FIGURE 12.
Image reconstruction to estimate penetration depth of fluorescent signal from tdTomato protein. Three-dimensional (3D) reconstruction is shown in center panel. False-colored contour map represents photon densities that have been assigned coloration on basis of multicolored bar spectrum (photons/mm3 × 10−3) shown at right of reconstruction. To obtain this map, program fills structure image (left panel) with cubic voxels and scores fluorescence density of each voxel. Red and dark circles within reconstruction indicate sources of fluorescence that lay on selected coronal (violet), sagittal (blue), and transaxial (green) planes. As seen from black-to-red bar spectrum at lower right of reconstruction, intensity of fluorescence from these sources is on order of 1012 photons/s. Enlarged sagittal and transaxial sections indicate that dorsal–ventral depth of animal was about 2.5 cm. Center (red dot) of fluorescent source from within rib cage cavity was about half way through animal or approximately 1.0 cm from ventral surface. 3D reconstructions were performed using 3D analysis software (Living Imaging; Xenogen Corp.) according to instruction manual from images collected at week 15, with 2.5-s exposure, and at emission wavelengths of 620 and 660 nm. 3D reconstruction program was designed for use with bioluminescence data and does not account for attenuation of fluorescence excitation signal by absorption and scatter. However, because emission wavelengths used (620 and 660 nm) are similar to those used during luciferase-based BLI, and photon densities and source intensities are similar to those observed with BLI, we have been advised that these results likely represent good first estimate of tissue penetration (communications with technical support at Xenogen Corp.).