System and method for processing specimens and images for optical tomography

• US 20050085721A1
• Filed: 10/13/2004
• Published: 04/21/2005
• Est. Priority Date: 04/19/2002
• Status: Active Grant

• Current Assignee: University of Washington, VisionGate, Inc.
• Original Assignee: University of Washington, VisionGate, Inc.
• Inventors: Nelson, Alan C., Fauver, Mark E., Seibel, Eric J., Rahn, John Richard
• Granted Patent: US 7,811,825 B2
• Time in Patent Office: Days
• US Class Current: 600/425
• CPC Class Codes: G02B 21/002 Scanning microscopes scanning near field optical microscopes G01Q60/18
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence

Abstract

A scanning method for scanning samples of biological cells using optical tomography includes preparing, acquiring, reconstructing and viewing three-dimensional images of cell samples. Concentration and enrichment of the cell sample follows. The cell sample is stained. Cells are isolated from the cell sample and purified. A cell/solvent mixture is injected into a gel by centrifugation. A cell/gel mixture is injected into a capillary tube until a cell appears centered in a field of view using a step flow method. An optical imaging system, such as a fixed or variable motion optical tomography system acquires a projection image. The sample is rotated about a tube axis to generate additional projections. Once image acquisition is completed, the acquired shawdowgrams or image projections are corrected for errors. A computer or other equivalent processor is used to compute filtered backprojection information for 3D reconstruction.

Claims

1. A scanning method for scanning samples of biological cells using optical tomography comprising the steps of:

concentrating and enriching a cell sample;

staining the cell sample;

isolating cells from the cell sample;

purifying the isolated cells;

injecting a cell/solvent mixture into a gel to produce a cell/gel mixture;

injecting the cell/gel mixture into a capillary tube until a cell appears in a field of view;

acquiring at least one projection image of at least one object in the field of view;

rotating the sample about a tube axis to generate additional projection images;

determining whether the projection data set is complete and, if not, repeating above steps of injecting into field of view, acquiring at least one projection image and rotating the sample for another object of interest;

correcting the at least one projection image for errors to produce corrected projection image information; and

from the corrected projection image information computing filtered backprojection information for 3D reconstruction.

2. The scanning method of claim 1 further comprising the steps of:

analyzing the 3D reconstruction images; and

viewing the 3D reconstruction images.

3. The scanning method of claim 1 wherein the step of injecting the cell/gel mixture into a capillary tube comprises using a step flow method.

4. The scanning method of claim 3 wherein the cell/gel mixture comprises at least one cell embedded in a liquid having a viscosity >

1 million cps.

5. The scanning method of claim 3 wherein the cell/gel mixture comprises at least one cell embedded in a thixotropic gel.

6. The scanning method of claim 1 wherein the step of acquiring at least one projection image further comprises the step of operating a pseudo-projection viewing subsystem for imaging the specimens, wherein the pseudo-projection viewing subsystem includes a fixed objective lens, and a plurality of mechanical scanning components mounted to engage opposing sides of the sample proximate to the fixed objective lens so as to vibrate the sample to allow the sample to be axially scanned by the fixed objective lens.

7. The scanning method of claim 1 wherein the step of acquiring at least one projection image further comprises the step of operating scanning apparatus comprising a pair of synchronized stepper motors, a microscope objective, a pair of mounting fixtures, a sample, and a pair of piezotubes, wherein the pair of stepper motors operates to rotate the sample and wherein the piezotubes are coupled at opposing ends of the sample to vibrate the sample so as to enable the objective lens to axially scan the sample.

8. The scanning method of claim 1 wherein the step of correcting projection images for errors further comprises the steps of

illuminating a portion of the sample;

locating at least one objective lens having a Fourier plane to receive light rays passing through the portion of the sample;

using a spatial filter mask in the Fourier plane to filter light rays projecting through the at least one objective lens to produce spatially filtered light;

detecting images in the spatially filtered light; and

reconstructing three-dimensional images from the spatially filtered light.

9. The scanning method of claim 8 wherein the spatial filter mask has a spatially varying optical density, OD_NA=q, which incorporates a quasi-ramp filter, said quasi-ramp filter functioning as a high-pass filter at low spatial frequencies.

10. A scanning system for scanning a sample, wherein the sample includes a series of objects within a container, the scanning system comprising:

a pseudo-projection viewing subsystem for imaging the objects, wherein the pseudo-projection viewing subsystem includes a fixed objective lens; and

a plurality of mechanical scanning components mounted to engage opposing sides of the sample proximate to the fixed objective lens so as to vibrate the sample to allow the sample to be axially scanned by the fixed objective lens.

11. The scanning system of claim 10 wherein the plurality of mechanical scanning components comprise piezoelectric pushers.

12. The scanning system of claim 10 wherein the sample comprises a series of objects embedded in a semi-rigid medium.

13. The scanning system of claim 12 wherein the semi-rigid medium is selected from the group consisting of a polymer thread, a capillary tube, cell beads and a frozen liquid stream.

