Extended depth of field imaging for high speed object analysis

• US 8,005,314 B2
• Filed: 12/11/2006
• Issued: 08/23/2011
• Est. Priority Date: 12/09/2005
• Status: Active Grant

• Current Assignee: Cytek Biosciences Inc
• Original Assignee: Amnis Corporation (DiaSorin S.p.A.)
• Inventors: Bauer, Richard, Hall, Brian, Basiji, David, Frost, Keith, Liang, Luchuan, Ortyn, William, Perry, David
• Primary Examiner(s): NEURAUTER, GEORGE C
• Application Number:
  US11/609,269
• Publication Number:
  US 20070146873A1
• Time in Patent Office: 1,716 Days
• Field of Search: 359 1- 35, 359368-398, 359/721, 359/725, 356457-458, 382209-210, 382/275, 382/279, 382/280, 348 79- 80
• US Class Current: 382/275
• 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 2015/1452 Adjustment of focus; Alignment
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence
• CPC Class Codes: G02B 27/0012 Optical design, e.g. procedures, algorithms, optimisation routines
• CPC Class Codes: G02B 27/0075 with means for altering, e.g. increasing, the depth of field or depth of focus

Abstract

A high speed, high-resolution flow imaging system is modified to achieve extended depth of field imaging. An optical distortion element is introduced into the flow imaging system. Light from an object, such as a cell, is distorted by the distortion element, such that a point spread function (PSF) of the imaging system is invariant across an extended depth of field. The distorted light is spectrally dispersed, and the dispersed light is used to simultaneously generate a plurality of images. The images are detected, and image processing is used to enhance the detected images by compensating for the distortion, to achieve extended depth of field images of the object. The post image processing preferably involves de-convolution, and requires knowledge of the PSF of the imaging system, as modified by the optical distortion element.

Claims

1. A method for producing an extended depth of field image of an object, comprising the steps of:

(a) deforming an optical wave front of light from the object to produce modified light, such that a point spread function (PSF) of an imaging system used to collect the modified light is substantially invariant across the extended depth of field, wherein an optical element is used to deform the optical wave front, the optical element comprising a cuvette having different optical thicknesses at different locations, such that imaging through the different locations of the cuvette induces different degrees of wave front deformation;

(b) dispersing the modified light, producing dispersed light;

(c) focusing the dispersed light to produce at least one dispersed image, the dispersed image being formed by light simultaneously collected from a plurality of different focal planes in the extended depth of field;

(d) detecting the dispersed image of the object to generate image data; and

(e) processing the image data to reduce artifacts introduced by deforming the optical wave front, to produce the extended depth of field image of the object.

2. The method of claim 1, further comprising the step of analyzing the extended depth of field image of the object to determine at least one characteristic of the object.

3. The method of claim 1, wherein there is relative motion between the object and an imaging system used to produce the extended depth of field image of the object.

4. The method of claim 3, wherein the object is entrained in a flow of fluid.

5. The method of claim 1, wherein the extended depth of field comprises less than about fifteen microns.

6. The method of claim 1, wherein the step of processing the image data comprises the steps of:

(a) determining the PSF of the imaging system configured to deform the optical wave front; and

(b) using the PSF function to produce the extended depth of field image of the object.

7. The method of claim 1, wherein processing the image data comprises the step of de-convolving the image data.

8. The method of claim 7, wherein the step of de-convolving the image data reduces spatial broadening and contrast loss induced by the step of deforming the optical wave front.

9. A method for producing an extended depth of field image of an object, while there is relative motion between the object and an imaging system used to produce the extended depth of field image of the object, comprising the steps of:

(a) using an optical element to induce an aberration in an optical wave front of light from the object to produce modified light, such that a point spread function (PSF) of an imaging system as modified by the optical element is substantially invariant across the extended depth of field, wherein the optical element comprises a cuvette having different optical thicknesses at different locations, such that imaging through the different locations of the cuvette induces different degrees of wave front deformation;

(b) focusing the modified light to produce an image of the object that includes light simultaneously collected from a plurality of different focal planes in the extended depth of field;

(c) detecting the image of the object to generate image data; and

(d) processing the image data to reduce artifacts introduced by deforming the optical wave front, to produce the extended depth of field image of the object.

10. The method of claim 9, wherein the step of detecting the image comprises the step of using a time delay integration detector to collect the image over a period of time, while the object moves relative to the imaging system.

11. The method of claim 9, further comprising the step of dispersing the modified light into a plurality of light beams, such that:

(a) the step of focusing the modified light comprises the step of focusing each of the light beams to produce a respective image corresponding to that light beam, each respective image being formed from the modified light simultaneously collected from the plurality of different focal planes in the extended depth of field;

(b) the step of detecting the image comprises the step of detecting each respective image of the object to generate image data; and

(c) the step of processing the image comprises the step of processing the image data for each respective image to reduce artifacts introduced by deforming the optical wave front, to produce the extended depth of field image of the object for each respective image.

