Detection of Changes in Macrophage Polarization as a Result of 5-Aminolevulinic Acid Photodynamic Therapy Using Fluorescence-Lifetime Imaging Microscopy

Round 1

Reviewer 1 Report

The manuscript titled-Detection of changes in macrophage polarization as a result of 2 5-aminolevulinic acid photodynamic therapy using fluores-3 cence-lifetime imaging microscopy- is reviewed. 

the comments are as follows-

what do you mean by "bed" in line 11 ?

write down full form of NADH in line 13.

author can merge the section 1.1, 1.2 and 1.3 in introduction. 

what is the specification of the laser 561 nm in line 135 ? 

which objective lens used for image acquizition ?

what is y axis in fig 1 ?

The standard deviation in fig. 1 are high. comment on it. 

include the scale bar in fig. 2

what is the rational to choose irradiation power ?

author could plot the FLIM images for free and bound NADH separately in fig 3. 

check the unit of "3.7±0.9 mg/kg " in line 333.

what is the thickness of tissue slice in section 3.3?

Is the photodamage due to irradiation checked for both in vitro and in vivo experiment ?

there are some important articles on FLIM measurement. cite some of the chapters from this book -https://link.springer.com/book/10.1007/978-3-319-14929-5

Author Response

Thank you dear reviewer. We really appreciate all the comments made and tried to answer them.

Answers to Reviewer 1

What do you mean by "bed" in line 11? – We meant «the vascular and stromal tissue that surrounds a cancerous tumor cells». However, for oncologists, this term can also carry a meaning «the tissue that envelopes a tumor site, following the surgical removal of the tumor». To avoid ambiguity "tumor bed" was replaced with “tumor”.

Write down full form of NADH in line 13. – Has been added “reduced form of nicotinamide adenine dinucleotide (NADH)”.

Author can merge the section 1.1, 1.2 and 1.3 in introduction. – Sections are merged.

What is the specification of the laser 561 nm in line 135? – Laser specification has been added: “561 nm 20 mW DPSS laser (LASOS Lasertechnik GmbH, supplied by Zeiss, Germany)”.

Which objective lens used for image acquizition? – “The Plan-Apochromat 63x/1.4 Oil objective (Zeiss, Germany) was used" was added to the section 2.5. FLIM procedure.

What is y axis in fig 1? – Thanks for your attention to detail; these are PpIX concentration units, now corrected to mg/kg.

The standard deviation in fig. 1 are high. Comment on it. – This is a very good note. Thanks for it. Explanation has been added: “It is worth noting the high standard deviation for PpIX accumulation in polarized macrophages. This is due to the large heterogeneity of accumulation across the cell’s population. Despite specific activation by minimal amounts of cytokines, cells within the same subpopulation are very heterogeneous, they can be in a state of high and low activation [40. Muldoon JJ, Chuang Y, Bagheri N, Leonard JN. Macrophages employ quorum licensing to regulate collective activation. Nat Commun. 2020 Feb 13;11(1):878. doi: 10.1038/s41467-020-14547-y.]. For each polarization type, all cells in the field of view at several images were taken into account, and their fluorescence intensity varied greatly.“

Include the scale bar in fig. 2. – Scale bars of 50 µm have been added.

What is the rational to choose irradiation power? – A text was added to the discussion about the effect of irradiation power density on the effectiveness of ALA-PDT for different types of cancer, and specific parameters were added to the conclusions at which M2-polarized macrophages can already die and be replaced by new M0 macrophages:

“Results on macrophage cultures show that moderate 5-ALA-PDT (10 mg/kg 5-ALA, 10–50 J/cm²) biases nonpolarized M0 macrophages toward M1. At the same time, M1 and M2 macrophages die and are replaced by unpolarized cells.

Tumor models in vivo have demonstrated an increase in the proportion of inflammatory macrophages in the tumor node in response to 5-ALA-PDT in the tumor node.

High doses of ALA-PDT (100 mg/kg 5-ALA, 50 J/cm²) that kill tumor cells may not be ideal for the development of an immune response, probably due to the powerful vasoconstriction that prevents immune cells from entering the tumor area.

Low doses of light irradiation (≤ 10 J/cm²) cause diffuse damage and promote an in-creased inflammatory response, but may result in a slower treatment rate.”