14. The scanning system of claim 10 further comprising at least one stepper motor coupled to the sample for rotating the sample.

15. The scanning system of claim 14 wherein the at least one stepper motor operates in cooperation with the plurality of mechanical scanning components to allow scanning of the sample through multiple angles.

16. The scanning system of claim 10 wherein the objective lens comprises an oil immersion lens and piezoelectric pushers are located around the objective lens, so that the sample is immersed within a liquid environment for refractive index matching.

17. The scanning system of claim 10 wherein the objective lens comprises an air immersion lens.

18. The scanning system of claim 10 wherein the plurality of mechanical scanning components comprises means for scanning by electrostatic attraction.

19. The scanning system of claim 10, wherein the plurality of mechanical scanning components comprise electrostatic actuators.

20. A scanning apparatus comprising a pair of synchronized stepper motors, a microscope objective, a pair of mounting fixtures, a sample, and a pair of piezotubes, wherein the pair of stepper motors operates to rotate the sample and wherein the piezotubes are coupled at opposing ends of the sample to vibrate the sample so as to enable the objective lens to axially scan the sample.

21. The scanning apparatus of claim 20 wherein the sample is contained in a tube having a plurality of flat surfaces.

22. The scanning apparatus of claim 20 wherein the sample is restrained by a vertical tube guide used to restrict the sample within a viewing area under the objective.

23. The scanning apparatus of claim 20, wherein the sample comprises a biological specimen.

24. The scanning apparatus of claim 20 wherein the sample comprises at least one human cell.

25. A system for pre-processing projection images from a sample including objects of interest, the system comprising:

a light source;

a portion of the sample located to be illuminated by the light source;

at least one objective lens having a Fourier plane, the at least one objective lens being located to receive light rays passing through the portion of the sample;

a spatial filter mask located in the Fourier plane;

at least one detector located to receive radiation from the sample when the sample is illuminated; and

a computer including an image analysis algorithm for producing three-dimensional images wherein the computer receives imaging information from the at least one detector.

26. The system of claim 25 wherein the spatial filter mask has an optical density, OD_NA=q(r) defined by the convolution of a condenser function, C(f), with a mapping function, r(f), multiplied by a Hanning window function and a deblurring function according to the relationship

OD_NA=q(r)=OD_NA=0(r){circle over (x)}C(r)

where OD_NA=0(r) is defined for the case of the condenser NA=0.

27. The system of claim 25 wherein the spatial filter mask has a spatially varying optical density, OD_NA=q, which incorporates a quasi-ramp filter, said quasi-ramp filter functioning as a high-pass filter at low spatial frequencies.

28. The system of claim 25 wherein the image analysis algorithm comprises a tomographic backprojection reconstruction algorithm.

29. A method for pre-processing projection images from a sample including objects of interest, the system comprising:

illuminating a portion of the sample;

using at least one objective lens having a Fourier plane receive light rays passing through the portion of the sample;

filtering light rays transmitted through the at least one objective lens with a spatial filter mask located in the Fourier plane to generate spatially filtered light;

detecting the spatially filtered light to generate imaging information; and

producing three-dimensional images from the imaging information using a computer including an image analysis algorithm.

30. The method of claim 29 wherein the spatial filter mask has a spatially varying optical density, OD_NA=q, which incorporates a quasi-ramp filter, said quasi-ramp filter functioning as a high-pass filter at low spatial frequencies.

31. The method of claim 29 wherein the spatial filter mask has an optical density, OD_NA=q(r) defined by the convolution of a condenser function, C(f), with a mapping function, r(f), multiplied by a Hanning window function and a deblurring function according to the relationship

OD_NA=q(r)=OD_NA=0(r){circle over (x)}C(r)

where OD_NA=0(r) is defined for the case of the condenser NA=0.

32. The method of claim 29 wherein the image analysis algorithm comprises a tomographic backprojection reconstruction algorithm.

33. A method for preparing samples of biological cells for analysis comprising the steps of:

concentrating and enriching a cell sample;

staining the cell sample;

isolating cells from the cell sample;

purifying the isolated cells; and

injecting a cell/solvent mixture into a gel to produce a cell/gel mixture.

34. The method of claim 33 further comprising the step of injecting the cell/gel mixture into a capillary tube having a glass surface, wherein the cell/gel mixture substantially lubricates the cell to reduce the tendency to adhere to the glass surface.

35. The method of claim 33 further comprising the step of injecting the cell/gel mixture into a capillary tube having a glass surface, wherein the cell/gel mixture substantially lubricates the cells to reduce the tendency to adhere to the glass surface.

36. The method of claim 33 further comprising the step of injecting the cell/gel mixture into a capillary tube, wherein the cell/gel mixture reduces cell adherence to the capillary tube.

37. The method of claim 33 wherein the cell/gel mixture has reduced cross contamination. because specimen retrieval is very high

38. The method of claim 33 wherein the cell/gel mixture comprises at least one cell embedded in a liquid having a viscosity >

1 million cps.

39. The method of claim 33 wherein the cell/gel mixture comprises at least one cell embedded in a thixotropic gel.