Imaging the Morphological Structure of Silk Fibroin Constructs through Fluorescence Energy Transfer and Confocal Microscopy

Round 1

Reviewer 1 Report

This paper presents the optical characteristics of silk fibers. It is an interesting research topic, however the methodology needs to be changed as follows:

1- The fluorescence contours are very confusing. Did you try one excitation of 290 nm or tunable range of excitation? If one excitation only, then you have to show the emission graph and then contours are useless. If you applied tunable excitation spectrum, so why did you focus on 290 nm which has no maximum emission intensity from the contour?

2- The comparison of fluorescence emission between silk films and silk nanofibers is needed to be clarified.

3- Caption of Figure 1 is needed to be revised.

4- Scale bar of SEM is needed to be written.

5-Why did the author stop by 1% PPO?

6- English language has to be clearly revised in the whole manuscript.

7- More literature survey about the applications of optical fluorescence of silk fibers is still needed.

Author Response

This paper presents the optical characteristics of silk fibers. It is an interesting research topic; however the methodology needs to be changed as follows:

1. The fluorescence contours are very confusing. Did you try one excitation of 290 nm or tunable range of excitation? If one excitation only, then you have to show the emission graph and then contours are useless. If you applied tunable excitation spectrum, so why did you focus on 290 nm which has no maximum emission intensity from the contour?

We thank the reviewer for his/her comment. We apologize for the lack of clarity. We exited the samples with different wavelength in the considered range, with a difference in frequency of 10nm between one excitation and the other. This have been clarified in the following part in the materials and methods: “The fluorescence 3D spectra were acquired with a Jasco FP-6300 spectrofluorometer. The bare fibroin film and the films with PPO where excited between 250 nm and 320 nm and the emission was collected between 270 nm and 450 nm. The films samples with the addition of LV where excited between 250 nm and 500 nm and the emission was collected between 300nm and 550nm. The 3D emission spectrum was done by collecting an emission spectrum at a specified excitation wavelength then moving the excitation wavelength of 10 nm and collecting the emission again”. We focus on the excitation at 290nm because it was value in which the autofluorescence of silk fibroin had the maximum emission, we clarify this point by the following sentence: “The combined effect of tryptophane and tyrosine resulted in an absorption that had its maximum at 290nm with an emission centered at 330nm, as reported in Figure 2A in the 3D fluorescence contour plot of pure fibroin”. Then we focused on that wavelength to understand if by exiting at 290nm we were able to suppress the fibroin autofluorescence emission at 330nm shifting it to red by using FRET, this have been clarified in the following sentence: “With the increasing in the percentage of added PPO the emission of fibroin was progressively suppressed while the fluorescence emission of PPO increased to a point in which the emission of fibroin not visible and only the large PPO emission peak was present with two maxima centered at 370nm and 380nm. This effect was better observed by slicing the surfaces at 290nm excitation (red lines in Figure 2A-2D) and plotting the emission (Figure 2E) where the clear effect is the reduction of the Fibroin emission and a contemporary in-crease in the PPO emission”. We were also seeking a second energy transmission to the second fluorophore always exiting the fibroin autofluorescence we wanted to transmit the energy from PPO to LV: “This effect results even more evident by slicing the fluorescence surfaces (Figure 3A-3C) along the excitation at 290nm (red lines). The result is shown in Figure 3E, whit the increment in the LV percentage the emission of PPO increased, and the emission of LV decreased”.

2. The comparison of fluorescence emission between silk films and silk nanofibers is needed to be clarified.

We thank the reviewer for his/her comment we explained the reason between the initial optimization on films in the material and methods, by adding the following part: “The optimization of the fluorophore’s concentrations has been done on silk fibroin films more suitable for the analysis on the spectrofluorometer. The same optimized composition has been then used to produce the fluorescent silk fibroin fibers with the electro-spinning. It should be noticed that the choice of the solvent (Formic Acid, FA, Fisher Scientific) was strategic to ensure the proper viscosity during the electrospinning”.

3. Caption of Figure 1 is needed to be revised.

Following the reviewer suggestion, the caption have been modified as follows: “Chemical structure of (A) Tryptophan, (B) Tyrosine, (C) 2,5-diphenyloxazole (PPO), and (D) Lumogen F Violet. (E) The absorption and emission spectra of the used fluorophores. The emission of PPO widely overlaps with the absorption of LV. This allows, if the distance between the two fluorophores is small enough, to excite the PPO and observe the LV emission through the non-radiative fluorescence energy transfer”.

