Three Dimensional Lifetime-Multiplex Tomography Based on Time-Gated Capturing of Near-Infrared Fluorescence Images

Round 1

Reviewer 1 Report

The manuscript “Three Dimensional Lifetime-Multiplex Tomography Based on 2 Time-Gated Capturing of Near-Infrared Fluorescence Images” by Umezawa  et al. reports featured applications of NIRF-TGI-CT for multiplex 3D observation of deep tissues in biology.  The study describes optical properties of  NIR fluorophore (NaYF4:Nd3 and Yb3+). The data presented, concurrent analysis and discussion is interesting, and it would be worth to have some real invitro/ invivo imaging analysis.

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1.       Manuscript’s abstract needs to be thoroughly revised and the scope of the study must be properly highlighted.

2.       The “Introduction” section requires revision. The authors need to expand the review of literature that is relevant to their study. And probably most importantly, the aim of the study needs to be properly highlighted and justified. I would suggest that the authors attempt to present the key objectives of their study with regards to what is currently known (i.e. literature), thus highlighting the added value of the paper.

3.       The conclusion “Time-gated NIRF-CT provides a novel choice for multiplex 19 3D observation of deep tissues in biology.”—Do author have any supporting preliminary data or relevant reported literature.

Author Response

Response to Reviewer 1

 

The manuscript “Three Dimensional Lifetime-Multiplex Tomography Based on Time-Gated Capturing of Near-Infrared Fluorescence Images” by Umezawa et al. reports featured applications of NIRF-TGI-CT for multiplex 3D observation of deep tissues in biology.  The study describes optical properties of NIR fluorophore (NaYF₄:Nd³⁺ and Yb³⁺). The data presented, concurrent analysis and discussion is interesting, and it would be worth to have some real in vitro/ in vivo imaging analysis.

➡ Thank you for your kind review and valuable comments. In this paper, we present fluorescence lifetime multiplex tomographic imaging using a conventional near-infrared (NIR) camera. This technique using NIR light is applicable to deep in vivo sample observation, but demonstration on such samples is a subject for future study.

1. Manuscript’s abstract needs to be thoroughly revised and the scope of the study must be properly highlighted.

We described the first sentence of the Abstract as follows to state clearly that the paper demonstrates fluorescence lifetime multiplex tomography using a conventional near-infrared camera.

➡ We report a computed tomography (CT) technique for mapping near-infrared fluorescence (NIRF) lifetime as a multiplex three-dimensional (3D) imaging method using a conventional NIR camera. This method is achieved by using a time-gated system composed of a pulsed laser and an NIR camera synchronized with a rotatable sample stage for NIRF-CT imaging. (The 1^st-2^nd sentences of Abstract)

 

2. The “Introduction” section requires revision. The authors need to expand the review of literature that is relevant to their study. And probably most importantly, the aim of the study needs to be properly highlighted and justified. I would suggest that the authors attempt to present the key objectives of their study with regards to what is currently known (i.e. literature), thus highlighting the added value of the paper.

First, we updated to expand the Reference for review of the research of 3D lifetime imaging and NIR fluorescence tomography techniques. Then, the advantage of the NIRF-TGI-CT reported in this paper is clearly described as below in Discussion section.

 

➡ The most advantage of the NIRF-TGI-CT method shown in this paper is 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.

 

 

3. The conclusion “Time-gated NIRF-CT provides a novel choice for multiplex 3D observation of deep tissues in biology.”—Do author have any supporting preliminary data or relevant reported literature.

➡ Although we do not have such preliminary “time-gated NIRF-CT” data, the availability of such NIR camera for in vivo NIRF-CT (not time-gated) of targets has been reported in mice (Umezawa et al., J. Biophotonics 2020). Combining this with time-gated technique, the NIRF-TGI-CT of this paper is expected to be applicable too in vivo samples. This sentence was inserted in Discussion section of our revised manuscript.

Reviewer 2 Report

1. The authors should revise the introduction to highlight the novelty of this work.

2. More characterization data are suggested to be provided, such as TEM images of nanoparticles.

3. The manuscript should be carefully checked and some mistakes need to be revised.

Author Response

Response to Reviewer 2

 

1. The authors should revise the introduction to highlight the novelty of this work.

In contrast to previous studies that reported fluorescence lifetime depiction on tomographic images using a high-speed camera, this paper reports the depiction of the lifetime contrast of fluorophores by combining a time-gated technique using a conventional NIR camera with fluorescence CT. This novelty is highlighted in the Introduction of our revised manuscript.

