Dual-modality Optical Tomography

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					Dual-modality Optical Tomography: Fluorescent and
Bioluminescent Imaging Tomography of Embedded Sources In vivo
Chaincy Kuo, Muhammad H. Ahsan, Angel Ang, Steven Smith, Dusty Whittemore, Heng Xu, and Brad W. Rice,
Xenogen Corporation - now part of Caliper Life Sciences, Inc., 2061 Challenger Dr, Alameda, CA 94501 USA

Abstract                                                                                                             Results                                                                                                                                   Results (continued)
  The development of instrumentation which captures both in vivo biolumines-                                          Fluorescence Imaging Tomography
  cent and fluorescent signals on a cooled, sensitive charge-coupled device has                                                                                                                                                                                 Dual Modality Optical Imaging in vivo
  offered flexibility in reporter detection for functional molecular imaging. The ad-                                   Algorithm                                                                                                                                 Implantation study
  dition of transillumination capabilities for fluorescence excitation provides im-
  proved detection sensitivity to deep fluorescent sources whose signal at the                                         •   Acquire photographic, and fluorescent images at a number of trans-illumination source positions.
  surface may be obscured by autofluorescence excited with reflectance-mode                                                Include a structured light image.
  imaging. Images of fluorescence for varying transillumination source positions                                       •   Use structured light image to reconstruct surface vertices of mouse.
  offer constraining information for quantification and localization of fluorescent                                                                                                                                                                                                                               Fluor Pillow (from CT)
  sources deep within tissue.                                                                                          •   From surface radiance images, determine the photon density just inside the surface on every
                                                                                                                           element of the surface vertices.                                                                                                                                                       Lumin Bead (from CT)
  In this work, a new fluorescence imaging tomography algorithm (FLIT) based
                                                                                                                       •   Parameterize the light sources in the object volume as voxels.                                                                                                                         Bioluminescence
  on diffusion theory in a homogeneous medium is described. Boundary condi-
                                                                                                                       • ρ = S exc Gexc Gem [δx3Q.E. σn]
                                                                                                                                                                                                                                                                                                                  Imaging Tomography                     (all estimated sources displayed)
  tions for photon propagation between tissue and air are appropriately modeled
  with surface topography extraction from structured light images of the animal.                                                                                                                                                                                  Bioluminescence Imaging Tomography reconstruction of luminescent bead registered with segmented CT data
  Separate Green’s functions model the propagation of photons in tissue after in-                                      •   Solve the linear system of equations to determine the fluorescent source strengths.
  jection of the excitation light into the body at the surface opposite the camera                                          - Non-negative least squares                                                                                                        Dual Modality Optical Tomography
  lens, and the subsequent fluorescence emission to the imaged portion of the                                                  • Regularization can be used to treat inexact theory.
  surface. The excitation and emission Green’s functions are combined to form                                               - Voxel gridding
                                                                                                                                                                                                                                                                  In vivo study
  a functional to solve for the fluorophore distribution and quantification. Treat-
                                                                                                                               • Initially coarse for well-posed linear problem.                                                                                  • 5x105 PC3M-luc cells injected orthotopically to the prostate
  ment of tissue autofluorescence with spectral unmixing will be discussed.
                                                                                                                               • Adaptively refined in size to converge on location and strength.                                                                 • 50 μg AF750 dye Herceptin conjugate injected IV on Day 20
  We will present results of our fluorescence tomographic algorithm with confir-                                                                                                                                                                                  Fluorescence Data                                                                                    Bioluminescence Data
  mation from co-registered CT scans. We show tomographic solutions of a ho-                                           Tissue Phantom
                                                                                                                                                                                                                                                                   Ex: 745nm Em: 800 nm                                                                                                                    560             580                600
  mogenous tissue phantom embedded with fluorescent dye for validation of our                                          • Resin with scatterer and absorber                                                                           Fluorescent imaging
  algorithm. To demonstrate the accuracy of our fluorescence imaging tomogra-                                          • Two rod positions                                                                                           of AF750 dye in
  phy and diffuse bioluminescence imaging tomography (DLIT)1 algorithms in                                             • Rod tip embedded with AlexaFluor 750 dye
  vivo, animals were implanted with both calibrated luminescent beads and si-                                                                                                                                                        Rod Tip A (4x1013
  lastic pillows filled with fluorescent dyes and imaged in an dual modality optical                                                                                                                                                 dye molecules)
  imaging system and a micro-CT system. Co-registration of the tomographic                                                                    B                                                                                      trans-Exc: 745 nm,           50 μg 750 dye
  solutions with segmented CT shows excellent superposition with submillimeter                                                                                                                                                       Em: 800 nm                   Herceptin conjugate
                                                                                                                                                                                                                                                                  injected intraveneously
  accuracy in the phantom case.                                                                                                                                                                                                                                                                                                                                                                            620             640
                                                                                                                                                                                                                                                                  Imaged on Day 22,
                                                                                                                                                                                                                                     Fluorescent images
  With these validations, dual optical tomographic analysis was performed on in                                                                                                                                                                                   T=48 hours post-
                                                                                                                                                                                                                                     acquired with trans-         injection of probe
  vivo studies in which animals were orthotopically injected with PC3M-luc cells                                                                                                Software panel                                       illuminated excitation
  and administered a fluorescent tumor probe intraveneously. There is good                                                                                                   indicating possible                                                                  Excitation source
                                                                                                                                                                                                                                     sources. Excitation
                                                                                                                                                                                                                                                                  locations indicated
  agreement between the bioluminescent and fluorescent solutions.                                                                                                             trans-illumination                                     source locations are
                                                                                                                                             A                                excitation source
                                                                                                                                                                                                                                                                  by green circle
                                                                                                                                                                                                                                     indicated as crosshairs
                                                                                                                                                                                   locations.                                        in each image.