4. Scale bar of SEM is needed to be written.

As requested from the reviewer, the values on the scale bars have been added.

5. Why did the author stop by 1% PPO?

We thank the reviewer for his/her insightful comment. Silk fibroin is commonly used with cells and biological tissues, and fluorophores as well as other materials developed for different applications than the biological are usually not well tolerated. The concentration of 1% allowed to quench the fibroin intrinsic fluorescence while maintaining a low amount of fluorophore. So, the less material added the better the interaction with biological tissues. This have been clarified in the following point: “The best result was obtained by the addition of 1% of PPO that was the minimum concentration that allowed to completely suppress the fibroin intrinsic fluorescence”.

6. English language has to be clearly revised in the whole manuscript.

We thank the reviewer, following his/her suggestion the manuscript have been completely revised.

7. More literature survey about the applications of optical fluorescence of silk fibers is still needed.

We thank the reviewer for his/her comment. Several references on the topic have been implemented at the end of the Discussion in the following sentence: “The produced fibers might find an application as optical sensor whenever the interaction with biological tissues is required [1,2] as for example the case of wound healing [3,4] or for visualization purposes both In-vitro and In-vivo [5,6]. In addition, the proposed methods could be easily coupled with other addictive manufacturing techniques [7,8] to produce 3D constructs [9]”. And in the conclusion as follows: “The proposed method could be used to produce fluorescent fibers with applications in optical sensing in biological tissues as well as a method to visualized silk constructs whenever other methods are inconvenient”.

Reviewer 2 Report

The authors Alessio Bucciarelli et al in here present a method based on the fluorescence energy transfer (FRET) to suppress the fibroin intrinsic fluorescence moving it to higher wavelength accessible to the confocal microscopy for a direct imaging. Silk fibroin is a well-known biopolymer used in several applications in which the interaction with biological tissue is required. In fact, fibroin is extremely versatile and can be shaped to form several constructs useful in tissue engineering applications. And confocal imaging is usually performed to test the cells behavior on the construct and in this context the fibroin intrinsic fluorescence is regarded as a problem. What’s more, the intrinsic fluorescence is not intense enough to provide useful morphological images. In view of this is a topic of an incessant research interest, I recommend to publish after very careful revision.

1. Compared to preciously published papers such as Kim, Soon Hee, et al. "Precisely printable and biocompatible silk fibroin bioink for digital light processing 3D printing." Nature communications9.1 (2018): 1-14.; Hu, Fan, Naibo Lin, and X. Y. Liu. "Interplay between Light and Functionalized Silk Fibroin and Applications." Iscience23.4 (2020): 101035.; Hadisi, Zhina, et al. "Hyaluronic acid (HA)‐based silk fibroin/zinc oxide core–shell electrospun dressing for burn wound management." Macromolecular bioscience 20.4 (2020): 1900328., the principle and innovation of this paper should be explained more in details.
2. There are many abbreviations in use. Please show the full names before use them.
3. There are many grammar errors which should be revised carefully.
4. In figure 2E, the signal is collected from around 300 nm, so it’s not recommended to show data from 250 nm. And showing a sharp signal in around 300 nm can not give any significant information.
5. The funding information is missing.
6. This part needs to be re-written:

‘Acknowledgments: In this section, you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments).’

Author Response

The authors Alessio Bucciarelli et al in here present a method based on the fluorescence energy transfer (FRET) to suppress the fibroin intrinsic fluorescence moving it to higher wavelength accessible to the confocal microscopy for a direct imaging. Silk fibroin is a well-known biopolymer used in several applications in which the interaction with biological tissue is required. In fact, fibroin is extremely versatile and can be shaped to form several constructs useful in tissue engineering applications. And confocal imaging is usually performed to test the cells behaviour on the construct and in this context the fibroin intrinsic fluorescence is regarded as a problem. What’s more, the intrinsic fluorescence is not intense enough to provide useful morphological images. In view of this is a topic of an incessant research interest, I recommend publishing after very careful revision.