However, there is no definitive answer to the question of which irradiation power is rational. It seems that PDT should be several stages: for killing cancer cells and for stimulating M1 macrophages.

Author could plot the FLIM images for free and bound NADH separately in fig 3. – The provided phasor diagrams show the distribution of fluorescent lifetimes in the entire FLIM image. In our work we have observed that plotting independent FLIM images for bound and free NADH is less informative than plotting pseudocolor NADH a1/a2 images (Fig.10) or amplitude-weighed mean fluorescence lifetime images.

Check the unit of "3.7±0.9 mg/kg" in line 333. – Thanks for this comment, but there is no error in units here. It is a fact that PpIX is formed an order of magnitude less than the administered 5-ALA. The phrase in the text has been clarified as: “Four hours after intravenous administration of 5-ALA, the intratissue accumulation of PpIX in the tumor area, as measured by fluorescence spectra, was 3.7±0.9 mg/kg for the administered 100 mg/kg of 5-ALA and 0.4±0.1 for the administered 10 mg/kg of 5-ALA, respectively.”

What is the thickness of tissue slice in section 3.3? – Thank you for your attention, we missed this moment to the description. Added text to the section 2.4: “50 µm thick sections were prepared using a freezing microtome Microm HM 560 Cryostat (Thermo Scientific, USA) and were further processed for FLIM procedure and immunofluorescent staining. For FLIM procedure, the sections were placed in saline under a coverslip and examined immediately.” and to the section 3.3: “50 µm thick sections…”.

Is the photodamage due to irradiation checked for both in vitro and in vivo experiment? – Thanks, that's a good question. We did not directly control photodamage. Nevertheless, a significant cell death at high PDT doses can be judged by a small number of surviving cells in vitro and by apoptosis-like fragmentation of nuclei on sections in vivo. It appears that quantifying photodamage doses is very important for all types of macrophage polarization, with great detail on 5-ALA doses and light doses. This is a subject for a separate study.

There are some important articles on FLIM measurement. Cite some of the chapters from this book -https://link.springer.com/book/10.1007/978-3-319-14929-5 – Thank you; this is indeed a very comprehensive book. The following chapters have been cited in the introduction and discussion:

11. Klaus Suhling, Liisa M. Hirvonen, James A. Levitt, Pei-Hua Chung, Carolyn Tregido, Alix le Marois, Dmitri A. Rusakov, Kaiyu Zheng, Simon Ameer-Beg, Simon Poland, Simon Coelho & Richard Dimble. Fluorescence Lifetime Imaging (FLIM): Basic Concepts and Recent Applications. In Advanced Time-Correlated Single Photon Counting Applications; Becker, W. Eds.; Springer Series in Chemical Physics, Springer; Cham, Switzerland, 2015; Volume 111, pp. 119–188
12. Walsh, A.J.; Shah, A.T.; Sharick, J.T.; Skala, M.C. Fluorescence Lifetime Measurements of NAD(P)H in Live Cells and Tissue. In Advanced Time-Correlated Single Photon Counting Applications; Becker, W. Eds.; Springer Series in Chemical Physics, Springer; Cham, Switzerland, 2015; Volume 111, pp. 435–456.
13. Becker, W.; Shcheslavskiy, V.; Studier, H. TCSPC FLIM with Different Optical Scanning Techniques. In Advanced Time-Correlated Single Photon Counting Applications; Becker, W. Eds.; Springer Series in Chemical Physics, Springer; Cham, Switzerland, 2015; Volume 111, pp. 65–117

Reviewer 2 Report

The manuscript by Ryabova et al. presents an exploration of fluorescence lifetime measurements of metabolic cofactors NADH and FAD and also PpIX to report differences in cellular metabolism of normal tissues, tumour, inflammatory and anti-inflammatory macrophages.

Without considering the scientific merit of this work, in general, I found the results presented in this manuscript to be quite difficult to interpret. I believe there are a few reasons for that: 1) the introduction covers many topics but does not explain how these relate to each other and what is their relevance and how they tie with the study objectives – it does not have a logic flow; 2) the experimental work is poorly structured and described (section 2) and it is challenging to understand exactly what the authors did - I believe this section should be rewritten to clarify the experimental work carried out by the authors; 3) presentation of results lacks clarity; 4) the discussion is vague and is not well tied to the introduction and results presented in section 3; 5) conclusions are also not well tied to the introduction.

