Peer review of: NIR-II bioluminescence for in vivo high contrast imaging and in situ ATP-mediated metastases trac  (2020)

REVIEWER COMMENTS 
Reviewer #1 (Expertise: design of chemical imaging probes, Remarks to the Author):  

This manuscript describes a self-illuminating NIR-II probe by using BRET-FRET-FRET cascade process. 
The authors designed an NIR-II fluorophore (FD1029) based on heptamethine cyanine with 
hydrophilic sulfonate groups. Then they prepared luciferase-modified micelles containing with Cy5, 
Cy7.5, and FD1029 as a self-illuminating NIR-II probe. By adding luciferase substrates, luminescence 
signals were observed at NIR-II regions beyond 1000 nm. The comparative experiment with 
fluorescence imaging revealed that NIR-II bioluminescence imaging has superior signal-to-noise ratio 
because of the reduced background signals. The authors also showed in vivo imaging of blood 
vessels, lymphatics, and tumor metastases with higher imaging contrast.
 

This study first demonstrates the utility of self-illuminating NIR-II probe for in vivo imaging. Owing to 
the absence of external excitation light, the images were obtained with high signal-to noise ratio. 
The enhanced contrast enables high-resolution imaging of blood vessels, lymphatics, and tumors. 
Although there are still challenges in current bioluminescence imaging system, this work would be 
acceptable for publication in Nature Communications after addressing the following concerns. 


The authors rationally explained the reason of less self-aggregation of FD1029 by the large dihedral 
angle in ipsilateral indole groups and the increased hydrophilicity. The experimental results in Figure 
2B-C and Supplementary Figures 1A-D showed the concentration dependency of the absorption and 
fluorescence signals. However, the local concentrations of the dyes in the micelle of NIR-II-BP were 
significantly high (9147 nmol/mL in Supplementary Table 2). I am concerned that the quenching 
effect still occurs in such a high local concentration. I suggest that the authors can measure the 
fluorescence spectra of FD1029 at higher concentration ranges, which would show the aggregation 
effect in a micelle. 

Related to previous comment, the authors should describe how to determine the dye concentrations 
in a micelle (Supplementary Table 2) from spectroscopic data. 

In tumor imaging experiments in Figures 7A and F, the timing of D-luciferin injection should be 
optimized. The bioluminescence images of xenograft tumor (Supplementary Figure 13) show 24 h is 
the best, while the images of peritoneal metastases (Supplementary Figure 16) show 8 h is the best. 
The requirement of the repetitive injection would be one limitation in the current system. The 
authors should mention the cause of the differences (the probe delivery rate by EPR, the probe 
stability in the local environment in vivo, or other effects). 



Reviewer #2 (Expertise: Cancer imaging, probe development, Remarks to the Author): 

This manuscript authored by Lu L et al. reports novel NIR-II bioluminescence molecular probes for in 
vivo imaging. NIR-II imaging is a highly promising technology. It is great to see that the authors 
further expand this field and achieved NIR-II bioluminescence imaging. The study is highly novel and 
interesting. The whole study includes the design of the probes, chemical synthesis, and in vitro and 
in vivo evaluation. The conclusion is well supported by the data presented. The manuscript can be 
accepted after addressing the following minor concerns. 


1). Luciferases are on the surface of the nanoparticle. While Cy5, Cy7.5, and FD-1029 are 
encapsulated insides of the nanoparticles. Short space distance between donor-acceptor molecules 
is usually required for FRET and BRET. It is interesting to see the probes can still produce good NIR-II 
signals even using several bridge molecules. It would be helpful to discuss this. 

2). How’s the stability of the nanoprobes prepared in mouse serum or blood, besides in PBS? 

3). It would be helpful to discuss the potential limitations of the probes designed. 

4). NIR-II imaging has been evaluated in clinical studies and demonstrates advantages over 
traditional NIR-I imaging technique. It would be helpful to cite the relevant work (Nature Biomedical 
Engineering, 2020) to highlight the potential of NIR-II imaging. 

5). The quality of some Figures can be further improved. 

6). For animal imaging results shown in Figures, add the number of mice used in the studies.