1. Compared to preciously published papers such as Kim, Soon Hee, et al. "Precisely printable and biocompatible silk fibroin bioink for digital light processing 3D printing." Nature communications1 (2018): 1-14.; Hu, Fan, Naibo Lin, and X. Y. Liu. "Interplay between Light and Functionalized Silk Fibroin and Applications." Iscience23.4 (2020): 101035.; Hadisi, Zhina, et al. "Hyaluronic acid (HA)‐based silk fibroin/zinc oxide core–shell electrospun dressing for burn wound management." Macromolecular bioscience20.4 (2020): 1900328., the principle and innovation of this paper should be explained more in details.

We thank the reviewer for his suggestion. The papers reported have been cited in the main text of the manuscript the references numbers are 42, 39 and 37.

2. There are many abbreviations in use. Please show the full names before use them.

We thank the reviewer for his/her comment. We recognized that several abbreviations were not explained. Following the suggestion, the article was accurately revised to ensure that all the abbreviations have been written extensively the first time in the paper.

3. There are many grammar errors which should be revised carefully.

We thank the reviewer; the manuscript has been completely revised to correct the grammatical errors.

4. In figure 2E, the signal is collected from around 300 nm, so it is not recommended to show data from 250 nm. And showing a sharp signal in around 300 nm cannot give any significant information.

We thank the reviewer for his comment the figure has been modified as requested.

5. The funding information is missing.

We thank the reviewer for his/her comment. This project was originally part of my PhD graduation and it was not founded. So, we did not report any founding information.

6. his part needs to be re-written: ‘Acknowledgments: In this section, you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments).’

We thank the reviewer for his/her comment, the paragraph was erased because there was no founding to list.

Reviewer 3 Report

In this work, the authors describe a FRET-based method to improve upon confocal imaging of silk fibroin. They describe how the inclusion of a FRET pair (donor: PPO; acceptor: lumogen F violet (LV)) significantly improves image signal to noise ratios, enabling morphological analysis of silk fibroin fibers. The study is well conceived, and the motivation for the work is clearly articulated.  Scientifically, this work is fine, however there are a number of typographic and grammatical errors that distracted from the quality of the work. I recommend publication, provided the following items are corrected:

Line 16: “to control study” change to “study”

Line 28: insert “fibroin” before “is extremely versatile”

Line 40-43: This sentence is confusing. Needs to be reworded.

Line 75: “two fluorophore” change to “two fluorophores”

Line 104: “Praparation” change to “Preparation”

Line 135: “The bare fibroin film and the films with PPO where between 250 nm and 320 nm” insert “excitation” somewhere in this sentence

Line:158: “fluorophore” change to “fluorophores”

Line 178: “whit” change to “with”

Line 212: “Figure 5E” change to “Figure 4H”

Line 248: “ant” change to “and”

Author Response

In this work, the authors describe a FRET-based method to improve upon confocal imaging of silk fibroin. They describe how the inclusion of a FRET pair (donor: PPO; acceptor: lumogen F violet (LV)) significantly improves image signal to noise ratios, enabling morphological analysis of silk fibroin fibers. The study is well conceived, and the motivation for the work is clearly articulated.  Scientifically, this work is fine, however there are a number of typographic and grammatical errors that distracted from the quality of the work. I recommend publication, provided the following items are corrected:

1. Line 16: “to control study” change to “study”

Line 28: insert “fibroin” before “is extremely versatile”

Line 40-43: This sentence is confusing. Needs to be reworded.

Line 75: “two fluorophore” change to “two fluorophores”

Line 104: “Praparation” change to “Preparation”

Line 135: “The bare fibroin film and the films with PPO where between 250 nm and 320 nm” insert “excitation” somewhere in this sentence

Line:158: “fluorophore” change to “fluorophores”

Line 178: “whit” change to “with”

Line 212: “Figure 5E” change to “Figure 4H”

Line 248: “ant” change to “and”

We thank the reviewer for his/her comment the manuscript have been revised and the errors listed by the reviewer carefully corrected. The sentence of line 40-43 have been corrected as follows: “The fibroin intrinsic fluorescence has been always regarded as problematic because it interfere with the emission of some typical staining for cells imaging [10] and additionally it has a low intensity that does not allow the effective visualization of fibroin morphology”.

Reviewer 4 Report

In the current article, the authors try to explain the structure of silk fibroin using fluorescence energy transfer and confocal imaging. The article is very well written; the quality and English of the article are very good. I think the article can be accepted after some corrections. However, I have a few comments on the article.