With respect to the presentation of results, authors should clearly identify in the text which figures and panels a specific part of the text refers to. For example, line 276, authors write: “For M1 inflammatory macrophages polarized with IFN-γ and LPS, the distribution in the phasor diagram is shifted toward bound NADH with a long fluorescence lifetime. M0 and M2 macrophages polarized with IL-4 and IL-13 show a similar distribution of pixels in the phase diagram shifted toward protein-bound NADH with a fluorescence lifetime of about 2 ns”. I believe this text refers to Fig. 3 top row (control) but this is not totally clear and makes interpretation of the results much more challenging. This repeats in many other parts of the manuscript and thus should be addressed.

One additional point refers to the lack of quantification of fluorescence lifetime data, derived from phasor plots, which makes interpretation of the data far more challenging for the reader, as all interpretation falls in the visual analysis of trends across phasor clouds. Fluorescence lifetime measurements comprises a substantial part of the work and therefore require clear presentation of the data (excitation wavelength for FLIM measurements is also missing). For example, in different parts of the text, the authors mention that the lifetime changes towards a shorter or longer value (for example, lines 349-350: “(…) NADH appears biased toward 4 ns”). But how are these changes quantified? In some cases, these changes are difficult to observe from the data presented (e.g. line 285 “For M2 macrophages, the appearance of a third component in the fluorescence lifetime distribution around 5 ns is observed”.

To conclude, I believe this study is scientifically sound and the results could merit publication in the future. However, considering the issues raised above, I cannot recommend this manuscript as is for publication in Photonics. I strongly recommend to authors to review the structure of the paper and enhance the clarity of the message to be conveyed, presentation of results, and discussion.

 

Other minor comments:

-          Are data from Figure 7 obtained from a single image? If so, how are they representative if there is no measure of variability? Y-axis missing label and units.

-          Is Figure 9 adding relevant information? Would redox ratio give better results?

-          For PpIX fluorescence measurements, the authors refer to fluorescence spectral measurement in line 227-228: “(…) macrophages and tumor cells was assessed by fluorescence spectra.” No spectral data are provided and there is no reference to spectrally-resolved acquisition in the methods.

-          What is the excitation wavelength used for FLIM measurements (NADH, flavins and PpIX)?

-          The authors do not use plot labels consistently, e.g. Figure 1 (LLC) and Figures 3-5 (tumor).

Author Response

Thank you dear reviewer. We really appreciate all the comments made and tried to answer them.

Answers to Reviewer 2

Without considering the scientific merit of this work, in general, I found the results presented in this manuscript to be quite difficult to interpret. I believe there are a few reasons for that:

1) the introduction covers many topics but does not explain how these relate to each other and what is their relevance and how they tie with the study objectives – it does not have a logic flow; - Thank you for this note. Perhaps this is due to the fact that in the introduction we tried to be concise, but we wanted to cover all aspects of the study. Now it's a little more expanded.

2) the experimental work is poorly structured and described (section 2) and it is challenging to understand exactly what the authors did - I believe this section should be rewritten to clarify the experimental work carried out by the authors; - Thanks, we've added more details to section 2 that we missed when submitting the manuscript.

3) presentation of results lacks clarity; - The description of the results has also been written in more detail.

4) the discussion is vague and is not well tied to the introduction and results presented in section 3; - We have tried to improve the discussion section as much as possible.

5) conclusions are also not well tied to the introduction. - We have tried to bring the conclusion into the best correspondence with the abstract.

With respect to the presentation of results, authors should clearly identify in the text which figures and panels a specific part of the text refers to. For example, line 276, authors write: “For M1 inflammatory macrophages polarized with IFN-γ and LPS, the distribution in the phasor diagram is shifted toward bound NADH with a long fluorescence lifetime. M0 and M2 macrophages polarized with IL-4 and IL-13 show a similar distribution of pixels in the phase diagram shifted toward protein-bound NADH with a fluorescence lifetime of about 2 ns”. I believe this text refers to Fig. 3 top row (control) but this is not totally clear and makes interpretation of the results much more challenging. This repeats in many other parts of the manuscript and thus should be addressed. - We have added references to the relevant phasor plots.

