Rapid Autofocus Method Based on LED Oblique Illumination for Metaphase Chromosome Microscopy Imaging System

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

 

Line 153

In illumination system, our platform employs a programmable green LED array, serving as a substitute for conventional Kohler illumination and aperture.

Replace with

In illumination system, our platform employs a programmable green LED array, serving as a substitute for conventional Kohler illumination and aperture.

 

Line 157

The green spectrum is particularly effective in en- hancing the contrast of chromosome samples stained with Giemsa, facilitating clearer imaging.

Replace with

The green spectrum is particularly effective in enhancing the contrast of chromosome samples stained with Giemsa, facilitating clearer imaging.

 

I do not like the format of figure 13. Words should be underneah the bars.

 

Line 527, on LED oblique illumi-nation

Your figure does not indicate oblique illuminaation. I do not believe that the illuminaation is oblique even if you place the the xis of LEDs at an angle with respect to the optical xid of the device.

 

Line 536

Eliminate

To our knowledge, this is the first study of a rapid autofocus method based on LED oblique illumination for automated chromosome imaging system.

 

It is not clear what the authors consider worthy of publication. It is not the illumination system which has aalready been described in other publications. In any case, the MS needs to be shortened and focused on what is new.

 

A. Ortega, “Near IR diodes as illumination sources to remotely detect under-drawings on century-old paintings,” Proc. SPIE 8011, 22nd Congress of the International Commission for Optics: Light for the Development of the World, 801177 (25 October 2011); https://doi.org/10.1117/12.902160

 

A. Ortega, Characterization of narrow-band Near IR diodes arranged in array patterns,” Infrared Remote Sensing and Instrumentation XIX, SPIE Proc. 8154, Bellingham, WA (2011). https://doi.org/10.1117/12.895997

 

 

If it is the control system, it is not described in adequate detail. If it is a complete instrument, it is not really new, just an improvement of the illumination system over the previous model.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This article introduces a rapid autofocusing method that utilizes a sequential activation of two LEDs from opposing angles to obtain the defocus distance through image cross-correlation. Illuminate with multiple LEDs in the array to obtain distinct illumination angles, enabling continuous autofocusing for dual-objective. This rapid autofocus method holds the potential for application in biomedical imaging, and can also be harnessed in high-throughput chromosome scanners utilizing LED array illumination. The manuscript could be published in Journal Photonics after minor reversion. Some questions and comments are referred to the authors in the following:

1) There are some errors in the numbering of the equations in the manuscript, please check for corrections. For example, the formula number in line 313 should be 5. The symbol ‘γ’ in Equation 3 is not defined. 

2) The symbol ‘(a)’ in Figure 2 is too far away from the first above figure. The capitalization of the words in the legend needs to be checked.

3) The manuscript utilizes LED array illumination instead of Kohler illumination, whether the LED lighting is uniform, will it affect the image quality? The authors should discuss this issue in the revised manuscript. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Karyotyping is an essential method for diagnosing genetic disorders. In this study, the authors present a rapid autofocusing technique that utilizes LED oblique illumination combined with image cross-correlation analysis. They applied this technique to a whole-slide microscopy imaging system for metaphase chromosomes. This research provides a good example for developing a next-generation high-throughput chromosome scanner using an LED array. I recommend this manuscript for publication after the authors add a scale bar for each chromosome image.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Changliang Yu et al., Rapid Autofocus Method Based on LED Oblique Illumination for Metaphase Chromosome Microscopy Imaging System

 

Particular comments

 

Line 10

Please shorten the abstract. The first sentence, for example can be eliminated without any loss of information.

 

Line 32

Insert “in”

Most optical microscopes have several objectives. Please do not emphasize the replacement of objective lens.

 

Line 33

“the advent of automated slide scanning and karyotype analysis systems has significantly alleviated the workload of medical professionals”

The workload is the same; it just takes a lot less of time.

 

Line 49

“The z-axis scanning and image quality evaluation technique, characterized by its straightforward technical principle, are readily integrated into conventional automated microscopes and is prevalent in autofocusing applications.”

Use the verb in plural form for better comprehension by readers.

 

Line 44, paragraph

It would be helpful if the ideas summarized in this paragraph were organized into several paragraphs.

 

Line 74

“as previously discussed,”

Please eliminate.

Also separate paragraphs should be used to organize the ideas.

 

Line 97

“imaging”

Replace with “image”

 

Line 119

“…system, distinguished…”

Replace the coma with a period. Start a new sentence.

