Picosecond Laser Processing of Photosensitive Glass for Generation of Biologically Relevant Microenvironments

Round 1

Reviewer 1 Report

Comments to the Author

1. The authors claim that “The microfludic structures efficiently fabricated by PLAE should provide the flow aspects, 3D characteristics, and possibility of producing functional structures to achieve the biomimetic microenvironments.” Please provide the potential application and how the structure mimics the biomicroenvironment in the study?

2. The femtosecond was mentioned in the abstract; however, there are only the picosecond laser-assisted etching (PLAE) and matrix-assisted pulsed evaporation (MAPLE) technique used in the manuscript. Please reorganize the Abstract.

3. Please provide some information about the MAPLE technique and the graphene oxide nanomaterials used in the introduction section.

4. Please reorganize the text regarding Figures 6 and 7 in manuscript.

5. It is suggested that the authors provide the schematic diagram of Figure 6 to help the reader to follow the fabrication process and magnify the image (for example, a single spot).

6. Please discuss why Phalloidin and Vinculin were chosen as growth markers and the meaning of the co-localization. In Figure 7, please provide a clear image of DAPI stain and scale bars. Why did the authors only calculate the 1 h post-seeding cell number but not after 24 h? The cell number in the image and that in histogram were not consistent. The spot in the histogram is 15, which is not consistent with the figure caption n=10? Please define the “Mander’s coefficient” used in the histogram.

7. The references layout looks inconsistent. Some citations do not provide pages. For example Ref. 3, 9, 10, 12 …

8. Please define abbreviations when they first appear in the article, such as CAD-CAM, Ag, LDW, and Nd:YAG.

Author Response

We are grateful to all reviewers for the observations and very useful comments. The corrections and clarifications allowed us to improve the quality of the paper. We did our best to improve the English of the manuscript text. We have added new Figure 2, we improved Figures 1, 6 (new Figs. 7 and 8) and 7 (new Fig. 9). We have included new Refs 43, 53, 54 and removed old Ref 18, as it was included in previous versions by error.

We provide in the following section a point by point list of answers to all the questions raised by the referees.

Reviewer 1.

1. The authors claim that “The microfludic structures efficiently fabricated by PLAE should provide the flow aspects, 3D characteristics, and possibility of producing functional structures to achieve the biomimetic microenvironments.” Please provide the potential application and how the structure mimics the biomicroenvironment in the study?

Thank you for the observation. We have provided supplementary text in the Abstract (lines 23-27) and Results Section (lines 468-478) about the potential biological relevant applications of such 3D dynamic environments. 

2. The femtosecond was mentioned in the abstract; however, there are only the picosecond laser-assisted etching (PLAE) and matrix-assisted pulsed evaporation (MAPLE) technique used in the manuscript. Please reorganize the Abstract.

We have reorganized the Abstract and eliminated FLAE technique. We also added potential applications in biology.

3. Please provide some information about the MAPLE technique and the graphene oxide nanomaterials used in the introduction section.

We have introduced a short paragraph in the Introduction Section about MAPLE and graphene oxide (page 2, lines 68-80) and a dedicated text in Materials and Methods Section, page 5, lines 197-208.

4. Please reorganize the text regarding Figures 6 and 7 inmanuscript.

We have reorganized Figures 6 and 7. Specifically, we have split the Figure 6 into two Figures (now new Figures 7 and 8) and clarify the aspects in Figure 7 (new Figure 9) by adding explicative text at page 13 and new Figure 9 k.

5. It is suggested that the authors provide the schematic diagram of Figure 6 to help the reader to follow the fabrication processand magnify the image (for example, a single spot).

A schematic representation of MAPLE technique is now introduced in the new version of the manuscript and the figure was enlarged (new Figure 8 a). The MAPLE process was also discussed in the Materials and Methods Section.

