SPECTRAL IMAGING OF BIOLOGICAL SAMPLES

• US 20090245605A1
• Filed: 02/23/2009
• Published: 10/01/2009
• Est. Priority Date: 09/23/2003
• Status: Active Grant

• Current Assignee: Cambridge Research & Instrumentation Incorporated (Revvity, Inc.)
• Original Assignee: Cambridge Research & Instrumentation Incorporated (Revvity, Inc.)
• Inventors: Cronin, Paul J., Levenson, Richard, Hoyt, Clifford C., Gossage, Kirk William
• Granted Patent: US 8,385,615 B2
• Time in Patent Office: Days
• US Class Current: 382/128
• CPC Class Codes: A61B 5/0059 using light, e.g. diagnosis by transillumination, diascopy, fluorescence A61B5/0093, A61B5/1455, A61B5/02416 and A61B5/0261 take precedence
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: G01J 3/32 Investigating bands of a spectrum in sequence by a single detector
• CPC Class Codes: G01J 3/4406 Fluorescence spectrometry
• CPC Class Codes: G01J 3/453 by correlation of the amplitudes
• CPC Class Codes: G01N 21/255 Details, e.g. use of specially adapted sources, lighting or optical systems
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/062 LED's
• CPC Class Codes: G01N 2201/125 Digital circuitry
• CPC Class Codes: G01N 33/4833 of solid biological material, e.g. tissue samples, cell cultures tissue in vivo A61B5/00; cell suspensions G01N33/48735

Abstract

The invention features a method including: (i) providing spectrally resolved information about light coming from different spatial locations in a sample comprising deep tissue in response to an illumination of the sample, wherein the light includes contributions from different components in the sample; (ii) decomposing the spectrally resolved information for each of at least some of the different spatial locations into contributions from spectral estimates associated with at least some of the components in the sample; and (iii) constructing a deep tissue image of the sample based on the decomposition to preferentially show a selected one of the components.

Claims

1. A method for spectrally unmixing light data corresponding to light emitted from multiple light sources internal to an animal, the method comprising:

selecting an excitation wavelength band for excitation light provided to the animal;

selecting an emission wavelength band that limits light wavelengths collected from the animal;

providing excitation light, into the animal, that is limited to the excitation wavelength band;

capturing a first image of at least a portion of the animal, where the first image includes light data that is limited in wavelength to the emission wavelength band and corresponds to light emitted by multiple fluorescent light sources internal to the animal;

changing the excitation wavelength band and/or the emission wavelength band;

capturing at least one additional image of at least the portion of the animal that uses a different combination of excitation wavelength band and emission wavelength band for each additional image, where each additional image includes light data that corresponds to light emitted by the multiple fluorescent light sources internal to the animal and is limited in wavelength to the emission wavelength band of the different combination; and

using an iterative solution process, unmixing spectra for the multiple fluorescent light sources internal to the animal to provide a spectrum for each light source.

2. The method of claim 1 further comprising determining the number of fluorescent light sources before unmixing spectra for the multiple fluorescent light sources.

3. The method of claim 1 wherein a total number of images captured by the camera is no less than the number of fluorescent light sources in the animal.

4. The method of claim 1 wherein the different combination for each additional image uses a common excitation wavelength band and multiple emission wavelength bands.

5. The method of claim 1 wherein the different combination for each additional image uses a common emission wavelength band and multiple excitation wavelength bands.

6. The method of claim 1 where the excitation light is provided into the animal from a portion of the animal that is visible to the camera during image capture.

7. The method of claim 1 where the excitation light is provided into the animal from a portion of the animal that is not visible to the camera during image capture.

8. The method of claim 1 wherein the iterative solution process also outputs a spatial distribution map for each of the multiple fluorescent light sources internal to the animal, where each spatial distribution map provides a two dimensional representation of a distribution of a fluorescent light source in the animal.

9. The method of claim 1 further comprising providing an initial estimate of each spectrum for the multiple fluorescent light sources.

10. The method of claim 1 further comprising applying a constraint to the iterative solution process.

11. The method of claim 10 wherein the constraint includes a spectral constraint.

12. The method of claim 11 wherein the spectral constraint includes a non-negativity constraint that limits the spectrum for each light source to a non-negative number.

13. The method of claim 11 wherein the spectral constraint includes a unimodality constraint that limits the spectrum for each light source to having a single peak.