 

Line 442

“…calculation, which is applied…”

Eliminate “which is”.

 

Line 448

“…the system can illuminate multiple LEDs…”

It is the LEDs that do the illumination. Please rephrase.

 

Line 450

“…process diverges from…”

Use “differs” / “is distinguished” if you like long words.

 

General Comments

 

This paper has been significantly improved. Publish implementing those corrections that you find useful.

 

Recommendation

 

Publish with some modifications.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I have no additional comments to your work. I accept the draft in the current form. Thank you for submitting your interesting research.

Reviewer 2 Report

Comments and Suggestions for Authors

Yu et al., Rapid autofocus…

 

Specific 

 

Line 43. Eliminate “lens”

Line 46. Replace “facilitate” with “accomplish”

Line 53. Replace “is” with “are”

Line 58. Is sentence starting with “current” carrying any information?

Line 59. Is sentence starting with “QUI” carrying any information?

Line 65. Replace “It was” with “Yilmaz” if this is meant.

Line 66. …”most optimal”…. Optimal is already superlative.

Line 66. …” optimal”…. By what criteria / ion?

Line 66. I do not understand what this sentence wants to say: “The focus value was determined by comparing the total pixel count to the count of white pixels, with the maximum focus value pinpointing the focus position [14].” Please kindly rephrase.

Line 80. By “this issue” you mean “slow focusing”? If yeas, please note that slow focusing is not an issue by itself.

Line 82. Multitude in “In recent years, a multitude of rapid autofocus methods based on real-time imaging have emerged.” requires at least five references.

Line 89. There is repetition in sentence, “FPM enhances imaging resolution through the use of oblique LED array illumination and sophisticated reconstruction algorithms, also harnessing the benefits of oblique illumination for autofocusing applications,” leading to much confusion in this reader’s mind.

Line 112. Please avoid using several nouns in a raw, as in “autofocus method research”. It is confusing in English.

Line 172. Explain “appearance dimensions”.

Line 184. Mixing of terminology in the same sentence is unacceptable.

Line 244. “Due to the limitations of optical imaging resolution, we have observed”. From where is this observation?

Line 244. The sentence “…the calculation results become unreliable or even erroneous…” is strange:

(i) Authors want to say: the results of calculations…

(ii) Authors are trying to say, “the method is not applicable…” and explain why.

Lines 255-259 do not really explain why authors’ method fails, they just note that the method has limited applicability. You may want to consult Born& Wolf, Principles of Optics, chapter on diffraction images and the recent papers of Yaujen Wang in Photonics about the effects of diffraction and the pixel size on the image quality.

Line 263. “The gradient of the focus curve”. Is it gradient, if it is only a curve in 1D? What is the focus curve, the graph on Figure 3c? 

Lines 297-310. Very confusing. Paragraph needs work, or authors need to study the reference above.

Lines 530 and beyond. This belongs at the beginning and should be presented as requirements.

 

 

Global

• What is the main question addressed by the research?

 

The authors fail to concisely describe what problem they are solving. 

The authors introduce an automated imaging system for metaphase chromosomes, distinguished from conventional microscopes by its employment of LED array illumination in lieu of Kohler illumination. they propose a rapid autofocus method based on symmetric LED oblique illumination and image cross-correlation calculation, enabling the determination of defocus distance and direction for each FOV with only two cycles of image acquisition. The authors conducted a thorough investigation into the interplay among the imaging system's depth of field (DOF), the focusable range, and the focusing error.

 

• Do you consider the topic original or relevant to the field? Does it address a specific gap in the field? Please also explain why this is/ is not the case. 

 

Authors contribute significantly to a field that is already established. They say that they are improving it. Their theoretical explanation is rather weak.

 

• What does it add to the subject area compared with other published material?

 

Authors share a worthwhile experience of one design team. 

• What specific improvements should the authors consider regarding the 
methodology? What further controls should be considered?

 

They should study the current publications on pixel size and F/# (NA) of an optical system and build on the established knowledge. 

 

• Are the conclusions consistent with the evidence and arguments presented 
and do they address the main question posed? Please also explain why this 
is/is not the case.

 

Authors summarize what they did in the MS.

 

• Are the references appropriate?

 

Too much about the biological technique, too little about imaging.

 

• Any additional comments on the tables and figures.

 

Each figure and its caption must be understood without referring to the text, in my opinion.