6. Please discuss why Phalloidin and Vinculin were chosen as growth markers and the meaning of the co-localization. In Figure 7, please provide a clear image of DAPI stain and scale bars. Why did the authors only calculate the 1 h post-seeding cell number but not after 24 h? The cell number in the image and that in histogram were not consistent. The spot in the histogram is 15, which is not consistent with the figure caption n=10? Please define the “Mander’s coefficient” used in the histogram.

We have discussed on page 13, lines 433-441 and 458-460 why we chose Phalloidin and vinculin as markers and clarify the co-localization aspect. More clear images of DAPI and scale bars have been introduced. As shown in Figure 7 (now new Figure 9, the tests were carried out both after 1 h and 24 h to evidence cell attachment and adhesion, respectively. We have corrected the value of n number in the Figure caption, we are sorry for the mistake. We renamed the “Mander’s coefficient” as co-localization coefficient, since Mander’s coefficient is a measure of the amount or degree of colocalization. It is proportional to the amount of fluorescence of the colocalizing pixels or voxels in each color channel.

7. The references layout looks inconsistent. Some citations do not provide pages. For example Ref. 3, 9, 10, 12 …

Thank you for the observation. All the references were corrected.

8. Please define abbreviations when they first appear in the article, such as CAD-CAM, Ag, LDW, and Nd:YAG.

Thank you for the observation. All abbreviations were explained when introduced for the first time in the manuscript text.

Reviewer 2 Report

Authors in their manuscript applsci-998328-peer-review-v1 titled „Picosecond Laser Processing of Photosensitive Glass for Generation of Biomimetic Microenvironments“, present novel results on the laser assisted chemical etching of glass and its usage for applications of microfluidic channels, structuring single-cell micro-chambers, and fabrication of glass photomasks. The results are novel, well-presented, thus deserves publication. However, several problems are indicated during the first peer-review. Thus, major revision is recommended before publication in the Applied Sciences journal by MDPI.

1. The word biomimetic has been used in the title of the manuscript and used many times in the text of the manuscript. Biomimeticsrefers to the artificial replication of functional structure found in nature to mimic its functional property. For example, the laser‐induced nanostructures were selectively textured on the glass surface in order to mimic the spatial randomness, pillar‐like morphology, as well as the remarkable antireflection properties found on the wings of the glass-wing butterfly, and various Cicada species [DOI: 10.1002/adma.201901123]. However, I could not find what kind of structure was replicated in the paper. I would recommend clarifying this point on remove the “biomimetic” term from the title and text of the manuscript.
2. The laser treatment followed by chemical etching has been used. The laser fluence and peak pulse intensity are crucial physical parameters for the characterization of the irradiation dose. The beam diameter of 4 um has been also declaring inlines 140 and 141. However, no characterization technique is described in the paper. There is a special technique called diameter squared D2 versus pulse energy [DOI: 10.1038/s41598-018-35604-z] for real characterization of spot radius on the sample. This technique can also be applied to transparent materials [DOI: 10.1038/s41598-017-05548-x]. I recommend including the characterization technique of beam size into the paper. It also can be CCD camera, or knife-edge method, or theoretical computation having the focal length, wavelength, and numerical aperture. I also recommend including the fluence [J/cm^2] and peal pulse intensities [TW/cm^2] for easier readability and comparison with scientific literature.
3. In reference [3] volume and page numbers are missing.
4. In reference [37] year and page numbers or volume are missing.

Author Response

We are grateful to all reviewers for the observations and very useful comments. The corrections and clarifications allowed us to improve the quality of the paper. We did our best to improve the English of the manuscript text. We have added new Figure 2, we improved Figures 1, 6 (new Figs. 7 and 8) and 7 (new Fig. 9). We have included new Refs 43, 53, 54 and removed old Ref 18, as it was included in previous versions by error.

We provide in the following section a point by point list of answers to all the questions raised by the referees.

Reviewer 2.

Authors in their manuscript applsci-998328-peer-review-v1 titled „Picosecond Laser Processing of Photosensitive Glass for Generation of Biomimetic Microenvironments“, present novel results on the laser assisted chemical etching of glass and its usage for applications of microfluidic channels, structuring single-cell micro-chambers, and fabrication of glass photomasks. The results are novel, well-presented, thus deserves publication. However, several problems are indicated during the first peer-review. Thus, major revision is recommended before publication in the Applied Sciences journal by MDPI.