One additional point refers to the lack of quantification of fluorescence lifetime data, derived from phasor plots, which makes interpretation of the data far more challenging for the reader, as all interpretation falls in the visual analysis of trends across phasor clouds. Fluorescence lifetime measurements comprises a substantial part of the work and therefore require clear presentation of the data (excitation wavelength for FLIM measurements is also missing). For example, in different parts of the text, the authors mention that the lifetime changes towards a shorter or longer value (for example, lines 349-350: “(…) NADH appears biased toward 4 ns”). But how are these changes quantified? - Thank you very much for this comment. At first, that's exactly what we wanted to do. However, to do decay curve fitting it is necessary to be sure of the exponential components. Under photodynamic exposure, contributions to the NADH fluorescence decay curves appear due to binding to new enzymes, and this greatly complicates the verification of the fit. In addition, it was also not easy to analyze large tables with numbers. So we came to the decision that with 'fit-free' phasor approach analyzing the pixel clouds on the phasor, it would be more optimal to find trends in the metabolism change.

In some cases, these changes are difficult to observe from the data presented (e.g. line 285 “For M2 macrophages, the appearance of a third component in the fluorescence lifetime distribution around 5 ns is observed”. - We have indeed noted a presence of lifetimes close to 5 ns, but the amount of pixels with such lifetimes was less than 1% and they were taken out of consideration when phasor diagrams were constructed with respect to pixel intensity. Sorry for this inconsistency in the text. Now we have removed it.

To conclude, I believe this study is scientifically sound and the results could merit publication in the future. However, considering the issues raised above, I cannot recommend this manuscript as is for publication in Photonics. I strongly recommend to authors to review the structure of the paper and enhance the clarity of the message to be conveyed, presentation of results, and discussion.

Other minor comments:

- Are data from Figure 7 obtained from a single image? If so, how are they representative if there is no measure of variability? Y-axis missing label and units. - Thank you for your comment. In section 2, there is about getting data for Fig. 7: «For each animal in the experimental point, data were averaged over 5 images of 425x425 μm.». Y-axis was added.

- Is Figure 9 adding relevant information? Would redox ratio give better results? - Thank you for this note. We tried building RR as well. But the images are noisier. Perhaps this is because the subcellular localization of the NADH and FAD fluorescence emission does not exactly match, therefore the intensities of their signals vary in different pixels.

- For PpIX fluorescence measurements, the authors refer to fluorescence spectral measurement in line 227-228: “(…) macrophages and tumor cells was assessed by fluorescence spectra.” No spectral data are provided and there is no reference to spectrally-resolved acquisition in the methods. - Yes, the spectral data obtained in the lambda-mode of confocal scanning, which were used to calculate the PpIX fluorescence intensity, are not shown so as not to overload the results with the same type of spectra. Nevertheless in 2.2. section there is a very detailed description: “Spectrally resolved fluorescence images were recorded using a laser scanning confocal inverted microscope LSM-710-NLO (Zeiss, Germany). PpIX fluorescence was excited by a 561 nm 20 mW DPSS laser (LASOS Lasertechnik GmbH, supplied by Zeiss, Germany), and the fluorescence signal was recorded in the 570-750 nm spectral range. To obtain statistically significant results, we performed mathematical processing of the obtained images. Contours of more than 50 cells were distinguished in the images obtained under the same conditions, and the average value of PpIX intensity was obtained for each cell...”

- What is the excitation wavelength used for FLIM measurements (NADH, flavins and PpIX)? – 740 nm, was added.

- The authors do not use plot labels consistently, e.g. Figure 1 (LLC) and Figures 3-5 (tumor). - Sorry for the inconsistency, everything has been corrected now.

Reviewer 3 Report

Ryabova et al. shows how the inner cell metabolic states are determined using FLIM. However, there are some points that can be improved.

Major comments:

1.       Since authors are trying to determine really fast decay process (< 1ns), what is the instrument response function of the detector? What is the approximate band width if it is not symmetric? What is the repetition rate of the laser?

 

2.       In the FLIM section, a representative fitting result is necessary to show the readers how reliable the fitting is, therefore, the quality of the lifetime result.

 

3.       In Figure 2, there is a yellow color labelled region. What does yellow color represent? Also, Line 258, authors said: “are 100...300 J/cm2.” What does this mean?

 

4.       The phasor diagrams in the manuscript are very hard to be understood, although authors cited reference 39. What is the x and y axes of the diagrams? In Figure 5, the data points in row 2 are out of the diagram region. What does this indicate? If you will not go through every figures, you can only put the important figures in the manuscript and put the rest in the supplementary.

