Optical Manipulation of Fibroblasts with Femtosecond Pulse and CW Laser

Round 1

Reviewer 1 Report

The paper builds an optical tweezers system that uses 2 laser beams: an 808nm cw laser and a 1030nm pulsed laser. The paper used the optical tweezers system to manipulate Ps microspheres and cells to carry out moving and rotational experiments. The following are the main comments:

1.The paper shows some experimental phenomena, as in Figs. 3-7, but the quantitative content is not enough. The context shown in Figs. 3-6 are not innovative, and Figure 7 manipulates two fibroblasts, which can be used as the innovative point of the paper if no one has ever manipulated this type of cells. It is recommended to measure the interaction force between the two cells or other quantitative measurements.

2. The abstract of the paper mentions that the pulsed laser reduces thermal damage, but damage is produced with bubbles in Figure 5 of the paper. What energy was used in this experiment should be given in the paper. In addition, the paper mentions that the 808nm laser power is 10-20mW, which basically does not cause thermal damage to the cells and PS microspheres according to the existing literature. The reason for using the pulsed laser should be explained in the paper.

3. Lines24-33 of the paper are redundant, and the introduction is not concise.

4. The results in Figure 2 show the capture force in the x, y direction. The paper needs to show how the xyz coordinate system is established. Typically, the laser propagation direction is set as the z-axis. I presumable that the light propagation direction is set as the x-axis in Figure 2.

5. Equation 1, it needs to describe what are E, B, and I in the paper.

6. In Figure 3. (I), is there a connection between the sub-figures abc? Why do the particles form a pentagram structure?

7. In the paper, the optical tweezers manipulate the particles to move at micrometers per second, and my question is that at what laser power is moving with this velocity? What is the maximum velocity of the particles at that laser power?

8. The source of fibroblasts should be given. Whether the study was approved by the ethics committee should be stated in the text.

Author Response

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Reviewer 2 Report

The authors have developed an experimental set-up for optical trapping based on 808 nm CW laser and 1030 nm femtosecond laser. Polystyrene particles and mouse fibroblasts have been trapped and the average moving speed has been measured. The possibility of prolonged trapping of fibroblasts (up to 25 minutes) has been demonstrated as well.

11. Despite the experiments performed, I suppose, that the conclusions made by authors should be proven by additional experiments. For example, based on experiments on fibroblasts manipulation and delaying their attachment to other fibroblasts, the authors concluded that fibroblasts attached to tumour cells could be removed with optical tweezers. I suppose, that it is easier to prevent fibroblast attachment to each other by optical trapping, than to detach adherent fibroblasts from each other only by optical forces. So, this statement is controversial, in my opinion, and should be supported by experimental results. If these experiments have been already performed, could you please add the detailed information about the results obtained.

22. Some details in the Materials and methods Section are missed. For example, the parameters of femtosecond laser source like the frequency rate, the pulse duration etc. should be added. Moreover, details on fibroblast culture and their handling during the experiments should be clarified. It is not clear, what culture medium was used when fibroblasts were optically trapped. What was the temperature of the medium during the experiments? I suppose, that these details are very important when the experiments are performed on living biological specimens. If the special culture medium differed from the deionized water (as for polystyrene particles) was used for fibroblast trapping and velocity measurements, this information should be clearly presented in the paper.

33. As a minor mistake I recommend to clarify the abbreviation “PS” (line 60) at first use.

1The manuscript is required significant editing by a professional English editing service, in my opinion. It is hard to understand the science when there are a lot of grammar and spelling mistakes (like  “different laser source (lines 120-121)”, “tow-particles (line 141)”, “ad shown (line 183)”, “mul-particles group” (line 247)) in the text. The sentences like “At higher power, bubbles were generated on the surface of the microspheres and fibroblasts near the focal point due to the larger thermal gradient force, and subsequently the collapse of the bubbles causing the microspheres and fibroblasts to produce undirected displacements, while the fibroblasts will be damaged (lines 56-60)” should be checked for grammar and rewritten in more clear and direct manner. Some terms like “biological atoms” (line 36) and “cell-biological interaction” (line 39) are hard to understand as well as the sentence “Therefore, the exclusion of fibroblasts is the key to tumor cell culture.” (lines 64-65).