1. The word biomimetic has been used in the title of the manuscript and used many times in the text of the manuscript. Biomimetics refers to the artificial replication of functional structure found in nature to mimic its functional property. For example, the laser‐induced nanostructures were selectively textured on the glass surface in order to mimic the spatial randomness, pillar‐like morphology, as well as the remarkable antireflection properties found on the wings of the glass-wing butterfly, and various Cicada species [DOI: 10.1002/adma.201901123]. However, I could not find what kind of structure was replicated in the paper. I would recommend clarifying this point on remove the “biomimetic” term from the title and text of the manuscript.

Indeed, in many cases, the biomimetics is related the artificial replication of functional structure found in nature to mimic its functional property. In our case, we identified technical challenges in several biology study areas and tried to propose some methods and tools to solve them. In particular, present 2D testing platforms can be replaced by new 3D devices. A principal motivation is to replicate the adaptation ability of cells to permanently changing environments. Microfluidic devices can precisely control the flow and delivery of liquids (cell media or other) to 3D materials and biological systems making them responsive to external stimuli in a manner similar with sensing processes that happen in our organism. Thus, we propose different engineered configurations of biocompatible materials in order to find alternative solutions to various biological problems. One can then mimic diffusion processes in true 3D microfluidic platforms or imitate 3D characteristics of surfaces and materials to dimensions similar to single cell sizes. However, since the word “biomimetic” seems rather confusing, we have decided to replace it in most of the manuscript text with “biologically relevant” environment.

1. The laser treatment followed by chemical etching has been used. The laser fluence and peak pulse intensity are crucial physical parameters for the characterization of the irradiation dose. The beam diameter of 4 um has been also declaring inlines 140 and 141. However, no characterization technique is described in the paper. There is a special technique called diameter squared D2 versus pulse energy [DOI: 10.1038/s41598-018-35604-z] for real characterization of spot radius on the sample. This technique can also be applied to transparent materials [DOI: 10.1038/s41598-017-05548-x]. I recommend including the characterization technique of beam size into the paper. It also can be CCD camera, or knife-edge method, or theoretical computation having the focal length, wavelength, and numerical aperture. I also recommend including the fluence [J/cm^2] and peal pulse intensities [TW/cm^2] for easier readability and comparison with scientific literature.

Thank you for suggestion. We have added new information about the laser processing, in particular new text at page 5, lines 182-188. A new Figure was also introduced (new Figure 2). We have also provided theoretical computation for the evaluation of beam diameter, page 4, lines 166-168.

1. In reference [3] volume and page numbers are missing.
2. In reference [37] year and page numbers or volume are missing.

We are grateful to the Reviewer for these observations. All the references were reconsidered.

Reviewer 3 Report

The manuscript presents a study of picosecond laser processing of a photosensitive glass for bio-applications. The manuscript is written logically, convincingly addresses the state-of-the-art and novelty of the presented study which is in using picosecond laser pulses instead of femtosecond ones that secures a higher throughput in laser processing. A number of possible applications have been demonstrated. The manuscript is suitable for Applied Sciences but can be recommended for publishing only after a proper addressing some questions/critical points as summarized below:

(1) The most confusing part of the manuscript is Section 3.4 with Fig. 7. Although the authors seem to intend to show an advantage of the laser-processed surfaces over unprocessed ones, the section addresses inadequately authors' goals. The description of Fig. 7 in the text and in the Figure caption is vague. It might be understood that upper panel on the left represents cell adhesion on cover glass and the bottom panel on the left corresponds to laser-processed Futuran samples. From this viewpoint, laser processing does not look as an advantage. Covering by collagen does not look as related to this study as well as it does not bring a favor for laser-processed surfaces. Thus, it is recommended to rewrite this part with better addressing the advantages (if any) of laser processing for cell/adhesion.  

