Whole-body fluorescence imaging, especially FOT, is challenging because the probe often does not form unique spots but distributes widely throughout the body, particularly when systemic assessment is required, e.g., in a skeleton scan. Employing line-laser excitation enables one to perform whole-body fluorescence imaging at a much shorter scan time as compared to data acquisition strategies employing a point-laser source. Moreover, because of the extended excitation pattern throughout the axial direction generated by the line laser, the approximation of Eq. (2) could be locally held in practice. It is, however, not the case for point-laser illumination. Two-dimensional image reconstruction of tomographic slices becomes, therefore, practically available over the entire animal following line-laser excitation. In comparison to 3-D FOT reconstruction with point-laser excitation, this method allows a much larger FOV given the same acquisition time. In the mouse phantom study with two point-like fluorescence inclusions, the 2-D FOT result from the line-laser excitation shows broader distributions of the resolved point sources, especially in the axial direction, as compared to the 3-D reconstruction result from the point-laser excitation (cf. Fig. 5). Although, in this phantom study, 3-D FOT with point-laser outperforms 2-D FOT with line-laser excitation. Note, however, that the data acquisition time of the latter case was seven times faster. The in vivo experiment performed in this study is in fact an extremely challenging example for FOT as the hydroxyapatite-targeting probe has an affinity to the entire skeleton system in the animal which is very different to the case of spatially distinct point sources as are being used in the mouse phantom. The 2-D FOT reconstruction is applied slice by slice along the whole axial FOV. Intrinsically coregistered SPECT results with , a probe with high bone-specificity as well, provides additional information for cross-validation. Two-dimensional FOT results are shown exemplarily in three different transaxial slices (cf. Fig. 7). It can be seen from “Slice 1” that the small bone structures close to surface are well resolved, while the spine, which shows a high activity uptake in SPECT, is not visible in FOT. In “Slice 2,” the 2-D FOT image shows not only the spine structure near to the surface but also probe uptake in the kidneys, while no tracer activity can be seen in SPECT. “Slice 3” includes spine and knee joints, showing similar information in FOT and SPECT. Employing point-laser excitation, however, only a small portion of the animal is within the active FOV. The reconstruction results show a more pronounced convergence at the surface area (see the last row of Fig. 7).