Phase-sensitive flow cytometer

• US 5,270,548 A
• Filed: 07/31/1992
• Issued: 12/14/1993
• Est. Priority Date: 07/31/1992
• Status: Expired due to Term

• Current Assignee: United States Department of Energy
• Original Assignee: United States Department of Energy
• Inventors: Steinkamp, John A.
• Primary Examiner(s): Hannaher, Constantine
• Assistant Examiner(s): Glick, Edward J.
• Application Number:
  US07/922,841
• Time in Patent Office: 501 Days
• Field of Search: 250/459.1, 250/461.2, 250/458.1, 250/461.1, 250/302
• US Class Current: 250/461.2
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence

Abstract

A phase-sensitive flow cytometer (FCM) provides additional FCM capability to use the fluorescence lifetime of one or more fluorochromes bound to single cells to provide additional information regarding the cells. The resulting fluorescence emission can be resolved into individual fluorescence signals if two fluorochromes are present or can be converted directly to a decay lifetime from a single fluorochrome. The excitation light for the fluorochromes is modulated to produce an amplitude modulated fluorescence pulse as the fluorochrome is excited in the FCM. The modulation signal also forms a reference signal that is phase-shifted a selected amount for subsequent mixing with the output modulated fluorescence intensity signal in phase-sensitive detection circuitry. The output from the phase-sensitive circuitry is then an individual resolved fluorochrome signal or a single fluorochrome decay lifetime, depending on the applied phase shifts.

Claims

1. A phase-sensitive flow cytometer for resolving fluorescence emissions from fluorochrome labeled cells into two components, comprising:

flow cytometer means for providing a flow steam containing said labeled cells;

an excitation light for exciting said labeled cells to fluoresce in said flow stream;

modulation means for modulating said excitation light and generating a reference signal at a selected modulation frequency;

detector means for receiving fluorescence emission spectra from said labeled cells as a modulated fluorescence signal and outputting a modulated intensity signal functionally related to said fluorescence emission spectra from said labeled cells; and

phase detector means for resolving said modulated intensity signal into two signal components, each functionally related to a different fluorescence decay lifetime of said fluorescent emission spectra.

2. A phase sensitive flow cytometer according to claim 1, wherein said phase detector means includes:

a phase shifter for phase shifting said reference signal an amount effective to null one of said two signal components in said modulated intensity signal; and

a mixer for mixing said modulated intensity signal with said reference signal after said phase shifting to output a first signal functionally related one of said two signal components.

3. A phase sensitive flow cytometer for determining the fluorescence decay lifetime of a fluorescence emission, comprising:

flow cytometer means for providing a flow stream containing cells labeled with a fluorochrome;

an excitation light for exciting said fluorochrome to fluoresce in said flow stream;

modulation means for modulating said excitation light and generating a reference signal at a selected modulation frequency;

detector means for receiving a fluorescence emission spectra from said fluorochrome as a modulated fluorescence signal and outputting a modulated intensity signal functionally related to said fluorescence emission spectra from said fluorochrome; and

phase detector means for inputting said modulated intensity signal and outputting two phase shifted signals whose ratio is functionally related to said fluorescence lifetime.

4. A phase sensitive flow cytometer according to claim 3, wherein said phase detector comprises:

a quadrature phase circuit for outputting said reference signal that is phase shifted by 0° and

by 90°

to form two phase shifted reference signals;

two phase sensitive detector means for mixing said modulated fluorescence signal from said fluorochrome with one of each said two phase shifted reference signals and outputting two quadrature signals; and

means for forming the ratio of said two quadrature signals and outputting a signal functionally related to the fluorescence lifetime of said fluorochrome.