12. The method of claim 11, further comprising the step of analyzing the extended depth of field image of the object for each respective image to determine at least one characteristic of the object.

13. The method of claim 11, wherein the step of processing the image data comprises the steps of:

(a) determining the PSF of the imaging system as modified by the optical element configured to deform the optical wave front; and

(b) using the PSF function to de-convolve each respective image, thereby reducing spatial broadening and contrast loss induced by the PSF.

14. An imaging system adapted to perform extended depth of field imaging of an object, comprising:

(a) an optical element configured to deform an optical wave front of light from the object, such that a point spread function (PSF) of the imaging system as modified by the optical element is substantially invariant across the extended depth of field, wherein the optical element is selectively positionable, such that in a first position, the optical element deforms the optical wave front, thereby enabling extended depth of field imaging, while in a second position, the optical element does not deform the optical wave front, thereby enabling non-extended depth of field imaging;

(b) a collection lens disposed so that light traveling from the object passes through the collection lens and travels along a collection path;

(c) a light dispersing element disposed in the collection path so as to disperse the light that has passed through the collection lens, producing dispersed light;

(d) an imaging lens disposed to receive the dispersed light, producing a dispersed image from the dispersed light;

(e) a detector disposed to receive the dispersed image produced by the imaging lens, producing an output signal that is indicative of the dispersed image; and

(f) a processor configured to manipulate the output signal to reduce artifacts introduced by the optical element configured to deform the optical wave front of light from the object, to produce an extended depth of field image of the object.

15. The system of claim 14, wherein the optical element comprises one element selected from the group consisting essentially of:

(a) a phase plate configured to induce a phase deviation in an optical wave front;

(b) an optical element configured to induce a spherical aberration in light from the object; and

(c) a cuvette having different optical thicknesses at different locations, such that imaging through the different locations of the cuvette induces different degrees of wave front deformation.

16. The system of claim 14, wherein the optical element configured to deform the optical wave front of light from the object comprises an adjustable objective lens, such that the aberration of the objective lens can be modified to deform the optical wave front of light from the object.

17. The system of claim 14, wherein the processor is configured to use the PSF function as modified by the optical element to de-convolve the output signal, thereby reducing spatial broadening and contrast loss induced by the PSF as modified by the optical element.

18. An imaging system adapted to perform extended depth of field imaging of an object, while there is relative movement between the object and the imaging system, comprising:

(a) an optical element configured to deform an optical wave front of light from the object, thereby producing modified light, such that a point spread function (PSF) of the imaging system as modified by the optical element is substantially invariant across the extended depth of field, wherein the optical element is selectively positionable, such that in a first position, the optical element deforms the optical wave front, thereby enabling extended depth of field imaging, while in a second position, the optical element does not deform the optical wave front, thereby enabling non-extended depth of field imaging;

(b) a collection lens disposed so that modified light passes through the collection lens and travels along a collection path;

(c) a dispersing component disposed in the collection path so as to receive the light that has passed through the collection lens, dispersing the light into a plurality of separate light beams, each light beam being directed away from the dispersing component in a different predetermined direction;

(d) an imaging lens disposed to receive the light beams from the dispersing component, producing a plurality of images, each image corresponding to one of the light beams and being projected by the imaging lens toward a different predetermined location;

(e) a detector disposed to receive the plurality of images produced by the imaging lens, producing an output signal that is indicative of imaging at the plurality of different focal planes; and

(f) a processor configured to manipulate the output signal to reduce artifacts introduced by the optical element configured to deform the optical wave front of light from the object, to produce an extended depth of field image of the object for at least one of the plurality of images.

19. The system of claim 18, wherein the optical element is a phase plate configured to induce a phase deviation in the optical wave front.

20. The system of claim 19, wherein the phase plate is disposed proximate a numerical objective of the imaging system.

21. The system of claim 18, wherein the optical element is configured to induce a spherical aberration in light from the object.

22. The system of claim 18, wherein the optical element is a cuvette having different optical thicknesses at different locations, such that imaging through the different locations of the cuvette induces different degrees of wave front deformation.

23. An imaging system adapted to perform extended depth of field imaging of an object, comprising:

(a) an optical element configured to deform an optical wave front of light from the object, such that a point spread function (PSF) of the imaging system as modified by the optical element is substantially invariant across the extended depth of field, wherein the optical element is a cuvette having different optical thicknesses at different locations, such that imaging through the different locations of the cuvette induces different degrees of wave front deformation;

(b) a collection lens disposed so that light traveling from the object passes through the collection lens and travels along a collection path;

(c) a light dispersing element disposed in the collection path so as to disperse the light that has passed through the collection lens, producing dispersed light;

(d) an imaging lens disposed to receive the dispersed light, producing a dispersed image from the dispersed light;

(e) a detector disposed to receive the dispersed image produced by the imaging lens, producing an output signal that is indicative of the dispersed image; and

(f) a processor configured to manipulate the output signal to reduce artifacts introduced by the optical element configured to deform the optical wave front of light from the object, to produce an extended depth of field image of the object.