➡ A methodology for fluorescence lifetime tomography has also been proposed [15] and experimentally validated by microscopic tomography using FLIM 3D fluorescence lifetime imaging using high-speed cameras [16,17]. (In the end of the 2^nd paragraph of Introduction)

➡ The objective of this study is to develop a lifetime-based multiplex 3D imaging method for NIRF in deep tissues by synchronizing the TGI system using a conventional NIR camera with a rotatable stage for controlling the detection angles. (In the end of Introduction)

 

2. More characterization data are suggested to be provided, such as TEM images of nanoparticles.

In this study, the fluorescent particles were used as composites by embedding in agar gels and TEM images of the particles were not obtained. The fluorescent particles were prepared in size such that their fluorescent properties were not affected by anything other than the host material (NaYF₄ or YPO₄). To clearly state this point, we revised our sentences as below in the 1^st paragraph of Results section.

➡ NIR fluorophores with different fluorescence lifetimes were prepared by doping guest ions into two different hosts with different phonon energies; NaYF₄ and YPO₄, and were used as composites by embedding in agar gel. …. The fluorescent particles were prepared in sizes so that their fluorescence properties are not affected by anything other than the host material (NaYF₄ and YPO₄) [23].

 

3. The manuscript should be carefully checked and some mistakes need to be revised.

Thank you very much for your kind review and comments. We rechecked it and submitted the revised version of our manuscript.

Reviewer 3 Report

The authors present a new work investigating multiplexed fluorescence lifetime tomography using near infrared fluorophores in the NIR-II optical window. Having previously illustrated NIR fluorescence tomography in this window in their 2020 work, the primary contributions are extending this technique to fluorescence lifetime imaging with a time-gated imaging setup and using the proposed sparse sampling slope linearity analysis (SLA) method. While the paper shows promising results, I have some major concerns about the work before suggesting publications. I have listed my concerns below. 

Main concerns:

1. The overall impact of SLA could be better discussed. The SLA method reduces the required image quantity for fluorescence lifetime measurements, but there is little discussed about the benefits of this decision. For example, it is unclear whether the sparser sampling of SLA provides significant speed improvements over more conventional FLIM methods or if SLA enables the use of more cost-effective imaging hardware. Section 3.4 briefly touches on SLA providing speed improvements with only a qualitative discussion. Furthermore, it is unclear why three samples at these particular time points were chosen and what occurs to the accuracy if alternate time points or additional time points were chosen. The reader can infer the arguments behind these choices but a discussion and evidence supporting these design choices to get the most accurate lifetime measurements are lacking. Additional quantification of these factors behind SLA and greater discussion of the work's achievements would better highlight the trade-offs, benefits, and limitations of the proposed approach.

2. A main concern from this manuscript is the evaluation of the method's tomographic recovery. Figure 4 provides qualitative visualizations of the proposed technique's tomographic reconstruction but provides little information regarding the system's resolution and reconstruction error from the known fluorescent structure dimensions. Additional discussion on these aspects would highlight the capabilities and limitations of this system when imaging biological systems.

3. In line with the above comment, this system should be evaluated on more rigorous, biologically realistic samples. In vivo measurements like those of Ref. [25], measurements adding tissue to the agar gel sample like Ref. [22], or phantoms incorporating additional scattering elements (for example, https://doi.org/10.1073/pnas.1014501108) would help illustrate the technique's performance under more challenging conditions. Specifically, showing whether the sparse sampling of SLA causes accuracy degradations in more turbid media and whether spatial resolution of 3D structures is lost would be beneficial for showing the technique's applicability robustness to its proposed use in deep tissue imaging.

Minor concerns:

1. Energy level diagram appears to be Figure 2c, not 2b as listed on line 170 (Page 5, section 3.1, first paragraph)

Author Response

Response to Reviewer 3

The authors present a new work investigating multiplexed fluorescence lifetime tomography using near infrared fluorophores in the NIR-II optical window. Having previously illustrated NIR fluorescence tomography in this window in their 2020 work, the primary contributions are extending this technique to fluorescence lifetime imaging with a time-gated imaging setup and using the proposed sparse sampling slope linearity analysis (SLA) method. While the paper shows promising results, I have some major concerns about the work before suggesting publications. I have listed my concerns below. 

Main concerns:

1. The overall impact of SLA could be better discussed. The SLA method reduces the required image quantity for fluorescence lifetime measurements, but there is little discussed about the benefits of this decision. For example, it is unclear whether the sparser sampling of SLA provides significant speed improvements over more conventional FLIM methods or if SLA enables the use of more cost-effective imaging hardware. Section 3.4 briefly touches on SLA providing speed improvements with only a qualitative discussion. Furthermore, it is unclear why three samples at these particular time points were chosen and what occurs to the accuracy if alternate time points or additional time points were chosen. The reader can infer the arguments behind these choices but a discussion and evidence supporting these design choices to get the most accurate lifetime measurements are lacking. Additional quantification of these factors behind SLA and greater discussion of the work's achievements would better highlight the trade-offs, benefits, and limitations of the proposed approach.

