## 4 Discussion  
This study aimed to develop a lifetime-based multiplex 3D imaging method for NIRF in deep tissues by synchronizing an NIRF-TGI system [23] with a rotatable stage controlling the detection angles for NIRF-CT [26]. Lanthanide-doped ceramic nanoparticles were used as probes with different fluorescence lifetimes to be supplied for TGI. Nd\({}^{3+}\) and Yb\({}^{3+}\) were used as dopants to obtain emissions at 1000\(-\)1040 nm under 808 nm excitation. A multiplex scheme with two different lifetimes was provided by different host materials, NaYF\({}_{4}\) and YPO\({}_{4}\). We developed an SCM as an NIRF lifetime analysis method using conventional NIR cameras with reduced delay time points and demonstrated its effectiveness. While fluorescence lifetime-based tomography has been previously reported [15; 33; 34], the present study first reports in detail the relationship between the fluorescence decay at the projection plane, where images can be obtained directly with a detector (NIR camera), and the decay depicted on cross-sectional tomograms. Furthermore, the accuracy of the mapped fluorescence lifetimes on the tomograms is examined. Although we do not have preliminary NIRF-TGI-CT data of in vivo tissues, the availability of such NIR camera for in vivo NIRF-CT (not time-gated) of targets has been reported in mice [26]. Combining this with the time-gated technique, the NIRF-TGI-CT of this paper is expected to be applicable to in vivo samples. NIRF-CT results of fluorescence located in deep tissue in larger samples, which is observable with NIRF imaging, are affected by the refraction of fluorescence at the sample surface. However, this problem can be solved by immersing the sample in a liquid with a refractive index close to that of the sample [35]. The influence of light scattering by tissue samples, reported in NIRF-CT [36] and NIRF-TGI [23] imaging techniques, has not been examined in the present NIRF-TGI-CT, and is still an important issue of future work. The basic strategy of NIRF-TGI-CT is similar to that of MRI, that is, it depicts a cross-sectional image of the inside of a sample by contrasting the signal decay rate [37]. As with MRI, NIRF-TGI-CT currently requires more than ten minutes of the imaging time. However, the number of imaging angles and delay times can be modified according to the required accuracy for each case. Therefore, the imaging time can be shortened by reducing the number of images. The greatest advantage of the NIRF-TGI-CT method shown in this paper is to allow tomographic imaging of contrasts of the fluorescence lifetime of rare-earth phosphors (on the order of hundreds of microseconds), using a conventional NIR camera in combination with time-gated technique, instead of FLIM, which is commonly used to visualize the lifetime of molecular dyes (nanosecond order) with a high-speed camera. Although the time required for imaging using the NIRF-TGI-CT method is not as short as the FLIM method, the time-gated NIRF-CT for lifetime-based multiplex 3D imagingtechnique presented in this study is expected to be applied to the acquisition of information on the deep interior of samples, especially biological tissues possessing refractive index interfaces that cause light scattering.