Minor comments:

1.       In Figure 1, the y label is PpIX (Mr/Kr), what is the unit?

2.       Figure 2 is missing scale bars.

Author Response

Thank you dear reviewer. We really appreciate all the comments made and tried to answer them.

Answers to Reviewer 3

Major comments:

1. Since authors are trying to determine really fast decay process (< 1ns), what is the instrument response function of the detector? What is the approximate band width if it is not symmetric? What is the repetition rate of the laser? – The following details have been added to the FLIM technique description: FWHM of the TCSPC instrument response function (IRF) was <20 ps (Transit Time Spread, with SPC-150); laser pulse width 140 fs, repetition rate 80 MHz.
2. In the FLIM section, a representative fitting result is necessary to show the readers how reliable the fitting is, therefore, the quality of the lifetime result. – There was added text: “Approximation examples for one M0 macrophages cell are shown in Fig.1. NADH fluorescence lifetime curve fitting is two-component. FAD fluorescence lifetime curve fitting is three-component.” and Figure 1. Fitting results for lifetime of fluorescence in NADH (blue line, a) and in FAD (green line, b) spectral range. Red line is the IRF.
3. In Figure 2, there is a yellow color labelled region. What does yellow color represent? – This is the simultaneous presence of red and green signals on confocal images. Now the describing text is: …macrophage remnants displaying markers characteristic of M1 and M2 types simultaneously, it can be seen as yellow in the images.

Also, Line 258, authors said: “are 100...300 J/cm²”. What does this mean? – The ellipsis denoted the variability of the light dose for ALA-PDT. Thanks to your comment, this value has been clarified: 50–200 J/cm² [42. Kim, M. M., & Darafsheh, A. (2020). Light Sources and Dosimetry Techniques for Photodynamic Therapy. Photochemistry and photobiology, 96(2), 280–294. https://doi.org/10.1111/php.13219; 43. Figueira, J. A., & Veltrini, V. C. (2017). Photodynamic therapy in oral potentially malignant disorders-Critical literature review of existing protocols. Photodiagnosis and photodynamic therapy, 20, 125–129. https://doi.org/10.1016/j.pdpdt.2017.09.007].

4. The phasor diagrams in the manuscript are very hard to be understood, although authors cited reference 39. What is the x and y axes of the diagrams? – Diagram scales have been added to figures 3, 4, 5 along the bottom and left edges so as not to overload the figures. Added explanatory text: “Phasor diagram represents the fluorescence decay curve in terms of phase parameters G (x axis) and S (y axis) – real and imaginary parts of the first element in the Fourier series of the repeating decay signal. The distributions were obtained using kernel-density estimation weighted by pixel intensity.”

In Figure 5, the data points in row 2 are out of the diagram region. What does this indicate? – Explanations have been added to the text: “The semicircle on the phasor diagram represents the values for monoexponential decay. The points inside the semicircle are a superposition of several lifetimes, and the points outside the circle represent subexponential decay possibly due to the long lifetime decay curve overlap.”

If you will not go through every figures, you can only put the important figures in the manuscript and put the rest in the supplementary. – This remark is very true, but phasor plots are used as a method of visualization of the general shift of the metabolic state towards glycolysis or oxidative phosphorylation and have to be considered as series of figures. We thought about the representativeness of these data, and decided that it would be more interesting for the reader to perceive them as a single image of all experiments in each spectral range (NADH, FAD, PpIX).

Minor comments:

1. In Figure 1, the y label is PpIX (Mr/Kr), what is the unit? – Thanks for your attention to detail; these are PpIX concentration units, now corrected to mg/kg.
2. Figure 2 is missing scale bars. – Scale bars of 50 µm have been added.