Materials and Methods                                                                                                                                                                The surface (B) of the subject is determined from structured
                                                                                                                                                                                                                                                                                                                                                                        5x105 PC3M-luc cells injected
                                                                                                                                                                                                                                                                                                                                                                        orthotopically to the prostate
                                                                                                                                                                                     light images1 (A) for appropriate modeling of boundary
  Dual Modality In vivo Optical Imaging Instrument                                                                                                                                                                                                                                                                                                                      imaged on Day 22
                                                                                                                                                                                     conditions at the air-tissue interface.
                                                                                                                                                                                                                                                                  Fluorescence Imaging Tomography                                         Bioluminescence Imaging Tomography
         CCD, TE-
         Cooled to
           -90 ºC
                                                        Emission                                                                                                                                                        (C) Fluorescent dyes are
                                                        Filter (18) Wheel
                                                                                                                           (A) Structured        (B) 3D surface topography                                              placed in hollow ends of rods                  Non-Specific dye     binding at
                                                                                                                            light image                                                                                 made of the same material as                                        tumor site
         Assembly                                                                                                                                                                                                       the tissue phantom. Caps at
                                                        Filter (10) Wheel                                                                                                                                               the rod ends are 5 mm in length.
                                                                                                                                                  5.5 mm
         Scanning                                                                                                                                               Rod (a)
            Laser                                       Optical                                                                                                                                          5 mm

           Heated                                                                                                          (D) Sources above 15% of max source displayed                                                                                              All voxels      Voxels above 54% of                                                                                             All voxels
                                                                                                                                                                                     (D) Reconstruction of dye in rod inserted into Bore A, shown as
           Sample                                       Scanning                                                                                                                                                                                                                      max source displayed
            Stage                                       Transillumination
                                                                                                                                                                                     red voxels. The long, narrow yellow surfaces indicate the
                                                        Fiber Bundle                                                                                                                 location of the bores. Note that the voxel location is                                                        The tomographic reconstruction from fluorescent images
                                                                                                                                                                Rod (b)
                                                                                                                                                                                     approximately 5 mm from the end of the bore, the dye’s                                                        matches the animal left cluster of voxels from the
                                                                                                                                            5.0 mm                                   location in the rod due to the length of the rod end cap.                                                     bioluminescent tomography estimation. Fluorescent images
                                                                                                                                                                                     (E) Reconstruction of dye in rod inserted in Bore B.                                                          from both transmission and reflectance excitation indicate
                                                                                                                           (E) All sources displayed                                                                                                                                               that the probe concentrated in the abdominal area on animal left.

        In vivo Bioluminescence Imaging Acquired without excitation light                                             Dual Modality Optical Imaging In vivo                                                                                                     Spectral Unmixing Prior to Fluorescence Imaging Tomography
                                                 Emission Filter
                                                    24 Positions
                                                                                   CCD                                 Implantation study                                                                                                                         Purpose
                                                (18 band-pass filters)