1. IF authors provide additional images of fibroin using SEM/TEM/microcomputer tomography to support their results it will be more helpful for readers.
2. Authors should give some of the previous literature surveys about FRET and confocal imaging which discuss structures like fibroin.  
3. The authors should discuss fluorescence energy transfer and confocal microscopy advantages in the second paragraph of the introduction.
4. Authors should analyze the electronic properties of fibroin so that the article will be more appropriate to the journal.
5. Authors should show electronic applications.

Author Response

In the current article, the authors try to explain the structure of silk fibroin using fluorescence energy transfer and confocal imaging. The article is very well written; the quality and English of the article are very good. I think the article can be accepted after some corrections. However, I have a few comments on the article.

1. IF authors provide additional images of fibroin using SEM/TEM/microcomputer tomography to support their results it will be more helpful for readers.

We thank the reviewer for his/her suggestion. A SEM analysis have been performed and integrated inside the manuscript. The following part has been added to materials and methods: “SEM analysis (FE-SEM, Carl-Zeiss Supra 40) have been conducted as validation of the proposed technique. The same sample used for the Confocal analysis has been coated with Pt/Pd by sputtering and attached to a stub with the use of carbon tape.  An image analysis was conducted by ImageJ [11], the intensity profile of 70 fibers on both SEM and Confocal images were evaluated and the diameter profiles extrapolated”. In the results the following part have been added: “As comparison we performed the same measurement on a SEM image (Figure 4H). The distributions, visible in Figure 5 within the descriptive statistic, resulted to be skewed towards the lower dimeter values in both cases and well fitted by a log-normal function. It should be noticed that in this case the standard deviation as well as the interquartile range should be regarded as measures of the distribution width. As can be clearly seen the distribution obtained had a mean value slightly higher that the value obtained by SEM (0.456μm versus 0.310μm) while the standard deviations were almost comparable (0.128μm versus 118μm). This could be attributable to the broadening of the of the diameter due the fluorescence emission and to the fact that using SEM the dept of field was higher, so the image included fibers that were on different planes. However even if the distributions resulted to be different, they largely overlap indicating an agreement of the two methodology. It should be noticed that for skewed distributions, as in our case, the median and the interquartile range (IQR) are the correct statistical descriptor. The median and the corresponding IQR resulted to be 0.426μm and 0.13μm for the confocal distribution, and 0.293μm and 0.12μm for the SEM distribution”. In addition, we reported in Figure 5 the comparison between the two distributions.

2. Authors should give some of the previous literature surveys about FRET and confocal imaging which discuss structures like fibroin.  

We thank the reviewer for his/her suggestion. We added in the introduction a better explanation of both FRET and confocal microscopy. The principle of FRET has been explained in the following sentence: “Fluorescence Resonance Energy Transfer (or Förster Resonance Energy Transfer, FRET) is a physical phenomenon that describe a mechanism of energy transfer between two light sensitive molecules that occurs through a non-radiative dipole-dipole coupling [12]. These two molecules are usually referred as donor and acceptor. FRET occurs throught an electronic phenomenon that involve excitation and relaxation. The donor is the molecule that initially absorbs the energy passing to an excited state, the subsequent relaxation is accompanied to a non-radiative energy transfer that allows the acceptor molecule to move to an excited state that relax throught a florescence emission [12]. This resonance interaction occurs on a higher scale than the interatomic distances, without conversion to thermal energy, and without any molecular collision [12]. The transfer of energy leads to a reduction in the donor’s fluorescence intensity and excited state lifetime, and an increase in the acceptor’s emission intensity”, while the confocal microscopy in the following sentence: “In fact, confocal microscopy offers several advantages compared with OM, including the ability to control depth of field, the reduction of background information away from the focal plane and the possibility to collect serial optical sections from thick specimens eventually reconstructing the 3D morphology. The key principle to the confocal approach is the use of spatial filtering (throught the pinhole) to eliminate out-of-focus light or glare in specimens whose thickness exceeds the immediate plane of focus [13].”

3. The authors should discuss fluorescence energy transfer and confocal microscopy advantages in the second paragraph of the introduction.

We thank the reviewer for his/her comment. To address the request the following sentence have been insert in the introduction: “Among the different techniques reported above, confocal microscopy offers several advantages, including the ability to control depth of field, the reduction of background information away from the focal plane and the possibility to collect serial optical sections from thick specimens eventually reconstructing the 3D morphology. The key principle to the confocal approach is the use of spatial filtering (throught the pinhole) to eliminate out-of-focus light or glare in specimens whose thickness exceeds the immediate plane of focus” to describe the advantages of confocal microscopy, while the following: “The fluorescence intensity is then related to distance with an extremely high sensitivity and FRET can be effectively used as an accurate measurement method in of molecular proximity at angstrom distances (10–100 Å)” describe the advantage in the use of FRET.

