High throughput analysis of samples in flowing liquid

• US 6,309,886 B1
• Filed: 11/24/1999
• Issued: 10/30/2001
• Est. Priority Date: 06/04/1999
• Status: Expired due to Fees

• Current Assignee: Los Alamos National Security LLC (Government of the United States of America)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Goodwin, Peter M., Keller, Richard A., Jett, James H., Grace, W. Kevin, Ambrose, W. Patrick, Orden, Alan Van
• Primary Examiner(s): SNAY, JEFFREY R
• Application Number:
  US09/449,280
• Time in Patent Office: 706 Days
• Field of Search: 436/63, 436/172, 422/82.08, 356/72, 356/73, 250/458.1, 250/459.1, 250/461.2
• US Class Current: 436/63
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16

Abstract

Apparatus and method enable imaging multiple fluorescent sample particles in a single flow channel. A flow channel defines a flow direction for samples in a flow stream and has a viewing plane perpendicular to the flow direction. A laser beam is formed as a ribbon having a width effective to cover the viewing plane. Imaging optics are arranged to view the viewing plane to form an image of the fluorescent sample particles in the flow stream, and a camera records the image formed by the imaging optics.

Claims

1. Apparatus for imaging multiple fluorescent sample particles in a flow channel comprising:

a flow channel defining a flow direction for samples in a flow stream and having a viewing plane perpendicular to the flow direction;

a laser beam formed as a ribbon having a width effective to cover the viewing plane; and

imaging optics arranged to view the viewing plane to form an image of the multiple fluorescent sample particles in the flow stream; and

a camera to record the image formed by the microscope objective.

2. Apparatus according to claim 1, wherein the camera is a CCD camera.

3. Apparatus according to claim 2, wherein the CCD camera is a frame transfer CCD camera.

4. Apparatus according to claim 1, further, including beam shaping optics for forming the ribbon laser beam.

5. Apparatus according to claim 1, wherein the imagining optics comprises a microscope objective.

6. Apparatus according to claim 4, where the beam shaping optics comprise crossed cylindrical lenses.

7. A method for imaging multiple fluorescent sample particles in a flow stream, comprising the steps of:

flowing the flow stream throughout a flow area of a flow channel;

forming a ribbon laser beam that is perpendicular to and illuminates the flow area along an image plane;

passing the sample particles through the ribbon laser beam to excite the sample particles; and

collecting the florescence light emitted from each sample particle within the image plane to form an image of the multiple fluorescent sample particles in the flow area.

8. A method according to claim 7, further including the step of recording the image on a CCD camera.

9. A method according to claim 8, further including the steps of:

integrating the image on pixels of the CCD camera for a first image period; and

reading out charge collected on the pixels to complete a first data acquisition cycle.

10. A method according to claim 7, further including the step of calibrating the image for particle velocity variations across the flow area.

11. A method according to claim 7, further including the step of calibrating the image to account for optical variations over positions in image field and particle transit times over the positions in image field.

12. A method according to claim 7, further including the step of labeling different particles with different fluorophores that fluoresce at different characteristic wavelengths.

13. A method according to claim 12, further including the step of forming the ribbon laser beam with a plurality of laser excitation wavelengths.