(Response) The achievement of this research is to present a method for tomographic imaging of contrasts of the lifetime of rare-earth phosphors (on the order of hundreds of microseconds) using a conventional near-infrared (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. The time required for imaging (several minutes to several tens of minutes) using the NIRF-TGI-CT method with a conventional camera is not as short as the FLIM method. The limitation in clearly shown in Discussion section of our revised manuscript.
➡The most advantage of the NIR-TGI-CT method shown in this paper is 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 FLIM method, the time-gated NIRF-CT for lifetime-based multiplex 3D imaging technique presented in this study is expected to be applied to the acquisition of information on the deep interior of samples, … (Discussion)

 

(Response) The three particular time points for the SLA method are (1) the time immediately after the complete disappearance of excitation light, (2) a time slightly shorter than the expected fluorescence lifetimes, and (3) a time of 2.5–3 times behind the second time point. Although these were determined approximately based on the definition of fluorescence lifetime, the optimal setting way for the three time points is a subject for future study. This fact and limitation are clearly described in Results section in our revised manuscript. Further, “slope linearity analysis (SLA) method” was replaced to “slope comparison method (SCM)” because the method for determining the delay time range to be used in the lifetime calculation is based on “comparison” rather than “linearity” of the slope.
➡ The three particular time points for the SCM were determined approximately based on the definition of fluorescence lifetime as follows: (1) the time immediately after the complete disappearance of excitation light, (2) a time slightly shorter than the expected fluorescence lifetimes, and (3) a time of 2.5–3 times behind the second time point; while the optimal setting way for the three time points is a subject for future study. (Results; after Fig. 5)

 

2. A main concern from this manuscript is the evaluation of the method’s tomographic recovery. Figure 4 provides qualitative visualizations of the proposed technique’s tomographic reconstruction but provides little information regarding the system's resolution and reconstruction error from the known fluorescent structure dimensions. Additional discussion on these aspects would highlight the capabilities and limitations of this system when imaging biological systems.

(Response) Thanks for your kind review. The reconstruction error of the calculated lifetimes were shown as follows in Results section (3.3).
➡ The calculated lifetimes of YPO₄: Nd³⁺, Yb³⁺ and NaYF₄: Nd³⁺, Yb³⁺ were 296 ± 3 μs and 516 ± 39 μs. Although accuracy of the calculation is still a challenge, the contrast of their fluorescence lifetime was mapped on the cross-sectional images by the NIRF-TGI-CT technique. (Results, 3.3)

 

3. In line with the above comment, this system should be evaluated on more rigorous, biologically realistic samples. In vivo measurements like those of Ref. [25], measurements adding tissue to the agar gel sample like Ref. [22], or phantoms incorporating additional scattering elements (for example, https://doi.org/10.1073/pnas.1014501108) would help illustrate the technique's performance under more challenging conditions. Specifically, showing whether the sparse sampling of SLA causes accuracy degradations in more turbid media and whether spatial resolution of 3D structures is lost would be beneficial for showing the technique's applicability robustness to its proposed use in deep tissue imaging.

(Response) The highlight of this paper is the demonstration of fluorescence CT combined with time-gated technique using a conventional near-infrared (NIR) camera resulting in depicting the lifetime contrast of rare-earth phosphors onto tomographic images. This technique using NIR light can be applied to deep tissue observation of in vivo samples, but demonstration on such samples is a subject for future study. This limitation is clearly described in Discussion section of our revised manuscript as below.

➡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 time-gated technique, the NIRF-TGI-CT of this paper is expected to be applicable too in vivo samples. (Discussion)

 

Minor concerns:

1. Energy level diagram appears to be Figure 2c, not 2b as listed on line 170 (Page 5, section 3.1, first paragraph)

Thank you very much for your comments. The list was corrected in 3.1 of our revised manuscript.

Round 2

Reviewer 3 Report

The authors have provided revisions to their manuscript addressing the majority of my comments. They have provided greater emphasis on this work's focus on NIR fluorophore tomographic imaging to better highlight it's contributions. I do still believe that showing the application of this system to scattering tissues would greatly improve the strength of this work, but the added citation and mention that this technique has not been applied to such samples yet is sufficient. My only remaining concern is that the paper's grammar and sentence structure be checked again by the authors before publication.

Author Response

Thank you very much for your review and comments. We agree the analysis using scattering tissues has not been done and will be the major issue in future studies. This limitation of this paper was additionally described in Discussion section of our revised manuscript. The grammar and structures are also checked again.

• 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. (Discussion; Page 8, Lines 298–301)

23. Chihara, T.; Umezawa, M.; Miyata, K.; Sekiyama, S.; Hosokawa, N.; Okubo, K.; Kamimura, M.; Soga, K. Biological deep temperature imaging with fluorescence lifetime of rare-earth-doped ceramics particles in the second NIR biological window. Sci. Rep. 2019, 9, 12806.

36. Barber, W. C.; et al. Combined fluorescence and X-ray tomography for quantitative in vivo detection of fluorophore. Technol. Cancer Res. Treat. 2010, 9(1), 45-52.