4. Authors should analyze the electronic properties of fibroin so that the article will be more appropriate to the journal.

We thank the reviewer for his/her comment. FRET is indeed an electronic phenomenon because it involves the excited state of the valence electron of the donor and in the moving to the normal state the non-radiative energy transmission throught the acceptor. Indeed, optical, and electric properties of materials are strongly related. This point has been clarified explaining the nature of FRET in the following part of the introduction: “FRET occurs throught an electronic phenomenon that involve excitation and relaxation. The donor is the molecule that initially absorbs the energy passing to an excited state the subsequent relaxation is accompanied to a non-radiative energy transfer that allows the acceptor molecule to move to an excited state that relax throught a florescence emission [12]”.

5. Authors should show electronic applications.

We thank the reviewer for his/her comment; we insert a brief paragraph about the possible applications at the end of the discussion. FRET can be effectively used to as sensor at the end of an electronic apparatus or also as method to visualize In-vivo or In-vitro experiments. This have been implemented in the main manuscript by adding the following part: “The produced fibers might find an application as optical sensor whenever the interaction with biological tissues is required [1,2] as for example the case of wound healing [3,4] or for visualization purposes both In-vitro and In-vivo [5,6]. In addition, the proposed methods could be easily coupled with other addictive manufacturing techniques [7,8] to produce 3D constructs [9]”.

Reviewer 5 Report

In this work, entitled"Imaging the morphological structure of silk fibroin constructs through fluorescence energy transfer and confocal imaging" the authors designed a FRET molecular pair to image the Fibrioin in silks, which can be meaningful for the morphorgical observation. In the view of this point, I think this work can be accepted by this journal. However, I have some concernings about this work to help for the improvement of the qualitiy of the manuscript. 

(1) The authors seems not to describe how to prepare for the fluorescent specimen, e.g. specifically chemical labelling or electrostatic abstraction. 

(2) Judging from the main text, the FRET pair are not colvalently binding together, how to make sure the same effeciency of energy transfer between the fluorophores in different area of the silk? 

Author Response

In this work, entitled "Imaging the morphological structure of silk fibroin constructs through fluorescence energy transfer and confocal imaging" the authors designed a FRET molecular pair to image the Fibroin in silks, which can be meaningful for the morphological observation. In the view of this point, I think this work can be accepted by this journal. However, I have some concerns about this work to help for the improvement of the quality of the manuscript. 

1. The authors seem not to describe how to prepare for the fluorescent specimen, e.g., specifically chemical labelling or electrostatic attraction. 

We thank the reviewer for his/her comment, we apologize for the lack of clarity in the manuscript. The samples were prepared by mixing some lyophilized silk fibroin and the fluorophores in a common solvent, Formic Acid. We supposed that the fluorophore resulted to be dispersed into the fibroin matrix and not covalently bonded with it. The mixing of the fluorophore with silk fibroin solution was previously reported in several work in literature [2,14,15]. The interaction commonly reported is due to the presence of the amine group in silk and several side groups that can promote the formation of hydrogen bonding within the considered molecules [16], this also explain the uniformity of the produced films that did not present any visible dishomogeneity. To better clarify this, the following sentence has been added: “We could hypothesize, as previously proved in literature [16], that due to the uniformity of the produced films, hydrogen bonding might be present among the amine groups of silk fibroin and the oxygen present in both fluorophores”.

2. Judging from the main text, the FRET pair are not covalently binding together, how to make sure the same efficiency of energy transfer between the fluorophores in different area of the silk? 

We thank the reviewer for his/her insightful comment. The solution was mixed until both the fluorophore where completely solubilized, and the solution was clear. We obtained a solution, not a dispersion as commonly happens where quantum dots are added. This should ensure the uniformity of the presence of PPO and LV in the outcoming material. The same method has been previously used to produce fluorescent fibroin materials [2,14,15]. We clarify this point with the following sentence: “The fluorophores were mixed until their complete dissolution to ensure their uniform presence among the material prepared [2,14,15]”.  