Author Response

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Reviewer 3 Report

The manuscript of Xia Zhang at al. is presenting the experimental configuration of the optical tweezers system operating at two wavelengths (808 nm, 1030 nm) and two modes (808 nm: CW, 1030 nm: pulsed) which was used to manipulate the polystyrene spheres and fibroblast cells. The topic is interesting, and it is in the scope of the Photonics Journal. The use of the femtoseconds lasers in biomedical applications is recently widely investigated due to the limitation of the possible damage and photothermal destruction of cells. The language of the manuscript should be understandable for wide audience of readers. The logical structure of the manuscript and used references are mostly appropriate. However, in my opinion, the manuscript is very superficially written, there are a lot of generalisations, understatements, lack of relevant information, lack of explanation of the conclusions drawn. Therefore, in its current form, it is not suitable for publication. The manuscript should be thoroughly revised.

Major comments:

1) In the introduction, the authors should explain the novelty of their solution, the research carried out in the context of the current state of knowledge. What new developments could their research lead to? The use of femtosecond lasers in optical tweezers to manipulate fibroblasts and other cells is well known and has been reported:

https://doi.org/10.1007/s10103-023-03740-2

https://doi.org/10.1038/nprot.2013.071

https://doi.org/10.1016/j.jphotochemrev.2022.100554

https://doi.org/10.1117/12.2080872

Authors should explain how their research adds to the current state of knowledge in the context of previously published work.

2) Section 2.1: The description of the optical system is only a cursory one. Authors are requested to provide all necessary technical specification about used optical/optoelectronic elements: light sources, lenses, objectives, mirrors, beam splitters (eg, FWHM, powers, focal length, ratio of transmission/reflection coefficients). If the stated that they “line 13: built an optical tweezers experimental platform” its configuration should be described and presented in appropriate manner. At present, the description of this arrangement is residual, if not negligible.

3) The manuscript lacks explicit explanations and references to the results obtained for the given laser power values. The authors quite often use statements such as " large enough” “appropriate” related to the laser energy/power etc. without explicitly stating the values of these quantities. There is no demonstration of the influence of the power values on the manipulations carried out.  The results regarding the impact of beam power are described in a sloppy manner. We only learn about the exact appropriate value of the power in two passages (in the introduction and in the conclusion):

-lines 60-61: “Under 5 mW laser power, the stable manipulation of PS particles and fibroblasts was performed, and the average velocities of manipulating individual PS particle and fibroblast were calculated to be 2.7 μm/s and 2.5 μm/s, respectively.”

-lines 247-250: “Then, we used a 1030 nm femtosecond pulse laser to achieve stable manipulation of PS particles and fibroblasts at a small power under 5 mW, and the average movement speeds is 2.7 μm/s and 2.5 μm/s, 249 respectively.”

but nowhere in the part relating to the results is it explained why exactly this value is appropriate. Which results confirms that this power is sufficient for manipulation and is not causing the cell damage?

4) In my opinion, there is also a lack of thermal imaging studies demonstrating the temperature distribution during the manipulation of a beam with different powers. This is the only clear way to exclude the possibility of cell damage.

5) The manuscript is silent or vague about the specifications of the polystyrene beads used, the cell lines used, how the cells were cultured, what medium they were in during the measurements, how the samples were prepared, etc. (eg. lines 80-81: “A small quantity of particles (Tianjin BaseLine Chrom Tech Research Centre) was taken into deionized water” - What does that even mean? One, two or a handful of PSs have been taken.)

