System and method for total internal reflection enhanced imaging flow cytometry

• US 8,879,797 B2
• Filed: 05/25/2012
• Issued: 11/04/2014
• Est. Priority Date: 05/25/2012
• Status: Active Grant

• Current Assignee: Yokogawa Fluid Imaging Technologies, Inc.
• Original Assignee: Fluid Imaging Technologies, Inc. (Yokogawa Electric Corporation)
• Inventors: Sieracki, Christian K., Peterson, Kent A., Duplisea, Matthew A., Drake, Corie F.
• Primary Examiner(s): COUSO, YON JUNG
• Application Number:
  US13/480,999
• Publication Number:
  US 20130315447A1
• Time in Patent Office: 893 Days
• Field of Search: 382/107, 382/100, 250/458.1, 250/459.1, 250/461.2, 356/337, 356/338, 356/339, 356/341, 356/332, 356/343
• US Class Current: 382/107
• CPC Class Codes: G01N 15/1427 with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences
• CPC Class Codes: G01N 15/1436 the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
• CPC Class Codes: G01N 2021/6493 by alternating fluorescence/transmission or fluorescence/reflection
• CPC Class Codes: G01N 21/47 Scattering, i.e. diffuse reflection G01N21/25, G01N21/41 take precedence G01N21/55 takes precedence
• CPC Class Codes: G01N 21/53 within a flowing fluid, e.g. smoke
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/85 Investigating moving fluids or granular solids
• CPC Class Codes: G01N 2201/062 LED's
• CPC Class Codes: G06V 10/145 Illumination specially adapted for pattern recognition, e.g. using gratings
• CPC Class Codes: G06V 10/147 Details of sensors, e.g. sensor lenses fingerprint or palmprint sensors G06V40/13; vascular sensors G06V40/145; eye sensors G06V40/19

Abstract

An imaging flow cytometry system and method which includes a flow chamber, fluorescence analysis and imaging optics, image capturing system, device to regulate fluid flow through the chamber, and backlighting generator. The flow cell is configured so as to enhance the fluorescence signal collection by the system with total internal reflections. The fluorescence collection optics are configured to enhance the collection of the fluorescence from the side of the flow cell and concentrate it on light detectors.

Claims

1. A system for imaging particles in a fluid, the system comprising:

a. A rectangular flow chamber having a width dimension and a depth dimension, the flow chamber including a channel arranged to transport the fluid therethrough at a selectable rate, wherein the flow chamber is a low fluorescence structure with an index of refraction of about 1.5 and having a ratio of the width dimension to the depth dimension that is greater than 10;

1;

b. a device configured to create a controllable fluid flow rate in the flow chamber;

c. a backlighting generator arranged to illuminate the fluid in the flow chamber;

d. an objective arranged to receive incident optical radiation from the flow chamber;

e. a light source arranged to generate light scatter and/or fluorescence from particles;

f. one or more detectors to detect light scatter and/or fluorescence emitted from the particles upon illumination;

g. a signal processor configured to receive signals from the one or more detectors; and

h. an image capturing system including means to capture images of particles in the fluid.

2. The system of claim 1, wherein the backlighting generator is a light emitting diode flash.

3. The system of claim 1, wherein the backlighting generator generates a high intensity flash.

4. The system of claim 1, wherein the system further includes a computing device to receive signals from the image capturing system.

5. The system of claim 1, wherein the image capturing system includes a computing device.

6. The system of claim 1, wherein the image capturing system includes a digital camera or an analog camera and a framegrabber.

7. The system of claim 1, wherein the image capturing system includes a CCD or a CMOS camera.

8. The system of claim 1, wherein the light source is a laser.

9. An apparatus to assist in the imaging of particles in a fluid, the apparatus comprising a rectangular flow chamber having a width dimension and a depth dimension and including a channel arranged to transport the fluid therethrough at a selectable rate, wherein the flow chamber is configured with a ratio of the width dimension to the depth dimension that is greater than 10:

1 to enable fluorescence propagation from the fluid within the channel to an edge of the flow chamber for enhanced light collection of the fluorescence.

10. A method for imaging particles in a fluid which is transported through a channel of a rectangular flow chamber at a selectable rate and illuminated with a light source so that scatter and/or fluorescence signals are detected, the method comprising the steps of:

a. directing the fluid through the flow chamber, wherein the flow chamber is configured with a ratio of the width dimension to the depth dimension that is greater than 10;

1 to enable fluorescence propagation from the fluid within the channel to an edge of the flow chamber for enhanced light collection of the fluorescence; and

b. imaging the tracked particle and transferring the captured images to a computing device.

11. The method of claim 10, wherein the method further includes the step of analyzing the image for particles.

12. The method of claim 10, wherein the backlighting generator is a light emitting diode flash.

13. A method for imaging particles in a fluid, the method comprising the steps of:

a. transporting the fluid through a channel of a rectangular flow chamber at a selectable rate, wherein the flow chamber is configured with a ratio of the width dimension to the depth dimension that is greater than 10;

1 to enable fluorescence propagation from the fluid within the channel to an edge of the flow chamber for enhanced light collection of the fluorescence;

b. illuminating the fluid with a light source arranged to generate light scatter and/or fluorescence from the particles;

c. transmitting a signal from a scatter detector and/or a fluorescence detector to a signal processor and, if the signal meets a predetermined threshold, initiating a particle tracking interval including controlling a tracking mirror, activating a backlighting generator, and activating an image capturing system; and

d. imaging the tracked particle and transferring the captured images to a computing device.

14. The method of claim 13, wherein the method further includes the step of analyzing the image for particles.

15. The method of claim 13, wherein the backlighting generator is a light emitting diode flash.