Multi-modal molecular diffuse optical tomography system for small animal imaging

Abstract

A multi-modal optical imaging system for quantitative 3D bioluminescence and functional diffuse imaging is presented, which has no moving parts and uses mirrors to provide multi-view tomographic data for image reconstruction. It is demonstrated that through the use of trans-illuminated spectral near infrared measurements and spectrally constrained tomographic reconstruction, recovered concentrations of absorbing agents can be used as prior knowledge for bioluminescence imaging within the visible spectrum. Additionally, the first use of a recently developed multi-view optical surface capture technique is shown and its application to model-based image reconstruction and free-space light modelling is demonstrated. The benefits of model-based tomographic image recovery as compared to 2D planar imaging are highlighted in a number of scenarios where the internal luminescence source is not visible or is confounding in 2D images. The results presented show that the luminescence tomographic imaging method produces 3D reconstructions of individual light sources within a mouse-sized solid phantom that are accurately localised to within 1.5mm for a range of target locations and depths indicating sensitivity and accurate imaging throughout the phantom volume. Additionally the total reconstructed luminescence source intensity is consistent to within 15% which is a dramatic improvement upon standard bioluminescence imaging. Finally, results from a heterogeneous phantom with an absorbing anomaly are presented demonstrating the use and benefits of a multi-view, spectrally constrained coupled imaging system that provides accurate 3D luminescence images.

Keywords: Bioluminescence Imaging; Bioluminescence Tomography; Diffuse Optical Tomography; Image Reconstruction; Imaging Systems; Molecular Imaging; Multimodality; Small Animal Imaging; Surface Capture.

Figure 1

Visual representation of the system concept. A mouse is surface captured to obtain its geometry and is then imaged in spectral luminescence and in spectral near-infrared trans-illumination modes. Using NIRFAST[49], DOT is used to reconstruct chromophore, scattering and subsequently functional parameters which are additionally used to inform reconstructions of bioluminescent source distributions.

Figure 2

(a) Labelled schematic and (b) photograph of the developed imaging system.

Figure 3

General imaging run protocol. Note that whilst “Project Image” is only shown once, it represents a total of three parallel operations in which a projection is done with any or each of the three projectors in the system.

Figure 4

Surface capture raw data for a single data set: (a) maximum of bright images (full-field white projection from each projector); (b) highest frequency pattern projected with projector 1 and (c) with projector 2.

Figure 5

The geometry of the imaging system illustrated in terms of the positions and view-directions of the two projectors used for surface capture and the camera used for detection along with accompanying virtual cameras which are the reflection of the camera in each of the mirrors. Note that z = 0 is the height of the stage when the labjack is fully retracted.

Figure 6

(a) Example surface capture point cloud in which points acquired at different views are indicated by different colours; and (b) FEM mesh following registration to the surface capture point cloud; black elements indicate the location of the rod that is left protruding slightly from the main cylinder and used as a reference for finding the correct rotation. The point cloud appears truncated compared to the mesh because the back portion of the cylinder was mounted into the rotation mount thus obscuring it from view.

Figure 7

(a) Diffuse imaging protocol; (b) source grid illustrated in the form of the maximum intensity through the stack of all projected images with each Gaussian centrepoint labelled by the order of appearance in the imaging protocol; (c) the same image showing the effective raster scan order for the source patterns.

Figure 8

Normalised spectral system response functions: (a) system response for the NIR source; (b) system response for luminescent source.

Figure 9

Bioluminescence phantom imaging results for the on-axis data set: (a-d) phantom schematics; and (e-h) luminescence images at λ = 600nm shown overlaid on maximum-signal images from surface capture data sets as a spatial reference. Luminescence images are set as completely transparent at all points where the value is less than 10% of the maximum value in the image.

Figure 10

Bioluminescence imaging results for the off-axis data set at λ = 600nm.

Figure 11

Visualisation of (a) 3D reconstruction for the data set with the most shallow source (see figure 12(a)) along with (b) indication of how the following 2D slice representations of results are obtained

Figure 12

Summary of on-axis, homogeneous BLT experiment results showing: (a-d) schematics of source experimental locations in 2D projection; (e-h) BLI images (CCD measurement e^–/s) of the phantom at λ = 600nm with approximate phantom outlines; (i-l) slices through corresponding BLT reconstructions at the axial offset corresponding to the centre-of-mass of the reconstruction; and (m-p) the slice images thresholded at 75% of the maximum value.

Figure 13

Summary of off-axis, homogeneous BLT experiment results showing: (a-d) schematics of source experimental locations in 2D projection; (e-h) BLI images (CCD measurement e^–/s) of the phantom at λ = 600nm with approximate phantom outlines; (i-l) slices through corresponding BLT reconstructions at the axial offset corresponding to the centre-of-mass of the reconstruction; and (m-p) the slice images thresholded at 75% of the maximum value.

Figure 14

Summary of BLT results in quantitative terms for both the on-axis and off-axis (45° rotated) experiments ordered versus effective depth w.r.t. the top of the phantom: (a) the 2D error in reconstructed source position based on the centre-of-mass metric; (b) the 2D position error based on the max-valued node metric; (c) the total (summed) reconstructed source shown in arbitrary units; (d) and (e) the 3D versions of (a) and (b) respectively; and (f) the values of (c) shown as a percentage of the mean value across both sets.

Figure 15

(a) Spectral dye extinction coefficient. Spectral sampling is shown both in terms of where DOT data was acquired (red lines and crosses) and where the BLT data was acquired (black dashed lines and circles); (b-d) diffuse trans-illumination images at 750nm with NIR source positions 1, 15 and 36 repectively, visualised in the same manner as the BLI images in figures 9 and 10 with approximate source location (under the phantom) shown by the overlaid white circle.

Figure 16

Spectral DOT results shown as slices through the 3D reconstruction at an axial offset equal to the location of the centre of the NIR source grid: (a) target absorber concentration showing rod anomaly; (b) reconstructed absorber concentration scaled to the same color-scale as the target; (c) reconstructed absorber concentration scaled to its own extremal values.

Figure 17

3D renderings of a cropped section of the cylinder (approximately 20mm long centred axially around the centre of the source grid: (a) the target dye concentration anomaly location; and (b) the reconstructed concentration thresholded at 1.45

Figure 18

Reconstructions of luminescence source distribution in the case where the target is in the lower tunnel with an anomaly in the upper tunnel: (a) schematic of target slice showing anomaly and source positions; (b) BLT reconstruction where the absorber concentration is assumed to be the background level throughout the volume; (c) BLT reconstruction where the absorber concentration is assumed to be that obtained via DOT; (d) and (e) 75% thresholded versions of (b) and (c).

Figure 19

Reconstructions of luminescence source distribution in the case where the target is in the lower tunnel with an anomaly in the upper tunnel, with reconstruction performed using top-view (direct) data only: (a) schematic of target slice showing anomaly and source positions; (b) BLT reconstruction where the absorber concentration is assumed to be the background level throughout the volume; (c) BLT reconstruction where the absorber concentration is assumed to be that obtained via DOT; (d) and (e) 75% thresholded versions of (b) and (c).