6)  Line 95: Authors are requested to add the information about the version and software producer.

7) Lines 125-126: “When the laser power is appropriate, the device can stably control the motion position of 10-μm-diameter particles by moving  the spot position.” - imprecise statement: what does it mean? what is the appropriate level of laser power?

8) Lines 144-147: The authors state that they carry out the rotation and displacement of the multiparticle group in three-dimensional space for different laser power values (from 10 to 20 mW), which is shown in Fig. 4. However, on this figure there is no indication of the results obtained for specific values of laser power. The authors’ statements are not demonstrated on this figure. Moreover, why were these power values chosen? What was the reasoning behind this?

9) Lines 149-151: It will be valuable to validate this by thermal imaging measurements.

10) Line 179: “it is found that fibroblasts were damaged” – unclear statement. on what grounds did the authors come to this conclusion? How authors confirmed the cells damaged? For what power value did this occur? After all, the use of the femtosecond laser is supposed to reduce the possibility of cell damage.  Why fibroblast cells were round? Have the cells been cultured in a medium that prevents cell adhesion?

11) Line 181: “when the laser energy is large enough” - imprecise statement: i.e. how much should it be?

12) Lines 198-199: “The experiments of fibroblasts capture are also conducted, which had hardly been mentioned in previous studies.” - regarding the works cited above, I would disagree with the authors' statement.

13) Line 201: “at a lower laser power” - imprecise statement:  for which one exactly?

14)  Lines 207-209:  How was this speed determined? It is not described at all in the manuscript.

15) Fig.3- in figure caption there is no explanation about the meaning of a,b,c

Minor comments:

1) Lack of the spaces before citations (eg. line 28: “ tail[4].”)

2) I will be valuable to explain the abbreviation PS before introducing for the first time in line 60.

3) Fig.2a,2b – units of x and y axis are the same?

4) Fig.5 : the figure and its caption should be on the same page.

Author Response

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Round 2

Reviewer 1 Report

Most of my concerns have been addressed. I have only one comment for the manuscript. The velocity in the table 1 has been taken to three decimal places. Please confirm the accuracy of the data in the table.

Author Response

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Reviewer 2 Report

Comments for author File: Comments.pdf

1I  recommend the authors to edit the manuscript by a professional English editing service, as there are a lot of mistakes in the text and overloaded sentences that are hard to understand.

Author Response

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Reviewer 3 Report

I would like to thank the authors for the changes made and the clarifications sent. However, I am somewhat confused by some of the authors' explanations.

In response to question 4, the authors state that "we mentioned in the article that when the power was increased, very obvious cell membrane breakage could be observed in the experiment".

However, in response to question 10, the authors state: "Before our experiment, we have to digest the walled fibroblasts with trypsin so that the cells are no longer walled, and then after a series of operations such as centrifugation, the unwalled cells are placed in the culture medium. 

Firstly, it seems to me that fibroblasts do not have a cell wall but a cell membrane. Only plants, fungi and some bacteria have a cell wall.

Secondly, the manuscript does not describe the process of removing the cell membrane in the "Materials and methods" section. Please describe this stage of sample preparation directly indicating that cell membrane were removed. In this case, is it possible to use the method proposed by the authors to study fibroblast's cells or rather to study the nuclei of these cells?

Thirdly, since the membrane was removed from the cells (and only the nucleus remained), how could the destruction of the cell, manifested by the disruption of the cell membrane, be assessed? Was it the appearance of vesicles in the membrane of the nucleus?

Furthermore, can the authors clarify for which studies on fibroblasts, or indeed on their nuclei, optical tweezers with a femtosecond laser can be used?

Author Response

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Round 3

Reviewer 2 Report

Comments for author File: Comments.pdf

The quality of English language has been improved

Author Response

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Reviewer 3 Report

I would like to thank the authors for clearing up my doubts.  In my opinion, the manuscript in its present form can be accepted for publication.

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