Quantitative Phase Dynamics of Cancer Cell Populations Affected by Blue Light

Round 1

Reviewer 1 Report

The paper is well written, methodology is well described and the results are interesting. I think it will advance the field, so I would recommend publishing it.

Author Response

Thank you for review of our article.

Reviewer 2 Report

Review of Appl. Sci. Manuscript 757521
Title: Quantitative phase dynamics of cancer cell populations affected by blue light

Authors: A. Feith et al.

Authors have reported the quantitative phase imaging to monitor the response of cellular morphologic and dynamic parameters to irradiation of blue light (488nm) onto the cancer cell lines and benign one in vitro. Using image analysis methods with data obtained from 24 hour measurements of QPI, the cell motility, dry mass, density, CSD were statistically compared for four cell lines. The comparison for all measured parameters showed inter variability between each cell line.

I guess, this is the first paper to demonstrate the impact of blue light on cancer cell lines using QPI. In a way that authors’ have tried quantitative approach to investigate the blue light induced changes in the cells with multiple QPI derived parameters (cell motility, cell density cell dry mass so on), the paper has originality although it is not new in terms of methodology. Hence, the paper could be accepted provided that their replies to following comments satisfy the criterion of Journal:

1. In abstract, authors’ mentioned “multimodal holographic microscopy”. What kind of multi-modalities were used for this work? I think that you did use only QPI, not with any different imaging modalities such as fluorescence imaging. Please make it clear.
2. In section of 2.4, author’s used commercial holography microscope for QPI imaging. Please describe the experimental set-up using the microscope in detail with a figure of system. What kind of sources used for QPI? The light source used for illumination is a broadband plasma light source. How did authors’ pick up the 488nm light from the light source?
3. For illumination of 488nm light, it is not clear how often the cell lines were exposed for 24-hour-long measurements. It seems to me that the cell lines were exposed with just one shot as Fig.1 and then followed by QPI imaging. Is it right? If it is right, I would be really curious if the only once of light illumination (<48mW/cm²) for 1s is possibly enough to induce such changes in the cells. It is evidenced that many recent studies have exposed the cancer cells in dish to the light that was irradiated onto the cell with a light power of 15~30 mW/cm² and an exposure time of at least few minutes to a half of hour (T. Yoshimoto et al., Ann Gastroenterol. Surg. 2(2), 154, (2018)), (X. B. Zeng et al., Laser Physics 20(6), 1500, (2010)),(P.-S. Oh et al., J. Cellular Physiol. 232(12), 3444, (2017)), (N. Matsumoto et al., Anticancer Research 34(9), 4709, (2014)). Please explain for this reviewer’s curiosity.
4. In Fig.1, what does 485/25 nm in y-axis mean? And I think that the power(W) is Joule(J)/time(s) and thus, J is power(W)*time(s). In that way, all notations for light dose (ex, 48mW/cm²/500ms) in manuscript look wrong.
5. How about cell density, cell dry mass, CDS of the benign cell line PNT1A?

 

Author Response

1. In abstract, authors’ mentioned “multimodal holographic microscopy”. What kind of multi-modalities were used for this work? I think that you did use only QPI, not with any different imaging modalities such as fluorescence imaging. Please make it clear.

The Q-PHASE is coherence controlled holographic microscope equipped with a fluorescence module. In this work, the module was used as a source of blue light as a treatment for observed cell lines.

2. In section of 2.4, author’s used commercial holography microscope for QPI imaging. Please describe the experimental set-up using the microscope in detail with a figure of system. What kind of sources used for QPI? The light source used for illumination is a broadband plasma light source. How did authors’ pick up the 488nm light from the light source?

The optical setup of the microscope was added to the article. See fig.1

As a source of light, a halogen lamp is used (non-coherent light). The 488 nm light waves are emitted by the fluorescence light source of the attached module ("the reason for multimodality").       

3. For illumination of 488nm light, it is not clear how often the cell lines were exposed for 24-hour-long measurements. It seems to me that the cell lines were exposed with just one shot as Fig.1 and then followed by QPI imaging. Is it right? If it is right, I would be really curious if the only once of light illumination (<48mW/cm²) for 1s is possibly enough to induce such changes in the cells. It is evidenced that many recent studies have exposed the cancer cells in dish to the light that was irradiated onto the cell with a light power of 15~30 mW/cm² and an exposure time of at least few minutes to a half of hour (T. Yoshimoto et al., Ann Gastroenterol. Surg. 2(2), 154, (2018)), (X. B. Zeng et al., Laser Physics 20(6), 1500, (2010)),(P.-S. Oh et al., J. Cellular Physiol. 232(12), 3444, (2017)), (N. Matsumoto et al., Anticancer Research 34(9), 4709, (2014)). Please explain for this reviewer’s curiosity.