(2) Figure 7 is related to FLAE but not to PLAE judging by the caption. If so, it is not related to the manuscript scope focused on PLAE.

(3) More detailed data are necessary to be provided when describing laser processing otherwise the manuscript is not self-consistent. The necessary data are pulse duration (comment that it is smaller than 10 ps is not enough as 100 fs is also smaller than 10 ps); laser spot size; evaluated laser fluence; pulse overlap for different scanning speeds.

Author Response

We are grateful to all reviewers for the observations and very useful comments. The corrections and clarifications allowed us to improve the quality of the paper. We did our best to improve the English of the manuscript text. We have added new Figure 2, we improved Figures 1, 6 (new Figs. 7 and 8) and 7 (new Fig. 9). We have included new Refs 43, 53, 54 and removed old Ref 18, as it was included in previous versions by error.

We provide in the following section a point by point list of answers to all the questions raised by the referees.

Reviewer 3.

The manuscript presents a study of picosecond laser processing of a photosensitive glass for bio-applications. The manuscript is written logically, convincingly addresses the state-of-the-art and novelty of the presented study which is in using picosecond laser pulses instead of femtosecond ones that secures a higher throughput in laser processing. A number of possible applications have been demonstrated. The manuscript is suitable for Applied Sciences but can be recommended for publishing only after a proper addressing some questions/critical points as summarized below:

(1) The most confusing part of the manuscript is Section 3.4 with Fig. 7. Although the authors seem to intend to show an advantage of the laser-processed surfaces over unprocessed ones, the section addresses inadequately authors' goals. The description of Fig. 7 in the text and in the Figure caption is vague. It might be understood that upper panel on the left represents cell adhesion on cover glass and the bottom panel on the left corresponds to laser-processed Futuran samples. From this viewpoint, laser processing does not look as an advantage. Covering by collagen does not look as related to this study as well as it does not bring a favor for laser-processed surfaces. Thus, it is recommended to rewrite this part with better addressing the advantages (if any) of laser processing for cell/adhesion.

We thank the Reviewer for the suggestions to clarify the Section 3.4. We have not intended to compare laser-processed surfaces with cover glass, but to show that no significant influence on cell adhesion is visible. To this aim, we chose cover glass as the positive control of experiment. In the present version of the manuscript, we have discussed at page 13, lines 433-441 and 458-460  why we chose specific markers and clarify the co-localization aspect. We have introduced a new Figure (now Figure 9 k) to show that there are no visible changes in co-localization aspect between cover glass and laser processed surface. We have also added clarification of the use of collagen in our studies.

 (2) Figure 7 is related to FLAE but not to PLAE judging by the caption. If so, it is not related to the manuscript scope focused on PLAE.

Thank you for the observation; we have corrected the error in the text.

(3) More detailed data are necessary to be provided when describing laser processing otherwise the manuscript is not self-consistent. The necessary data are pulse duration (comment that it is smaller than 10 ps is not enough as 100 fs is also smaller than 10 ps); laser spot size; evaluated laser fluence; pulse overlap for different scanning speeds.

Most of discussion about comparison between ps and fs laser pulses were carried out in our previous study (ref. 27 in the present manuscript). In the present form of the manuscript, we have added new information about the laser processing, in particular new text at page 5, lines 182-188. Figure 1 was revised. A new Figure was also introduced (new Figure 2). We have also provided theoretical computation for the evaluation of beam diameter, page 4, lines 166-168. We have indicated a more exact value for the laser pulse duration and estimated pulse overlap.

Round 2

Reviewer 1 Report

Comments to the Author

1. The titles of the article and the abstract showed in the system are not the same as those in the new manuscript version.

2. Please improve the quality of the figure images, especially Figure 4, 8c, and 9. The DAPI image of Figure 9 was not clear.

3. Please rewrite the figure capture of Figure 9. The description of Figure 9 k is missing.  

4. Figure 9 describes that the MSC seeding on the PG sample developed by PLAE. Is the Fotouran glass (PG) demonstrated in Figure 2a?