Reviewer 6 Report

Bucciarelli et al image the fluorescence of 'native' silk fibroin, alongside with its dyed modifications, with  2,5-diphenyloxazole and 75 Lumogen F Violet 570. The advantage of the modified fibroin is its red-shifted fluorescence that stems from the FRET between  the aminoacids of fibroin and the two dyes. The work looks very elegant to me, although, from the point of view of bioengineering, it is the quenching of the scaffold fluorescence and not its promotion is what we are interested in. The paper is reasonably well written and fits the remit and the calibre of the Journal perfectly.

The title needs revision(imaging... through imaging)

The Abstract does not report any results/conclusions but consists of some points that would fit better in the Introduction. Ironically, the last paragraph of the Introduction contains the pieces missing in the Abstract. That paragraph is way too detailed and long to my taste.

Methods>characterization:  please add the details of the imaging device (objective), pinhole/pixel dwell time etc.

Figure 1: please check the legend in the panel E.

Figure 2, Figure 3: look very nice, informative

Figure 4: panel A is not very informative: please think what the message is and amend. What is the red frame for? This needs to be explained. Panels B-E,G are way too dark to see any details, panel H is not very clear either. Panels F and H present the imaging data with the linear resolution of 0.6 um, which is in contrast with the distribution in panel I that boasts the range all the way to 0.2 um. By the way, the latter assumes the linear Nyquist resolution of circa 0.1 um and I am very curious about the optics used to get that.

Figure 5: it would be useful to provide a close-up of the two elements of the structure that generate FRET instead of the presented polymer. 

Author Response

Bucciarelli et al image the fluorescence of 'native' silk fibroin, alongside with its dyed modifications, with 2,5-diphenyloxazole and 75 Lumogen F Violet 570. The advantage of the modified fibroin is its red-shifted fluorescence that stems from the FRET between the amino acids of fibroin and the two dyes. The work looks very elegant to me, although, from the point of view of bioengineering, it is the quenching of the scaffold fluorescence and not its promotion is what we are interested in. The paper is reasonably well written and fits the remit and the calibre of the Journal perfectly.

1. The title needs revision (imaging... through imaging)

Following the reviewer suggestion, the title was modified as follows: “Imaging the morphological structure of silk fibroin constructs through fluorescence energy transfer and confocal microscopy”.

2. The Abstract does not report any results/conclusions but consists of some points that would fit better in the Introduction. Ironically, the last paragraph of the Introduction contains the pieces missing in the Abstract. That paragraph is way too detailed and long to my taste.

We thank the reviewer for his/her comment. The abstract has been modified as follows: “Silk fibroin is a well-known biopolymer used in several applications in which the interaction with biological tissue is required. In fact, fibroin is extremely versatile and can be shaped to form several constructs useful in tissue engineering applications. Confocal imaging is usually performed to test the cells behaviour on the construct and in this context the fibroin intrinsic fluorescence is regarded as a problem. In addition, the intrinsic fluorescence is not intense enough to provide useful morphological images. In fact, to study the constructs morphology other techniques are used (i.e. SEM, Micro-CT). In this work we propose a method based on the fluorescence energy transfer (FRET) to suppress the fibroin intrinsic fluorescence moving it to higher wavelength accessible to the confocal microscopy for a direct imaging. This has been done by creating two FRET couples by dispersing two fluorophores (2,5-diphenyloxazole (PPO) and Lumogen F Violet 570 (LV)) into the fibroin matrix and optimizing their percentage to suppress the fibroin intrinsic fluorescence. With the optimized composition an electro spun mat have been produced and the dimension of the fibers accurately determined by confocal microscopy”.

3. Methods>characterization:  please add the details of the imaging device (objective), pinhole/pixel dwell time etc.

We thank the reviewer for his/her comment. The characterization section relative to confocal acquisition has been modified as follows: “Laser Scanning confocal microscopy was performed on the electrospun networks using a Nikon A1 confocal microscope. A 405 nm wavelength laser was used fluorescence excitation while emission was collected through a 20x, 0.75 N.A. apochromatic objective and a scanning zoom set at 5x to get a total magnification equal to 100x. To reconstruct the emission spectrum, a spectral detector with a 6 nm amplitude channel was used, for a total of 32 parallel channels spanning the range 405-596 nm. To permit spectra comparison, pinhole aperture (12.8 µm), laser intensity (~39 mW), PMT gain (43.1%) and dwell time (1 µs/pixel) were set constants for all the samplings”.

