Improved Optics for Super-Resolution Time-Lapse Observations of Biological Phenomenon Using Speckle Interferometry

Round 1

Reviewer 1 Report

In this paper, the author proposes a new optical system, which can surpass the diffraction limit and achieve long-term observation of live cell tissues. Based on speckle interferometry, the method achieves super-resolution by analyzing the phase distribution associated with the shape of the object under test. The author conducted a detailed study on this topic. I have the following questions that need to be answered by the author.

1. Figure 1, depicting the optical system of the speckle interferometer, is suggested to be illustrated as a 3D schematic diagram to enhance the readability of the diagram. Additionally, textual annotations should utilize arrows pointing to specific components.

2. The statement regarding 532 nm wavelength lasers typically being solid-state lasers rather than semiconductor lasers needs verification from the author.

3. Could the author provide a clearer SEM image of the diffraction grating in Figure 3 with higher resolution and more cycles?

4. In Figures 8(a) and 8(b), finer stripes can be observed along the tilted direction. The author needs to provide an explanation and description for these stripes.

5. Generally, the super-resolution imaging capability of a system is typically assessed by determining whether two closely spaced points can be well distinguished. In this paper, however, the author characterizes the system's performance by describing stripes with different line widths, which may seem less accurate.

6. The organization of the content in this paper needs improvement as it appears somewhat scattered, with many paragraphs consisting of only one or two sentences.

The English of the article can be modified and improved by a professional.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

This study may be of interest to the broader super-resolution microscopy community, especially those working in the area of live cell imaging. The paper would definitely benefit from a real-life example of some biological object imaging: even though the imaging of model objects proved the resolution beyond the diffraction limit, the static nature of these model object would not allow an unequivocal performance assessment in case when the objects are dynamic (e.g. mitotic cells). I strongly recommend the authors to discuss possible challenges related to the imaging of dynamic systems (in principle, the demonstrated imaging of the out-of-focus objects points toward this discussion).

Very low quality, in some sentences it is almost impossible to figure out the author's train of thoughts. Massive editing of the English language is therefore required.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

In this study, the author applied speckle interferometry to propose a new optical system capable of achieving super-resolution visualization. Additionally, the author experimentally tested this system and discovered that super-resolution can be realized by analyzing the phase distribution, which preserves the shape information of the measured object in the reflected light. Overall, the manuscript is clearly written and well-structured. The only concern is the claimed ability to observe live cultured cells, which, as stated in the title, is not supported in this manuscript.

1. As claimed in the title, this newly developed system can observe cells. However, there is no cell-related data in the manuscript. Therefore, the statement is incorrect. It should be "has the potential to observe cells," or the author should include actual cell data to support the title. The text should be carefully revised throughout the manuscript.

2. What is the temporal resolution of your system? Since live cells are highly dynamic, this could affect the resolution of your observations. What is the resolution when observing cell motion?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The revised manuscript is rigor and suitable for publication. This reviewer has no more questions.