                                                                                Laser Galvanometer                     • 1015 molecules AF750 fluorescent dye in a silastic tubing pillow implanted in abdominal cavity                                           • Improve the 3D localization and clarification when target fluorophore signal is at a level comparable to tissue
                                                                                                                                                                                                                                                                   autofluorescence or another fluorophore.
               Excitation Filter
                                                                                  for Structured Light
                                                                                and Surface Topography                 • Calibrated luminescent bead implanted in animal scruff
                                                  Epi (Top)
                  12 Positions
                                                Illumination                                                                                                                                                                                                      Method
              (10 band-pass filters)
                                                                                                                       Trans-illumination                                    Bioluminescence Imaging                             Micro-CT Data                    • Multi-spectral transillumination imaging at multiple positions
                                       Bundle                                                                          Fluorescence Imaging                                                                                                                       • Perform spectral unmixing at all the positions and separate target fluorophore from background
                                                                                                                       Excitation 710 nm/Emission 800 nm                                                                                                          • Execute FLIT reconstruction using unmixed fluorophore images
                                                                                                                                                                                                                                                                Fluorescence Imaging Tomography without Spectral Unmixing
                                                                                                                                                                                                                                                                                      FLIT Quantum Dot 605
                                                                                                                                                                                                                                                                                   FLIT data, data, Quantum Dot 605                     (reconstructed sources above
                                                                                                                                                                                                                                                                                    9 positions, ex605/em640                            20% of max source displayed)
                                                                               Trans (Bottom)                                                                                                                                                                                           9 positions, ex605/em640
                                                                                                                                                                              560 nm       580 nm       600 nm
                                                                                                                                                                                                                                                                                                                                                                                     Autofluorescence leads to
                                                                         x-y Translation Stage                                                                                                                                                                                                                                                                                       reconstructed sources
                                                                                                                                                                                                                                                                                                                              FLIT                                                   distributed throughout the
         In vivo Fluorescence Imaging Transmission-Mode Excitation                                                                                                                                                                                                                                                                                                                   illumination area.

  Boundary Conditions for Fluorescence Imaging Tomography
                                                                   The instrument is absolutely calibrated so
                                                                   that count units measured at the CCD are
                                                                   converted to radiance units at the surface.                                                                620 nm       640 nm       660 nm
                                                                   Appropriate boundary conditions convert                                                                                                                                                      Spectral Unmixing + Fluorescence Imaging Tomography
                                                                   the radiance data to photon density data ρ.                                                                            Photon Density Fit for single trans-illumination
                     F                                                                                                                                                                    source position of Fluorescence Data                                                                                                      Unmixed QD605
                                                                                                                                                                                                                                                                                                                                                                                                 (reconstructed sources above 20%
                                                                                                                                                                                                                                                                                                                                                                                                      of max source displayed)
                                                                                                                                                                                                       Measured      Simulated
                                                                         The Green’s functions are modelled
                                                                         with a combination of the partial current
                                                                         boundary condition and extrapolated                                                                                                                                                             Multi-spectral FLIT data, QD605
                                                                                                                                                 Lumin Bead (from CT)
                                                                         boundary condition.                                                                                                                                                                               9 positions, 4 wavelengths
                                                                                                                                                 Fluor Pillow (from CT)                                                                                                         ex605, em640-680                                                                 FLIT
    ρ = S exc Gexc Gem [δx3Q.E. σn]
                                           Estimate this
                                           quantity F                                                                                            Fluorescence
                                           [number • mm 2]                                                                                                                                                                                                                                                                                                       Improved reconstruction and localized source
                                                                                                                                                 Imaging Tomography                                                                                                                                                    Spectral
                                           using a Non-Negative
                                                                                                                                                                                          Photon Density Fit for single wavelength                                                                                     Unmixing
                                           Least Squares algorithm.                                                                                                                                                                                                                                                                                                                             1
                                                                                                                                                                                                                                                                                                                                                                                                            Unmixed Spectra

                                                                                                                                                                                          (620 nm) of Bioluminescence Data
                                                                                                                                                                                                                                                                                                                                                                        normalized intensity

                                                                                                                                                                                                       Measured      Simulated
                                                                                                                                                                                                                                                                                                                               Unmixed Autofluorescence                                        0.6
                                                                                                                                                                                                                                                                                                                                                                                                                          Unmixed QD605

                                                                                                                                                                                                                                                                                                                                                                                               0.4                        Unmixed AutoFluor


                                                                                                                                                                                                                                                                                                                                                                                                     640      660       680           700

References                                                                                                                       (sources above 30% of max source displayed)                                                                                         640nm :No.1-9             660nm :No.10-18                                                                                                wavelength(nm)

1. Kuo et al., “Three-dimensional reconstruction of in vivo bioluminescent sources based on multi-spectral              Fluorescence Imaging Tomography reconstruction                                                                                               680nm :No.19-27           700nm :No.28-36
   imaging,” JBO, 12, 024007, 2007.                                                                                     of AF750 fluorescent dye pillow registered with
Acknowledgements                                                                                                        segmented CT data
Philip Salmon, Ph.D. - SkyScan
Daniel Stearns, Ph.D.