High-Precision Detection of Cells and Amyloid-β Using Multi-Frame Brightfield Imaging and Quantitative Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors came up with the interesting issue seen in the most of the research works associated with the microscopic instruments, however i feel this manuscript need to be improved further and figures must have less background in order to consider for publication. 

1. In figure-1, the preparation of the slides for imaging seems to be very poor quality, could be due to poor hands in the fixation or quality of the cells looking for ither there will be Aβ aggregates doesn't looks great.
2. in figure-2, before and after corrections wasn't great, only looks better. Might be due to debris associated with cellular or stained materials.
3. in figure-4 also seems to be filled with cellular debris, authors need to provide how many cells fixed or unfixed were used and what are the confluency ratio when seeded for experiment along with hours of the incubation (for example 0.4 × 104 cells per well, might vary depending on the plate seeded). 
4. Authors may need to standardize the experiments with where coating is essential to get clean images (authors coated with 20 μg/mL fibronectin solution), did authors also tried with different concentrations of fibronectin or stick with particular amount?
5. the background issue might be due to overcoating? authors need to verify these issue to have a images with less background before going further. Thanks, and best 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study focuses on the quantitative analysis of interactions between cells and amyloid-β (Aβ) under time-lapse brightfield microscopy. Here are some suggestions for improving the manuscript:

1. The selection of the third-order polynomial background correction needs to be determined by comparing it with first-order, second-order or higher-order polynomials.
2. The preprocessing steps should be compared with existing methods (such as correction based on adaptive histogram equalization).
3. The author divided the Aβ concentration into low (≤5 μM) and high (≥10 μM) levels, but did not provide the experimental reason for the division between 5 μM and 10 μM. Thus, are samples with intermediate concentrations (such as 7.5 μM) applicable for the setting method?
4. The author mentioned that cell segmentation is based on local variance filtering, but it does not compare the segmentation accuracy with the recognized cell segmentation tools (such as Cellpose).
5. Does the current quantitative indicators have limitations by solely focusing on morphological aspects and failing to effectively correlate the substantive relationship between Aβ aggregation and cellular physiological functional changes?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors made necessary changes required to improve the manuscript, with no further changes, I would recommend the article to accept for further steps.

Author Response

Thank you very much for taking the time to review our manuscript. We sincerely appreciate your positive response and acceptance.

Reviewer 2 Report

Comments and Suggestions for Authors

The present manuscript have gained significant improvement after revision. Here are some suggestions for improving the manuscript:

1. Figures 5-9 are missing in the main text.

Author Response

Thank you very much for taking the time to review our manuscript.
We have revised Figures 5–9 in accordance with your comment.