Volumetric trans-scale imaging of massive quantity of heterogeneous cell populations in centimeter-wide tissue and embryo

Reviewer #1 (Public Review):

Summary:
The authors are trying to develop a microscopy system that generates data output exceeding the previous systems based on huge objectives.

Strengths:
They have accomplished building such a system, with a field of view of 1.5x1.0 cm2 and a resolution of up to 1.2 um. They have also demonstrated their system performance on samples such as organoids, brain sections, and embryos.

Weaknesses:
To be used as a volumetric imaging technique, the authors only showcase the implementation of multi-focal confocal sectioning. On the other hand, most of the real biological samples were acquired under wide-field illumination, and processed with so-called computational sectioning. Despite the claim that it improves the contrast, sometimes I felt that the images were oversharpened and the quantitative nature of these fluorescence images may be perturbed.

Reviewer #2 (Public Review):

Summary:
This manuscript introduced a volumetric trans-scale imaging system with an ultra-large field-of-view (FOV) that enables simultaneous observation of millions of cellular dynamics in centimeter-wide 3D tissues and embryos. In terms of technique, this paper is just a minor improvement of the authors' previous work, which is a fluorescence imaging system working at visible wavelength region (https://www.nature.com/articles/s41598-021-95930-7).

Strengths:
In this study, the authors enhanced the system's resolution and sensitivity by increasing the numerical aperture (NA) of the lens. Furthermore, they achieved volumetric imaging by integrating optical sectioning and computational sectioning. This study encompasses a broad range of biological applications, including imaging and analysis of organoids, mouse brains, and quail embryos, respectively. Overall, this method is useful and versatile.

Weaknesses:
The unique application that only can be done by this high-throughput system remains vague. Meanwhile, there are also several outstanding issues in this paper, such as the lack of technical advances, unclear method details, and non-standardized figures.

Author Response

We would like to thank the editors and reviewers who took their valuable time to evaluate the manuscript from various perspectives. We are delighted that our technique was found appealing to biologists and imaging technologists. However, we received several comments that the principles and effectiveness of our techniques are often vague and difficult to understand. They also pointed out that the explanations and representations for several figures were not appropriate. We will revise the manuscript to address these issues and make the manuscript more clear and rigorous.