HIGH THROUGHPUT LENS-FREE THREE-DIMENSIONAL TRACKING OF SPERM

• US 20150204773A1
• Filed: 07/12/2013
• Published: 07/23/2015
• Est. Priority Date: 07/13/2012
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Ozcan, Aydogan, Su, Ting-Wei
• Granted Patent: US 9,588,037 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G01B 11/00 Measuring arrangements characterised by the use of optical techniques
• CPC Class Codes: G01B 11/24 for measuring contours or curvatures
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1027 Determining speed or velocity of a particle
• CPC Class Codes: G03H 1/0443 Digital holography, i.e. recording holograms with digital recording means holobject computation G03H1/0866
• CPC Class Codes: G03H 1/0465 Particular recording light; Beam shape or geometry G03H1/06 takes precedence
• CPC Class Codes: G03H 1/0866 Digital holographic imaging, i.e. synthesizing holobjects from holograms
• CPC Class Codes: G03H 2001/0033 in hologrammetry for measuring or analysing
• CPC Class Codes: G03H 2001/0447 In-line recording arrangement
• CPC Class Codes: G03H 2210/30 3D object
• CPC Class Codes: G03H 2222/34 Multiple light sources
• CPC Class Codes: G06T 19/20 Editing of 3D images, e.g. changing shapes or colours, aligning objects or positioning parts
• CPC Class Codes: G06T 2200/04 involving 3D image data
• CPC Class Codes: G06T 2207/30004 Biomedical image processing

Abstract

A system for three dimensional imaging of motile objects includes an image sensor and a sample holder disposed adjacent to the image sensor. A first illumination source is provided and has a first wavelength and positioned relative to the sample holder at a first location to illuminate the sample. A second illumination source is also provided having a second wavelength, different from the first wavelength, and positioned relative to the sample holder at a second location, different from the first location, to illuminate the sample. The first and second illumination sources are configured to simultaneously, or alternatively, sequentially illuminate the sample contained within the sample holder. Three dimensional positions of the motile objects in each frame are obtained based on digitally reconstructed projection images of the mobile objects obtained from the first and second illumination sources. This positional data is connected for each frame to obtain 3D trajectories of motile objects.

Claims

1. A system for three dimensional imaging of motile objects contained within a sample comprising:

an image sensor;

a sample holder configured to hold the sample, the sample holder disposed adjacent to the image sensor;

a first illumination source having a first wavelength and positioned relative to the sample holder at a first location to illuminate the sample;

a second illumination source having a second wavelength, different from the first wavelength, and positioned relative to the sample holder at a second location, different from the first location, to illuminate the sample; and

wherein the first illumination source and the second illumination source are configured to simultaneously illuminate the sample contained within the sample holder.

2. The system of claim 1, wherein the motile objects comprise sperms.

3. The system of claim 1, wherein the first illumination source and the second illumination source comprise, respectively, a first LED and a second LED.

4. The system of claim 3, wherein the first and second LEDs are, respectively, butt-coupled to optical fibers.

5. The system of claim 1, further comprising a programmable power relay configured to cycle the image sensor between an ON state and an OFF state.

6. The system of claim 5, wherein the programmable power relay is programmed to maintain the sample holder within a temperature range of +/−

2°

C. of a target temperature.

7. The system of claim 1, further comprising a heat sink in thermal contact with the image sensor.

8. The system of claim 1, wherein the first location of the first illumination source is angled relative to the second location of the second illumination source.

9. The system of claim 8, wherein the angle between the first location and the second location is within the range of about 20°

to about 60°

.

10. The system of claim 1, further comprising at least one digital processor configured to reconstruct a three dimensional trajectory of the motile objects observed over a period of time.

11. The system of claim 10, wherein the at least one digital processor is configured to classify the three dimensional trajectory into one or more classes of pre-defined trajectories.

12. The system of claim 1, wherein the sample holder has a depth of field within the range of about 0.01 mm to about 5.0 mm.

13. The system of claim 1, further comprising a programmable relay configured to cycle the first illumination source and the second illumination source between an ON state and an OFF state.

14. A method for three dimensional tracking of motile objects contained within a sample comprising:

simultaneously illuminating a sample holder containing the motile objects with a first illumination source and a second illumination source wherein the first illumination source and the second illumination source are located at different positions from one another and emit light at the same or different wavelengths;

obtaining a plurality of image frames over time of the motile objects with an image sensor disposed adjacent to the sample holder while the sample holder is illuminated;

digitally reconstructing a projection image of the motile objects in each frame based on illumination originating from the first illumination source;

digitally reconstructing a projection image of the motile objects in each frame based on illumination originating from the second illumination source;

identifying the x, y, and z positions of the motile objects in each frame based on the digitally reconstructed projection images of the mobile objects obtained from the first and second illumination sources; and

connecting the x, y, and z positions of the motile objects over a plurality of frames to form a three-dimensional track of the motile objects within the sample.

15. The method of claim 14, wherein the motile objects comprise sperm.

16. The method of claim 15, wherein the sperm comprises human sperm.

17. The method of claim 15, wherein the sperm comprises equine sperm.

18. The method of claim 15, further comprising classifying the three-dimensional tracks of the sperm into one of a plurality of categories, wherein said classification is performed automatically by at least one processor.

19. The method of claim 18, wherein the categories comprise, immotile, typical, helical, ribbon, hyper-ribbon, hyper-activated, and hyper-helical.

20. The method of claim 19, wherein the ribbon category comprises one of a chiral ribbon, helical ribbon, or twisted ribbon.

21. The method of claim 14, wherein each image frame was divided into a plurality of regions-of-interest.

22. A method for three dimensional tracking of motile objects contained within a sample comprising:

sequentially illuminating a sample holder containing the motile objects with a first illumination source and a second illumination source wherein the first illumination source and the second illumination source are located at different positions from one another and emit light at the same or different wavelengths;

obtaining a plurality of image frames over time of the motile objects with an image sensor disposed adjacent to the sample holder while the sample holder is illuminated;

digitally reconstructing a projection image of the motile objects in each frame based on illumination originating from the first illumination source;

digitally reconstructing a projection image of the motile objects in each frame based on illumination originating from the second illumination source;

identifying the x, y, and z positions of the motile objects in each frame based on the digitally reconstructed projection images of the mobile objects obtained from the first and second illumination sources; and

connecting the x, y, and z positions of the motile objects over a plurality of frames to form a three-dimensional track of the motile objects within the sample.

23. The method of claim 22, wherein the motile objects comprise sperm.

24. The method of claim 23, wherein the sperm comprises human sperm.

25. The method of claim 23, wherein the sperm comprises equine sperm.

26. The method of claim 23, further comprising classifying the three-dimensional tracks of the sperm into one of a plurality of categories, wherein said classification is performed automatically by at least one processor.

27. The method of claim 26, wherein the categories comprise, immotile, typical, helical, ribbon, hyper-ribbon, hyper-activated, and hyper-helical.

28. The method of claim 27, wherein the ribbon category comprises one of a chiral ribbon, helical ribbon, or twisted ribbon.

29. The method of claim 22, wherein each image frame was divided into a plurality of regions-of-interest.