Observed cell lines were exposed to the illumination of 488 nm light in the same frame rate as they were captured by holographic imaging (3 mins/frame, 481 expositions during 24 h experiment). The reason for such setup is in the required „simulation“ of fluorescence time-lapse observation of the cell population.

4. In Fig.1, what does 485/25 nm in y-axis mean? And I think that the power(W) is Joule(J)/time(s) and thus, J is power(W)*time(s). In that way, all notations for light dose (ex, 48mW/cm²/500ms) in manuscript look wrong.

We appreciate this point, the reviewer is correct, all notations were changed as suggested to „48mW/cm²×500ms“ instead of „/500 ms“

the „485/25 nm“ annotates the wavelength/bandwidth. For clarity, only „485 nm“ was used in the figure legends and bandwidth was discussed in the Methods section: „Cells were irradiated with a 485 nm light with a 25 nm bandwidth“. The y-axis legend of Fig 1 is simplified as follows: „485 nm light power (mW/cm2)“

5. How about cell density, cell dry mass, CDS of the benign cell line PNT1A?

The PNT1A cell line results were not shown because their QPI parameters were not significantly changed during the 24h time-lapse observation. But if you want to see it, you can check it in the attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

Recommendation: Publish after minor revisions noted.

Comments:
In the manuscript entitled “Quantitative Phase Dynamics of Cancer Cell Populations Affected by Blue Light” (applsci-757521), Balvan and authors report an ultra-sensitive biophysical risk assessment of light effect on the different cancer cells. Manuscript is well-written and reports on results of interest to the readership of applied Science.  

Some minor correction needs to be done in the final manuscript as outlined below:

• Authors should explain why they choose only these specific cell lines PC-3, A2780, G361 and G361 for photo-physiology study?
• Authors should include details in each section why each measurement is important in terms of cellular functions and signaling. For example: why cell dry mass analysis is important on specific light does in section 3.3?
• Chemist and biologist commonly use NIR probes for real time and long-term time resolution cell signaling analysis. Did authors try to compare how NIR light (561/633) with the same intensity affect these parameters (cell motility, proliferation, cell mass etc) in comparison to blue light?

Author Response

• Authors should explain why they choose only these specific cell lines PC-3, A2780, G361 and G361 for photo-physiology study?

In this study, we wanted to compare the effect of blue light on the cell lines differing both by morphology (large PC-3 cells in comparison with small A2780 cells) and by their origin (melanoma G361 cells) and transformation state (benign PNT1A vs. malignant PC-3 and A2780).

 

• Authors should include details in each section why each measurement is important in terms of cellular functions and signaling. For example: why cell dry mass analysis is important on specific light does in section 3.3?

Cell dry mass reflects various cellular processes. An increasing cell mass is a typical event for anabolic processes connected with the cell growth, moreover, the steep decrease in cell dry mas sis a sign of cell death (cells are losing their mass during plasma membrane rupture or apoptotic decomposition).

Cell speed is an especially important parameter during various drug-discovery connected experiments because some substances are designed to decrease the movement of cancer cells and inhibit cell migration and bona fide creation of metastases. Here we wanted to demonstrate that doses of blue light can also affect cell speed and so change the results independently on used treatments.

Cell proliferation is important in the same way as cell speed. It is an important parameter for the monitoring of cell population response to some experimental treatment or for assessment of cell-friendly conditions (e. g. biocompatible materials for cell growth).

We decided to not include this information in the results section because they are mentioned in the discussion of the article.

 

• Chemist and biologist commonly use NIR probes for real time and long-term time resolution cell signaling analysis. Did authors try to compare how NIR light (561/633) with the same intensity affect these parameters (cell motility, proliferation, cell mass etc) in comparison to blue light?

Indeed the use for NIR probes is rational for the long-term experiments due to its relatively low phototoxicity. With this in mind, 650 nm light is used in the holographic microscope for the quantitative phase reconstruction and therefore these wavelengths cannot be used for fluorescence acquisitions. We, therefore, aimed to analyze the impact of the most commonly used wavelength – 488 nm.