Reviewer 4 Report

In this manuscript, authors have analyzed the fluorescence lifetime of the metabolic  NADH cofactors and flavins, to differentiate the  cellular metabolism of normal tissue, tumor, inflammatory and anti-inflammatory macrophages. They further studied changes inthe polarization of macrophages obtained from THP-1 monocytes in response to photodynamic  therapy with 5-aminolevulinic acid (ALA-PDT). Authors have nicely shown the phasor analysis plots for the NADH, flavins, and 5-ALA induced protoporphyrin IX (PpIX) fluorescence lifetime helps to determine the change  in metabolism in response to different modes of PDT at the cellular and tissue levels. This content of the manuscript is very interesting and it is generally written well. Authors are advised to amend the manuscript based on the following minor comments

1. Could you provide the decay time of the different cellular components in table ?

2. What does the error bar in Figure 1 and 9 represent? please mention it in the figure captions.

3.To make understand for the broad audience group, authors can mention briefly  couple of sentences about the typical fluorescence scheme in bio-imaging. Fluorescence imaging an inevitable research tool to investigate the different cellular component in a cells, where each cellular components including pigments could be detected and visualized by their intrinsic fluorescence (such as confocal fluorescence microscopy [1-2]  or fluorescence life time imaging (FLIM), which is an  advanced version of confocal fluorescence microscopy, based on differences in the fluorescence decay time. Authors can these statements with suggested references in the revised draft.( https://doi.org/10.1167/iovs.61.13.1 ;  https://doi.org/10.1038/s41598-021-95320-z )

4. Did authors ever noticed the changes in the pigmentary granules in tissues such as melanosomes and lipofuscin using FLIM for all these tissue conditions ?

 

Author Response

Thank you dear reviewer. We really appreciate all the comments made and tried to answer them.

Answer to Reviewer 4

1. Could you provide the decay time of the different cellular components in table? – Indeed, it is very convenient to present this data in a table. Table 1 “Specific fluorescence lifetimes for various cellular metabolites.” has been added to the 3.3 section.

Table 1. Specific fluorescence lifetimes for various cellular metabolites.

Spectral range	Cellular components	Decay time [ns]	References
Blue 410-490 nm	NADH	free NADH - ?1 = 0.4 bound NADH - ?2 = 2.5	[36]
	NAD(P)H	bound NAD(P)H - ?2 = 1.9–5.7	[42, 43, 44, 45, 46]
Green-orange 510-590 nm	FAD	bound FAD - ?1 = 0.25 free FAD - ?2 = 1.4	[36]
	FMN	5.0	[36]
Red 610-670 nm	PpIX	monomer, ?1 = 12.8–17.8 aggregates, ?2 = 3.5–3.9	[47]
	Uro-porphyrin I	1.7	[48]
	Uroporphyrin III	?1 = 8.4 ?2 = 16.5	[49]
	PpIX’ photoproducts	1.5–6, 2.6	[50]

2. What does the error bar in Figure 1 and 9 represent? Please mention it in the figure captions. – Has been added to Fig.2 (ex Fig.1) “Error bars signify the standard deviation”, to Fig.10 (ex Fig.9) “Error bars signify the standard deviation weighed by the fluorescence intensity of pixels”.
3. To make understand for the broad audience group, authors can mention briefly couple of sentences about the typical fluorescence scheme in bio-imaging. Fluorescence imaging an inevitable research tool to investigate the different cellular component in a cells, where each cellular components including pigments could be detected and visualized by their intrinsic fluorescence (such as confocal fluorescence microscopy [9-10] or fluorescence life time imaging (FLIM), which is an advanced version of confocal fluorescence microscopy, based on differences in the fluorescence decay time. Authors can these statements with suggested references in the revised draft. (https://doi.org/10.1167/iovs.61.13.1; https://doi.org/10.1038/s41598-021-95320-z ) – Thanks for that tip. This was indeed missing from our introduction. Text has been added: “Fluorescence imaging is an inevitable research tool for studying various metabolites in cells and tissues, including pigments. These fluorescent metabolites can be detected and visualized using their own fluorescence (for example, using confocal fluorescence microscopy) [9-10] or using FLIM, which is an advanced version of confocal fluorescence microscopy based on fluorescence time decay differences [11].

