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  1. eLife Assessment:

    This paper demonstrates an integrated labeling and block face fluorescence imaging method that enables the rapid evaluation of biological specimens as large as an E18 mouse embryo with single cell resolution. Such capabilities will likely be of great interest to developmental biologists and pathologists. While the approach can be considered a major step forward, additional experimental support is necessary to gauge how quantitative the method is.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    This manuscript presents a fast, and cost-effective multicolor 3D imaging system termed translational rapid ultraviolet-excited sectioning tomography (TRUST). TRUST combined sub-systems of sectioning, staining, and imaging to take the complexity out of preparation workload. The integrated system can provide high-contrast fluorescence images from the cellular level to the organ level in an automated series of protocols. Although the authors have well described the systematic feasibility of TRUST, its novelty is ambiguous from a biological point of view.

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  3. Reviewer #2 (Public Review):

    In this manuscript, Yu et al describe Translational Rapid Ultraviolent-excited Sectioning Tomography (TRUST), a fast and cost-effective 3D imaging system for organ and organism-scale samples. TRUST combines MUSE (microscopy with ultraviolet surface excitation) with real-time staining, imaging, mechanical sectioning of the tissues, and stitching of the data, thereby essentially circumventing lengthy and laborious sample preparation steps. Specifically, gelatin or agarose-embedded specimens are placed in a large chamber with fluidics for replenishing the labeling reagents and removing the previously imaged portions of the specimen after slicing. Imaging takes place in an upright geometry, with the entire imaging system placed on an automated XY stage, whereas focusing is performed manually. The sample remains stationary throughout the imaging procedure. To illuminate the specimen, ultraviolet light is launched at an oblique angle, which in addition to its wavelength, reduces its optical penetration into the tissue (improving optical sectioning). Images are acquired in a 24-bit RGB format, providing 8-bits per channel. As labels, they predominantly use propidium iodide and DAPI, which both are fluorogenic and therefore advantageously reduce the background signal unless bound to their molecular targets. Owing to their distinct localization in tissues, and the distinct autofluorescent signature that each tissue type natively has, they achieve 'content-rich' images that reveal boundaries between tissue regions and tissue types. The authors image every major mouse organ, and even an entire developing mouse at E15 and E18, which would be challenging to achieve otherwise. And, compared to other variants of MUSE, the use of a waterproof case is a good solution that improves the contrast by minimizing the illuminated volume while leveraging water dipping objectives. Given these advances, they employed the proposed tool for addressing biological questions that ranged from evaluating a single organ to visualizing entire mouse embryos.

    The biggest concern that we have is that the method is presented in relatively qualitative terms. As such, it remains difficult for us to ascertain how quantitative the method is, and whether it may be used to foster insight beyond qualitative differences in tissue architecture. And, for the data that were presented quantitatively, e.g., segmentation of the glomeruli and vasculature, the accuracy of the results could not be evaluated. Standard imaging metrics were also largely absent. For example, neither the axial nor lateral resolution is presented, and therefore it remains difficult to know whether the data is properly Nyquist sampled. Indeed, the slicing mechanism may prohibit Nyquist sampling in the axial dimension, which would be problematic in circumstances where one wants to quantitatively evaluate cell density, etc. And because TRUST is a volumetric imaging technique, it would be informative to present some of the results as XZ or YZ slices (perhaps as a supporting figure), rather than the more optically powerful XY orientation. In general, we believe that many statements would benefit from quantitative metrics. For example, while the stitching qualitatively appears excellent (as should be for such a system), and free of distortions introduced from slicing, it remains hard for a reader go truly gauge whether this is the case. Likewise, how do adjacent slices align in the axial direction? How deep does the UV penetrate the specimen, and how does this (and labeling duration) influence optical sectioning? Would such an approach be compatible with a genetically encoded mouse model with a fluorescent reporter, or does the bit-depth of the camera limit these sample types? Nonetheless, we believe TRUST provides a significant advance over previous versions of MUSE and enables scientists to evaluate biological events throughout development at the whole organism scale.

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