4. Figure 1: please check the legend in the panel E.

We thank the reviewer for his/her comment. The figure caption has been modified as follows: “Chemical structure of (A) Tryptophan, (B) Tyrosine, (C) 2,5-diphenyloxazole (PPO), and (D) Lumogen F Violet. (E) The absorption and emission spectra of the used fluorophores. The emission of PPO widely overlaps with the absorption of LV. This allows, if the distance between the two fluorophores is small enough, to excite the PPO and observe the LV emission throught the non-radiative fluorescence energy transfer”.

5. Figure 2, Figure 3: look very nice, informative.

We thank the reviewer for the nice comment.

6. Figure 4: panel A is not very informative: please think what the message is and amend. What is the red frame for? This needs to be explained. Panels B-E,C are way too dark to see any details, panel H is not very clear either. Panels F and H present the imaging data with the linear resolution of 0.6 um, which is in contrast with the distribution in panel I that boasts the range all the way to 0.2 um. By the way, the latter assumes the linear Nyquist resolution of circa 0.1 um, and I am very curious about the optics used to get that.

We thank the reviewer for his comment. We were not clear about the nature of the first panel, in fact we were able with our confocal to collect the emission spectra divided in channels and for each channel we collected per image per sample. Indeed, we had all the images, but we showed only the one related to the channel in which LV emitted (447-452 nm) and the one related to all the combined channels (from 405 to 596 nm). Panel B and panel C were related to the fibers prepared with the bare silk fibroin that cannot be seen by confocal imaging, the same results were obtained by adding only PPO while the addition of both PPO and LV allowed the effective imaging of the fibers structures (in case of the selection of the proper channel). The caption of Figure 4 was modified as follows: “Maintaining the same condition, the intensity of Fibroin with the addition of the single fluorophore (PPO) and the combination of two fluorophore (PPO+LV) was higher than the emission of the silk fibroin. The channel in which the emission of LV occurred (highlighted with a red rectangle) was chosen to produce the images of the fibers shown in (B) for the bare fibroin sample, (D) for the fibroin with 1% of PPO sample and (F) for the Fibroin with 1% of PPO and 0.01% of LV sample”.  To make clear that only some specific conditions allowed the imaging of the fibers in the caption the following sentence was added: “How can be noticed the structure of the fibers was well resolved only in the case of the addition of both fluorophore and the imaging only the portion of the emission spectra in which the emission of LV occurred”. It should be noticed in panel I that we were able to distinguish and measure fibers which dimensions was between 0.3 and 0.4μm. in particular the smallest fiber that we measured had a diameter of 0.37 μm. The bin size has been chosen to correctly show the lognormal distribution, superimposed to the figure. The explanation of the optical configuration used in the confocal microscope was reported in material and methods, as follows: “Laser Scanning confocal microscopy was performed on the electrospun networks using a Nikon A1 confocal microscope. A 405 nm wavelength laser was used fluorescence excitation while emission was collected through a 20x, 0.75 N.A. apochromatic objective and a scanning zoom set at 5x to get a total magnification equal to 100x. To reconstruct the emission spectrum, a spectral detector with a 6 nm amplitude channel was used, for a total of 32 parallel channels spanning the range 405-596 nm. To permit spectra comparison, pinhole aperture (12.8 µm), laser intensity (about 39 mW), PMT gain (43.1%) and dwell time (1 µs/pixel) were set constants for all the samplings.”

7. Figure 5: it would be useful to provide a close-up of the two elements of the structure that generate FRET instead of the presented polymer. 

We thank the reviewer for his/her comment. Following his/her request, we added a second part on the same image explaining the FRET phenomena among the different molecules that generate it.

Round 2

Reviewer 1 Report

All comments have been well addressed. 

Reviewer 2 Report

The authors Alessio Bucciarelli present interesting data about imaging the morphological structure of silk fibroin constructs through fluorescence energy transfer and confocal microscopy. The authors have carefully reversed their manuscripts and answer the reviewer’s questions. The manuscript is well written, has important scientific value, and should be of great interest to the readers. And the results are well presented and the statistical analysis would help a lot for related readers. Though, there are a couple of other minor issues that need further revision according to the requirement of Guide for Authors. Overall, it is an important study, and should be considered for publication after some minor revision with the help of editor.

Reviewer 4 Report

Article can be acceptable in the present form