Pigment analysis based on a line-scanning fluorescence hyperspectral imaging microscope combined with multivariate curve resolution

Lijin Lian; Xuejuan Hu; Zhenhong Huang; Liang Hu; Lu Xu

doi:10.1371/journal.pone.0254864

Peer Review History

Original SubmissionApril 23, 2021
22 May 2021 Decision Letter - Xiaowei Zhang, Editor PONE-D-21-07134 Pigment analysis based on a line-scanning fluorescence hyperspectral imaging microscope combined with multivariate curve resolution PLOS ONE Dear Dr. Hu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 06 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The research was funded by Natural Science Foundation of Guangdong Province (2017A030310568) and Science, Technology and Innovation Commission of Shenzhen Municipality (JCYJ20180301170959233) We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 1 and 2 in your text; if accepted, production will need this reference to link the reader to the Table. 6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this manuscript, the authors aim to algae detection that use hyperspectral fluorescence imaging to capture images and detect specific fluorescence signatures. Overall, the study provides a new approach to detect the algal pigment. This manuscript can be considered for publication after proper revisions. 1. “Another approach is to capture images based on specific fluorescence signatures [3]. That means that the value of fluorescent peaks is considered as the gold standard for pigment analysis” (lines 33 and 34). What does the gold standard mean? and is it another approach HSI? 2 The author argued that ‘HSI is easy to implement, fast and reliable (line 68). Why? 3. Sometimes, the authors use the abbreviation without definition. 4. In line 189 ‘Spectroscopy contains two gratings with a blaze wavelength of 600 nm and grooves of 300 mm-1 and 1200 mm-1’, the unit of grating is not correct. Reviewer #2: In this manuscript, the authors carry on the analysis in the algae based on fluorescent hyperspectral imaging microscope. The results realize the visualization of the microalgae pigment obtained from reconstruction image and demonstrates the feasibility of applying the method to analyze the algae. The results are interesting and reliable. In my opinion, the manuscript can be accepted for publication in PLOS ONE after some minor points are addressed. 1. the line 176 ‘For a very low algae concentration, the fluorescence intensity was linearly related to the concentration of the fluorescent substance’ is the basic principle to calculate the content of pigments. However, the value of low algae concentration could not be found from the whole manuscript, and it should be given in revised manuscript. 2. The description and presentation of results aren’t clear. For example, ‘For this work, hyperspectral imaging combined with the MCR algorithm simultaneously got the spectral features and pigment location. The spectral features of the pigment components are consistent with the fluorescence excitation spectrum in the verification experiment.’ It is also not clear for the data of verification experiment. Please make description in detail. (lines 338 and 339). 3. Some expressions should be refined by native English speakers. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0254864.r001
Revision 1
17 Jun 2021 Author Response Response to Reviewers1: Reviewer #1: 1. “Another approach is to capture images based on specific fluorescence signatures [3]. That means that the value of fluorescent peaks is considered as the gold standard for pigment analysis” (lines 33 and 34). What does the gold standard mean? and is it another approach HSI? Answer Q1: Another approach in this sentence(lines 33 and 34) is exactly the specialized microscope. The manuscript presents the hyperspectral imaging microscope based on the line-scanning method. The method acquires the spatial and spectral information and reconstructs the image according to the fluorescent feature. Therefore another approach in the paper is based on the fluorescent feature to catch the images of samples. 2 The author argued that ‘HSI is easy to implement, fast and reliable (line 68). Why? Answer Q2: Hyperspectral imaging microscope is a method with the advantages of acquiring spatial and spectral information simultaneously. The system contains two parts: the hardware and the software. We programmed the user interface to control the movement of stage and capture of images. It is concise and easily operated. As soon as just pressing the button, we acquire the hyperspectral images of samples. Moreover, the system is based on the line scanning, that is, scanning the sample each time we can get spectral information of a whole line. It costs time shorter than the point-scanning method. 3. Sometimes, the authors use the abbreviation without definition. Answer Q3: From the whole manuscript, I found the lines 60 and 99 has a abbreviation without definition. The GFP is the abbreviation of green fluorescent protein; the MCR-ALS algorithm is the abbreviation of multivariate curve resolution - alternating least squires. The MCR-ALS algorithm develops based on the MCR algorithm. 4. In line 189 ‘Spectroscopy contains two gratings with a blaze wavelength of 600 nm and grooves of 300 mm-1 and 1200 mm-1’, the unit of grating is not correct. Answer Q4: The unit of grating isn't mm-1 but lpmm, which is the abbreviation of line pairs per mini-meter. It means that the grating has two gratings. One is 300 line pairs per mini-meter in the surface. Anther is 1200 line pairs per mini-meter. Response to Reviewers2: Reviewer #2: 1.the line 176 ‘For a very low algae concentration, the fluorescence intensity was linearly related to the concentration of the fluorescent substance’ is the basic principle to calculate the content of pigments. However, the value of low algae concentration could not be found from the whole manuscript, and it should be given in revised manuscript. Answer Q1: Before the experiment, we diluted the samples with the original solution to culture medium at a ratio of 1: 1000 in the sample preparation. The sample has more than the original concentration of 10e6 cells per milliliter. Moreover, in order to verify the limited numbers of microalgal cells we can catch the images of algae under different concentration. It concludes that the detection limited is more than two cells has enough intensity that could be detected. The image in S3 figure is full of cells, which isn’t classified for several cells. What‘s more, we catch the image without overlapping cells that can be distinguished. 2. The description and presentation of results aren’t clear. For example, ‘For this work, hyperspectral imaging combined with the MCR algorithm simultaneously got the spectral features and pigment location. The spectral features of the pigment components are consistent with the fluorescence excitation spectrum in the verification experiment.’ It is also not clear for the data of verification experiment. Please make description in detail. (lines 338 and 339). Answer to Q2: In the verification experiment, we have got the data of the absorption spectrum and the emission spectrum using 95% alcohol to extracting the pigment in algae. Emission measurement excited at 512 nm (ex: 512 nm) after pigment extraction. The peaks of emission spectrum are at 671 nm, 587 nm, 492 nm and 438 nm(figure 8), which is consistent with the peaks of 675nm and sub-peak ranges from 470nm to 574nm respectively in the hyperspectral images(Fig S2). The fluorescent excitation spectrum of Chlorella sp. indicates that the pigment composition is Chl-a (671 nm) and carotenoid (492 nm). Therefore，the spectral feature of the pigment components are consistent with the fluorescence excitation spectrum. 3. Some expressions should be refined by native English speakers. Answer to Q3: The manuscript is revised through native speaker in spelling, English-grammar and language usage. I employed the professional scientific editing service to check the the text. Thanks for your good comments. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0254864.r002
6 Jul 2021 Decision Letter - Xiaowei Zhang, Editor Pigment analysis based on a line-scanning fluorescence hyperspectral imaging microscope combined with multivariate curve resolution PONE-D-21-07134R1 Dear Dr. Hu, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Xiaowei Zhang, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): The author's responses to the reviewer's queries are satisfactory. Now the revised version improves well with the better scientific discussions. I consider the manuscript is suitable for publication Reviewers' comments: https://doi.org/10.1371/journal.pone.0254864.r003
Formally Accepted
30 Jul 2021 Acceptance Letter - Xiaowei Zhang, Editor PONE-D-21-07134R1 Pigment analysis based on a line-scanning fluorescence hyperspectral imaging microscope combined with multivariate curve resolution Dear Dr. Hu: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Xiaowei Zhang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0254864.r004

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