5. Please mention the species of MSCs. Were they derived from mouse or human? 

6. What is the nuclei stained used in the cell experiment? The DAPI images are showed in Figure 9, but only Hoechst stain is listed in the Materials and Methods section? DAPI and Hoechst are different dyes.

7. There was a miss typing in Figure 3 as Figure 2 on page 8, line 306.

8. Please rewrite the result of Figure 2 on page 4, lane 182. The description is not clear, and please mention the final parameters for the glass processing and the reason of choosing such parameters?

9. Please define the abbreviations, such as CCD, VIS, VWR, Pen-Strep, and Nd:YAG.

Author Response

We are grateful to the reviewers for the observations and very useful comments. The supplementary corrections and clarifications allowed us to improve the quality of the paper. We further did our best to improve the English of the manuscript text.

We provide in the following section a point by point list of answers to all the questions raised by the two referees.

Reviewer 1.

We are grateful for your evaluation.

Thank you for the supplementary observations.

1. The titles of the article and the abstract showed in the system are not the same as those in the new manuscript version.

Thank you for the observation. I believe that this is a technical issue that we could not control. We will change the title of the manuscript in the system as soon as we are allowed.

2. Please improve the quality of the figure images, especially Figure 4, 8c, and 9. The DAPI image of Figure 9 was not clear.

All figures will be uploaded separately in the system at the highest possible resolution. We introduced herein in the new revised version Figures with improved quality.

3. Please rewrite the figure capture of Figure 9. The description of Figure 9 k is missing.  

Thank you for the observation. The figure caption was corrected by adding reference to Fig. 9 k.

4. Figure 9 describes that the MSC seeding on the PG sample developed by PLAE. Is the Fotouran glass (PG) demonstrated in Figure 2a?

Yes, the samples we have used for in vitro tests were similar to the processed sample in Figure 2 (actually Figure 3 in the latest revised version). We have added this information in caption of Figure 9.

5. Please mention the species of MSCs. Were they derived from mouse or human? 

We have added the required information in the Materials and Methods Section, lines 222-223.

6. What is the nuclei stained used in the cell experiment? The DAPI images are showed in Figure 9, but only Hoechst stain is listed in the Materials and Methods section? DAPI and Hoechst are different dyes.

Thank you for the observation. In our experiments we generally used either DAPI or Hoechst as they were many trial experiments. We have added this information in Materials and Methods Section, page 6, lines 239-240. We kept the information in Figure 9 as it is the correct one for the respective images.

7. There was a miss typing in Figure 3 as Figure 2 on page 8, line 306.

Thank you for the observation. We have made the correction.

8. Please rewrite the result of Figure 2 on page 4, lane 182. The description is not clear, and please mention the final parameters for the glass processing and the reason of choosing such parameters?

We have rewritten the description on page 4, lines 186-187 and added new information.

9. Please define the abbreviations, such as CCD, VIS, VWR, Pen-Strep, and Nd:YAG.

Thank you for the observation. We have defined all abbreviations.

Reviewer 2 Report

Authors in their manuscript applsci-998328-peer-review-v2 with the updated title „Picosecond Laser Processing of Photosensitive Glass for Generation of Biologically Relevant Microenvironments“, present novel results on the laser-assisted chemical etching of glass and its usage for applications of microfluidic channels, structuring single-cell micro-chambers, and fabrication of glass photomasks. The authors have performed a good job in revising the manuscript according to most of the reviewer’s comments and answered questions in great detail. The manuscript quality increased drastically compared to the first version of the paper. Now the results are novel, well-presented, and deserve publication. Thus, acceptance in its current form is recommended for publication in the Applied Sciences journal by MDPI.

Author Response

We are grateful to the reviewers for the observations and very useful comments. The supplementary corrections and clarifications allowed us to improve the quality of the paper. We further did our best to improve the English of the manuscript text.

We provide in the following section a point by point list of answers to all the questions raised by the two referees.

We are grateful for your evaluation.

We did our best to improve the English of the manuscript text.