the fluid to emit photon energy including energy scattered from said fluid and energy from fluorescent particles in said fluid,

means for causing fluorescent particles to transit through said area whereby said particles emit bursts of energy having a duration equal to their transit time through said area,

lens for receiving and focusing said energy,

a detector for receiving said focused energy and providing an output signal,

a spatial filter interposed between said detector and lens means to define the area from which energy is received, and

means for receiving the output signal and distinguishing bursts of photon energy emitted by said particles from background energy to provide an indication of the detection of a particle transiting said area.

10. Apparatus as in claim 9 in which said means for causing fluorescent particles to transit said area comprises means for moving the particles and fluid through said area.

11. Apparatus as in claim 10 in which said means for moving the particles and fluid through said area comprises a fluid stream.

12. Apparatus as in claim 10 in which said means for moving the particles and fluid through said area comprises placing the fluid on a stage and moving the stage relative to said area whereby to scan the fluid.

13. Apparatus as in claim 9 in which said means for causing the fluorescent particles to transit said area comprises placing the fluid on a stage and scanning the illumination over said stage.

14. Apparatus as in claim 13 in which the illumination is scanned in the X and Y direction by rotating mirrors.

15. An apparatus according to claim 13 further comprising a fluorescence microscope which focuses light on the sample through an objective lens and collects light from the sample through the same lens.

16. The method of detecting single fluorescent particles in a fluid comprising the steps of

illuminating a predetermined volume of the fluid,

transiting particles through said illuminated volume whereby to cause fluorescent energy to emit from said volume which includes background energy and bursts of energy from the transiting particles, and

processing said emitted energy to distinguish said bursts of energy from the background energy to provide an indication of particles transiting through said volume

17. The method of claim 16 in which said processing steps includes

detecting said emitted energy and generating representative electrical signals and

processing said representative electrical signals to reject electrical signals representative of background energy and providing an output when electrical signals having amplitude and duration corresponding to the transit of particles through said volume.

18. The method as in claim 16 including the steps of

choosing the illuminating energy and the transit time of the particles through the predetermined volume to provide the optimal ratio of emitted energy from particles with respect to background energy or energy fluctuation.

19. The method of claim 18 in which said steps include employing fluorescence emission lifetime and fluorescence photodestruction time of fluorophores as determining factors.