14. The method of claim 11 wherein the spectral constraint includes a bandpass constraint that limits spectral data input to the iterative solution process to within a desired wavelength range.

15. The method of claim 10 wherein the constraint includes a spatial distribution map constraint.

16. The method of claim 15 wherein the spatial distribution map constraint includes a region of interest constraint that limits spatial area for a fluorescent light source in a spatial distribution map produced by the iterative solution process.

17. The method of claim 1 wherein the multiple fluorescent light sources include tissue autofluoresence in the animal.

18. (canceled)

19. Logic encoded in one or more tangible media for execution and, when executed, operable to spectrally unmix light data corresponding to light emitted from multiple light sources internal to an animal, the logic including:

instructions for selecting an excitation wavelength band for excitation light provided to the animal;

instructions for selecting an emission wavelength band that limits light wavelengths collected from the animal;

instructions for providing excitation light, into the animal, that is limited to the excitation wavelength band;

instructions for capturing a first image of at least a portion of the animal, where the first image includes light data that is limited in wavelength to the emission wavelength band and corresponds to light emitted by multiple fluorescent light sources internal to the animal;

instructions for changing the excitation wavelength band and/or the emission wavelength band;

instructions for capturing one or more additional images of at least the portion of the animal, using a different combination of excitation wavelength band and emission wavelength band for each additional image, where each additional image includes light data that corresponds to light emitted by the multiple fluorescent light sources internal to the animal and is limited in wavelength to the emission wavelength band of the different combination; and

instructions for unmixing spectra, using an iterative solution process, for the multiple fluorescent light sources internal to the animal to provide a spectrum for each light source.

20-24. -24. (canceled)

25. A method for spectrally unmixing light data corresponding to light emitted from multiple light sources internal to an animal, the method comprising:

selecting an emission wavelength band that limits light wavelengths collected from the animal;

capturing a first image of at least a portion of the animal, where the first image includes light data that is limited in wavelength to the emission wavelength band and corresponds to light emitted by the multiple light sources internal to the animal;

changing the emission wavelength band;

capturing one or more additional images of at least the portion of the animal, using a different emission wavelength band for each additional image, where each additional image includes light data that corresponds to light emitted by the multiple light sources internal to the animal and is limited in wavelength to the different emission wavelength band; and

using an iterative solution process, unmixing spectra for the multiple light sources internal to the animal to provide a spectrum for each light source, wherein the iterative solution process implements a multivariate curve resolution and iteratively solves for a spectrum of each of the multiple light sources using finishing criteria.

26. The method of claim 25 wherein the multiple light sources include a bioluminscent light source and tissue autofluoresence in the animal.

27. The method of claim 25 further comprising selecting an excitation spectrum for excitation light provided to the animal and providing excitation light, into the animal, that is limited to the excitation wavelength band, when the multiple light sources include a fluorescent light source.

28. The method of claim 27 wherein the iterative solution process operates simultaneously on images for a) multiple illumination positions, b) multiple combinations of excitation wavelength bands, and c) multiple emission wavelength bands.

29. The method of claim 25 wherein the iterative solution process also outputs a spatial distribution map for each of the multiple light sources internal to the animal, where each spatial distribution map provides a two dimensional representation of a distribution of a light source in the animal.

30. The method of claim 25 wherein the finishing criteria uses an alternating least square method.

31. An imaging system for imaging an animal, the imaging system comprising:

an imaging chamber that includes

a set of walls and a door that enclose an interior cavity,

a stage configured to support the animal within the interior cavity,

a camera, and

a fluorescent excitation source; and

a processing system including a processor and memory, the memory including

instructions for selecting an excitation wavelength band for excitation light provided to the animal,

instructions for selecting an emission wavelength band that limits light spectra collected from the animal,

instructions for obtaining one or more fluorescent images using the camera, and

instructions for unmixing spectra, using an iterative solution process, for the multiple fluorescent light sources internal to the animal to provide a spectrum for each fluorescent light source.

32. The imaging system of claim 31 further comprising:

an excitation filter configured to intercept excitation light produced by the fluorescent excitation source before the excitation light reaches the animal; and

an emission filter configured to intercept emission light emitted from the animal before the emission light reaches the camera.

33. The imaging system of claim 31 further comprising a structured light projector.

34. The imaging system of claim 31 wherein the interior cavity is substantially light tight.

35. The imaging system of claim 31 wherein the fluorescent excitation source is below the stage.