Method and apparatus for quantitative hyperspectral fluorescence and reflectance imaging for surgical guidance
An imaging system includes an illumination device for illuminating a target. A surgical microscope receives light from the target, the surgical microscope comprising at least one optical output port at which at least a portion of the received light is provided as an output from the surgical microscope. A tunable filter receives the portion of the received light provided as the output from the surgical microscope, the tunable filter being tunable to pass a filtered portion of the received light, the filtered portion of the received light having a plurality of wavelengths selected by the tunable filter and provided as output from the tunable filter. A high-resolution, broad-bandwidth electronic camera receives the light of a plurality of wavelengths selected by the tunable filter, the electronic camera converting the light of a plurality of wavelengths selected by the tunable filter to a plurality of electrical signals. A processor processes the plurality of electrical signals to form an image of the target.
1. A hyperspectral imaging method for determining depth and concentration of fluorophores in tissue of a patient, comprising:
capturing a white-light hyperspectral image cube of the tissue as illuminated with white light, the capturing performed using a hyperspectral imaging device that passes light from the tissue through a tunable filter into a broad-spectrum electronic camera;
capturing, using the hyperspectral imaging device, a fluorescence-light hyperspectral image cube of the tissue as illuminated with fluorescence stimulus light;
analyzing spectral bands of the white-light hyperspectral image cube near a wavelength of the fluorescence stimulus light and a wavelength of associated fluorescence emissions to extract scattering and absorption parameters of the tissue at the wavelengths of the fluorescence stimulus light and the associated fluorescence emissions;
normalizing at least one selected pair of images of the fluorescence-light hyperspectral image cube, the selected pair of images being associated with two different respective wavelengths near a peak wavelength of the fluorescence emission;
deriving, based upon the scattering and absorption parameters, the depth of fluorophores in the tissue at pixels from a ratio between the two images of the at least one selected pair of images of the fluorescence-light hyperspectral image cube, as normalized; and
determining, using a voxel-based model and the extracted scattering and absorption parameters derived from the white-light hyperspectral image cube, and the depth of the fluorophores in the tissue at the pixels and at least one image of the fluorescence-light hyperspectral image cube, fluorophore concentration at voxels of the voxel-based model; wherein
the step of deriving comprises comparing the ratio between the two images of the at least one selected pair of images to a model of depth-dependent spectral properties of the fluorescence of the two images of the at least one selected pair of images.
2. The hyperspectral imaging method of claim 1 , further comprising, prior to the step of comparing, determining parameters of the model of the depth-dependent spectral properties from the scattering and absorption parameters.
3. The hyperspectral imaging method of claim 1 , the model of the depth-dependent spectral properties specifying the depth as d=[1n(Γ)−ln(D 1 /D 2 )]/[1/δ 2 −1/δ 1 ], wherein (a) D 1 and D 2 are diffusion constants for the two different wavelengths, respectively, (b) δ 1 and δ 2 are penetration depths for the two different wavelengths, respectively, and (c) Γ is the ratio, the hyperspectral imaging method further comprising determining each of D 1 , D 2 , δ 1 , and δ 2 from the scattering and absorption parameters.
4. A hyperspectral imaging method for determining depth and concentration of fluorophores in tissue of a patient, comprising:
capturing a white-light hyperspectral image cube of the tissue as illuminated with white light, the capturing performed using a hyperspectral imaging device that passes light from the tissue through a tunable filter into an electronic camera;
capturing, using the hyperspectral imaging device, a fluorescence-light hyperspectral image cube of the tissue as illuminated with fluorescence stimulus light;
analyzing spectral bands of the white-light hyperspectral image cube near a wavelength of the fluorescence stimulus light and a wavelength of associated fluorescence emissions to extract scattering and absorption parameters of the tissue at the wavelengths of the fluorescence stimulus light and the associated fluorescence emissions;
normalizing at least one selected pair of images of the fluorescence-light hyperspectral image cube, the selected pair of images being associated with two different respective wavelengths near a peak wavelength of the fluorescence emission;
deriving, based upon the scattering and absorption parameters, depth of fluorophores in the tissue at pixels of the selected pair of images from a ratio between the two images of the at least one selected pair of images of the fluorescence-light hyperspectral image cube, as normalized; and
determining, using a voxel-based model and the extracted scattering and absorption parameters derived from the white-light hyperspectral image cube, and the depth of the fluorophores in the tissue at the pixels and at least one image of the fluorescence-light hyperspectral image cube, fluorophore concentration at voxels of the voxel-based model.
5. The hyperspectral imaging method of claim 4 , comprising:
in the step of normalizing, selecting a plurality of pairs of images of the fluorescence-light hyperspectral image cube; and
in the step of deriving, collectively evaluating a plurality of ratios, each obtained from a respective one of the plurality of pairs.
6. The hyperspectral imaging method of claim 5 ,
the step of collectively evaluating comprising fitting, to the plurality of ratios, a model of depth-dependent spectral properties of fluorescence emissions, to determine the depth of fluorophores from said fitting; and
the hyperspectral imaging method further comprising, prior to the step of fitting, determining parameters of the model of the depth-dependent spectral properties from the scattering and absorption parameters.
7. The hyperspectral imaging method of claim 4 , the step of normalizing comprising dividing intensity of pixels of images in the at least a subset of the fluorescence-light hyperspectral image cube by (a) intensity of pixels in an image of the white-light hyperspectral image cube corresponding to wavelength of the fluorescence stimulus light and (b) intensity of pixels in an image of the white-light hyperspectral image cube associated with same wavelength as the respective image in the at least a subset of the fluorescence-light hyperspectral image cube.
8. The hyperspectral imaging method of claim 7 , further comprising capturing the white-light hyperspectral image cube and the fluorescence-light hyperspectral image cube at same set of wavelengths, the step of normalizing comprising normalizing at least two pairs of images of the fluorescence-light hyperspectral image cube.
9. The method of claim 4 wherein the step of analyzing comprises setting up the voxel-based model with the scattering and absorption parameters and refining the scattering and absorption parameters.