## 4 Discussion and Conclusions  
We achieved in vivo dual-scale fluorescent bacterial imaging using the developed DSFE system in an orthotopic mouse colon tumor model. The DSFE system successfully demonstrated the bacterial accumulation of tumor tissue from the gross level to the cellular level. To the best of our knowledge, this is the first case of imaging bacterial accumulation in WFE and CFEM simultaneously in vivo. The fused DSFE system is a promising imaging modality to evaluate the therapeutic response of colon cancer by bacterial therapy in vivo. The DSFE system has the advantage of compensating the limitation of the single WFE and CFEM system. WFE screened the colonic wall to identify tumor lesions with a large FOV. Additionally, it localized fluorescence-emitting suspicious lesions to contact the CFEM probe within a short examination time. Meanwhile, the CFEM probe exhibited a bacteria-specific fluorescence image at a cellular-scaled resolution, which WFE could not. As a result, the DSFE system enables fast bacterial fluorescence imaging with high sensitivity. The distribution of fluorescent bacteria in multiple organs was imaged with the CFEM probe ex vivo. The number of viable bacteria was higher in the tumor than in the vital organs. The trends of the fluorescence signal were correlated to the viable bacterial counting results. This bacterial distribution in the mouse tumor model is similar to those in previous reports [39]. Therefore, the fluorescent signal measured by CFEM can be used to monitor the viable bacterial accumulation in the tumor tissue. Longitudinal in vivo imaging with the DSFE system enables further understanding of bacteria-related cancer therapy. However, this study has two weaknesses. Firstly, our CFEM exhibited a green-dotted image without any specific structure of the colonic mucosa. To obtain high-sensitive imaging, we did not use other nonspecific mucosal staining dyes, such as fluorescein or acriflavine [40; 41]. The vascular imaging dye can improve the morphological characterization of the tumor lesion. Further multi-colored fluorescence imaging study with tissue-specific dye will be needed to maximize the usefulness of the DSFE system. Secondly, our WFE exhibited relatively low fluorescence signals on the tumor in comparison with the adjacent normal-looking tissue due to autofluorescence of the tissue (Figure S1). However, the additional CFEM probe exhibited bacteria-specific fluorescence at the tumor site. Figure 5: Ex vivo quantification of green fluorescent protein (GFP) bacteria in a mouse model with CFEM (n = 2). (**a**) Confocal fluorescence imaging of GFP bacteria in various extracted tissues from the orthotopic tumor mouse. (**b**) The quantified fluorescence signal of bacteria from each tissue. (**c**) Viable bacterial counting analysis of each extracted tissue. The bacterial fluorescence signal was much higher when comparing with autofluorescence of the normal mouse organ (Figure S2). This compensatory imaging is an advantage of the DSFE system. Further optimization of the bacteria-specific fluorescence system will improve the quality of the image. In conclusion, although there are some areas to be improved, the developed DSFE system has successfully demonstrated the feasibility of visualizing and quantifying the distribution of bacteria in orthotopic mouse colon cancer. Therefore, the DSFE system is expected to be successfully applied to research on the therapy of colon cancer using bacteria. The following are available online at [[http://www.mdpi.com/2076-3417/10/3/844/s1](http://www.mdpi.com/2076-3417/10/3/844/s1)]([http://www.mdpi.com/2076-3417/10/3/844/s1](http://www.mdpi.com/2076-3417/10/3/844/s1)). Figure S1: In vivo DSFE imaging of normal mouse colon without bacterial injection; Figure S2: Ex vivo autofluorescence image of normal mouse organ without bacterial injection using CFEM. Conceptualization: S.W.Y., C.L., C.L., and J.M. ; methodology: S.W.Y. ; experiment: S.W.Y., D.-h.N., S.P., and H.L. ; data analysis: S.W.Y., D.N., S.P., and C.L.