Far-Field Super-Resolution Optical Microscopy for Nanostructures in a Reflective Substrate

Round 1

Reviewer 1 Report

This paper focus on the effects of reflective substrate on resolution of a coherent illumination far-field super-resolution optical microscopy system, and the FWHM of PSF is served as a measurable quantity for the spatial resolution. I would like the authors to comment on some issues

1. In section 4.2, line 260, why the the gaps of 80 nm and 200 nm are selected to analyzed? Or the value of the gaps has great influence on the results?

2. In section 5, the key components for experiment set-up should be stated, including the type or parameters.

3. If the material of substrate, and the distance between the nanoparticles and the substrate have influences on the results?

4. In the introduction, the author has stated the FWHM of PSF is served as a measurable quantity for the spatial resolution (line 34-35), that means the nanoparticles next to each other can be distinguished is associated with FWHM. But in the section 5 has not discussed, and the relevant conclusions should be stated.

5. If the polarization state of the illumination light emitted by the laser has influence on the PSF?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this paper the authors present theoretical findings on the effect of a reflective substrate-assisted interference phenomenon, in confocal microscopy, that enhances optical resolution. The experimental results show that the technique works for the differentiation of not densely packed individual nanoparticles. However, when nanoparticles are positioned in close proximity to each other they appear unresolved in the experimental images.

The paper is well written and the topic, description and findings are of great interest for the microscopy research community. Therefore, I would recommend its publication in its present form.

Author Response

Thank you very much for taking the time to review this manuscript and giving the positive comments.

Reviewer 3 Report

The authors investigate how background light from objects can impact the resolution of an imaging system using the Point Spread Function (PSF). They have developed a model for a confocal microscope and demonstrated both theoretically and experimentally that interference between the background and scattered fields can potentially enhance spatial resolution, particularly evident in nanoparticles. While their study exhibits promising results for isolated particles, it falls short when densely distributed nanoscale particles are examined. The authors provide a detailed analysis of the spatial separation between particles and the reflective surface generating the background field. Overall, the manuscript is well-written, interesting, and the conclusions are well-supported by the obtained results. However, there are some minor issues that need addressing before publication.

Throughout the text, the authors consistently refer to their methodology as a "super-resolution technique." I would suggest that they adjust their terminology to reflect the preliminary nature of their findings and acknowledge the challenges that remain unresolved. For example, the authors themselves note instances where densely clustered particles remain unresolved ("However, when nanoparticles are positioned in close proximity to each other, such as the two particles arranged vertically in subgraph (a3) and the three particles clustered in images (a4), (a5), and (a6), they manifest as an unresolved spot in the experimental images"), contradicting the claims made in the abstract. Similarly, the limitations of their technique should be acknowledged, especially when dealing with more complex samples.

It is unclear from the model whether the authors strictly adhere to the setup depicted in Figure 1. A point scanning microscopy system typically operates with a de-scanning configuration, implying that the detected light passes through Lens 2, the Mask, and the Polarization Converter (PC) twice. The authors should clarify whether their model accounts for this double passage. This is specially important for the Mask. Alternatively, they should accurately describe their experimental setup and clarify if the Mask and PC are positioned outside the de-scanned path. Additionally, the absence of scanning mirrors for directing the beam in the sample plane raises questions about whether normal incidence is always assumed and how scanning would impact the results.

Lastly, while the manuscript mentions iSCAT, the authors could enhance the discussion by introducing other fluorescence-based super-resolution techniques and comparing their pros and cons against their proposed method. This would provide a broader context for understanding the significance of their findings within the field of super-resolution imaging.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed my comments properly. Now I would like to recommend its publication on Photonics.