…

9. Ratheesh K. Meleppat, Kaitryn E. Ronning, Sarah J. Karlen, Karuna K. Kothandath, Marie E. Burns, Edward N. Pugh, Robert J. Zawadzki; In Situ Morphologic and Spectral Characterization of Retinal Pigment Epithelium Organelles in Mice Using Multicolor Confocal Fluorescence Imaging. Invest. Ophthalmol. Vis. Sci.2020;61(13):1. doi: https://doi.org/10.1167/iovs.61.13.1.
10. Meleppat, R.K., Ronning, K.E., Karlen, S.J.; Burns, M.E.; Pugh Jr., E.N.; Zawadzki, R.J. In vivo multimodal retinal imaging of disease-related pigmentary changes in retinal pigment epithelium. Sci Rep11, 16252 (2021). https://doi.org/10.1038/s41598-021-95320-z
11. Klaus Suhling, Liisa M. Hirvonen, James A. Levitt, Pei-Hua Chung, Carolyn Tregido, Alix le Marois, Dmitri A. Rusakov, Kaiyu Zheng, Simon Ameer-Beg, Simon Poland, Simon Coelho & Richard Dimble. Fluorescence Lifetime Imaging (FLIM): Basic Concepts and Recent Applications. In Advanced Time-Correlated Single Photon Counting Applications; Becker, W. Eds.; Springer Series in Chemical Physics, Springer; Cham, Switzerland, 2015; Volume 111, pp. 119–188”
12. Did authors ever noticed the changes in the pigmentary granules in tissues such as melanosomes and lipofuscin using FLIM for all these tissue conditions? – An interesting question. In this study, we used white BALB/c mice, which lack pigmentation. However, in other cases, for example, in a black mouse with transplanted fluorescent glioblastoma (tracked mScarlet), bright granules were seen in the brain during intravital examination. Granules were not present in every cell, but in cells that formed a network characteristic of microglia. As well as in glioblastoma cultures (not stained with fluorescent proteins), bright fluorescent protein aggregates are noted inside and next to the cells. Yes, this can indeed be a problem for the use of FLIM in the clinic, since they give a strong signal in the entire visible spectrum.

The following text has been added to discussion:

“Segmentation of the image in vivo into individual cells with their phenotype identification would greatly help in the therapeutic approaches study. Currently biotissue image fragmentation into individual cells only by the autofluorescence signal seems to be a rather difficult task. However, using a vector approach to determine the metabolic index through the lifetime of NADH fluorescence, it is possible to study complex and heterogeneous fluorescence with subsequent image mapping [38]. In this study, we used white BALB/c mice, which lack pigmentation. However, in other cases difficulties with pigmentation and lipofuscin granules are possible. Using characteristics for specific pigments in non-invasive 'fit-free' phasor approach their contribution to the overall image can be taken into account, especially if apply phasor plot segmentation for different spectral regions [69].”

Round 2

Reviewer 1 Report

The author addressed all the concerns. 

Author Response

Dear reviewer, thank you very much! Your questions and comments have greatly improved the manuscript.

Reviewer 2 Report

I thank the authors for reviewing the manuscript and for their effort in restructuring it, namely the introduction. I believe the manuscript is more clear and easier to understand. The information added to the material & methods section also clarifies the methods and instrumentation used in the experiments.

A few comments/issues:

- text from l.358-361 about phasor plot interpretation must be added earlier in the manuscript, either in the methods or upon the first description of phasor data.

- Table 1. NADH protein-bound lifetime is known to change with binding partner. Therefore, it is misleading to suggest the lifetime of bound NADH is strictly one value. It is more accurate to report a range of values and there are plenty of studies to reference, for both single-photon and two-photon excitation.

- Fluorescence lifetime curves in Fig.1 should be plotted in log scale to facilitate visualization. In linear y-axis it is very challenging to appreciate the differences between the two curves.

- Quality of figure 6 must be improved

Author Response

Dear reviewer, thank you very much for your rating. We have tried to improve our work. Here are the answers to the comments of Round 2:

- text from l.358-361 about phasor plot interpretation must be added earlier in the manuscript, either in the methods or upon the first description of phasor data.  – We agree with your comment. This text and the text about phasor plot was moved from l. 308-311 to Materials and methods l.213-220.

- Table 1. NADH protein-bound lifetime is known to change with binding partner. Therefore, it is misleading to suggest the lifetime of bound NADH is strictly one value. It is more accurate to report a range of values and there are plenty of studies to reference, for both single-photon and two-photon excitation. - Thank you for this comment. We have clarified the values for bound NADH and provided additional references.

- Fluorescence lifetime curves in Fig.1 should be plotted in log scale to facilitate visualization. In linear y-axis it is very challenging to appreciate the differences between the two curves. – We agree with your comment. We have now presented the NADH and FAD fluorescence kinetics with log scale Y-axis.

- Quality of figure 6 must be improved. - Agree with your comment. The quality of figures 4, 5, 6, 7 and 9 have been improved.