ATUM-Tomo: A multi-scale approach to cellular ultrastructure by combined volume scanning electron microscopy and electron tomography

Reviewer #1 Public Review

Summary
This paper presents a new, but simple and low-cost technique for multimodal EM imaging that combines the strengths of both volume scanning electron microscopy (SEM) and electron microscopic tomography. The novel ATUM-Tomo approach enables the consecutive inspection of selected areas of interest by correlated serial SEM and TEM, optionally in combination with CLEM, as demonstrated here. The most important feature of ATUM-Tomo, particularly of correlative ATUM-Tomo, is that it can bridge scales, from the cellular to the high-resolution subcellular scale, from micrometer to low nanometer resolution. This is particularly important for ultrastructural analyses of biological regions of interest, which is demonstrated here for focal pathologies or rare cellular and subcellular events. Both imaging modalities are non-destructive, thus allowing re-imaging and hierarchical imaging at the SEM and TEM levels. This is particularly important for precious samples, including human biopsies and samples from complex CLEM experiments. Beyond the demonstrated neuropathology-related application, further use in investigating normal and pathologically altered brains, including human brain tissue samples that require high-resolution SEM and TEM in combination with immunohistochemistry, and virus or tracer injections, would be possible. Thus, ATUM-Tomo provides new possibilities in multimodal volume EM imaging for diverse areas of biological research.

Strengths
This paper is a very nice piece of work, bringing together modern high-end state-of-the-art technology that will allow us to investigate diverse biological questions in different areas of interest and at different scales. The paper is clear and well-written, although some additions are necessary to the methods section and the scientific results as exemplified by investigations of the blood-brain barrier. The discussion would benefit from an expansion of the part dealing with the scientific results. The paper is accompanied by excellent figures, supplemental information, and colored 3D-reconstructions, which makes it easy for the reader to follow the experimental procedure and the scientific context. The authors may consider moving the supplemental figures into the main body of the paper, which would then still contain 'only' eight figures.

Weaknesses
There is some imbalance between the description of the state-of-the-art methodology and the scientific context.

Reviewer #2 Public Review

Summary
Kislinger et al. present a method permitting a targeted, multiscale ultrastructural imaging approach to bridge the resolution gap between large-scale scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The key methodological development consists of an approach to recover sections of resin-embedded material produced by Automated Tape Collecting Ultramicrotomy (ATUM), thereby permitting regions of interest identified by serial section SEM (ATUM-SEM) screening to be subsequently re-examined at higher resolution by TEM tomography (ATUM-Tomo). The study shows that both formvar and permanent marker coatings are in principle compatible with the solvent-based release of pre-screened sections from ATUM tape (carbon nanotubule or Kapton tape). However, a comparative analysis of potential limitations and artifacts introduced by these respective coatings revealed permanent marker to provide a superior coating; permanent marker coatings are more easily and reliably applied to tape with only minor contaminants affecting the recovered section-tape interface with negligible influence on tomogram interpretation. Proof-of-principle is provided by integrating this novel ATUMTomo technique into a technically impressive correlated light and electron microscopy (CLEM) approach specifically tailored to investigate ultrastructural manifestations of trauma-induced changes in blood-brain barrier permeability (Khalin et al., 2022).

Strengths
Schematics and well-constructed figures clearly illustrate the general workflow, light and electron microscope image data are of excellent quality, and the efficacy of the ATUM-Tomo approach is documented by qualitative assessment of ATUM-SEM performance using coated tape variants and a convincing correlation between scanning and transmission electron microscopy imaging modalities. Potential ultrastructural artifacts induced via solvent exposure and any subsequent mechanical stress incurred during section detachment were systematically investigated using appropriate methods and transparently reported. In summary, the presented data are consistent with the study's claims. A major strength of this work includes its general applicability to a broad range of biological questions and ultrastructural targets demanding resolutions exceeding that obtained via serial section and block-face imaging approaches alone. Importantly, this relatively simple and cost-effective technique is widely adopted by electron microscopy laboratories. Its integration into existing ATUM-SEM workflows supports a versatile and non-destructive imaging regime enabling high-resolution details of targeted structures to be interpreted within anatomical and subcellular contexts.

Weaknesses
Given the identified importance of glow-discharge treatment of precoated tape to the flat deposition of sections during ATUM, a corresponding schematic or appropriate reference(s) providing more information about the custom-built tape plasma device would likely be a prerequisite for effective reproduction of this technique in other laboratories.

Author Response

We thank the Editors and Reviewers for the thorough assessment of our work. We are pleased that you agree with us that our proof-of-concept study of the ATUM Tomo technology advances volume electron microscopy and has the potential to solve research questions in diverse biological areas. Based on your comments, we are planning to revise the manuscript to optimize readability, clarify the fields of applicability of our approach more, and add some data related to questions you raised. We plan the following revisions:

Reviewer #1 The authors may consider moving the supplemental figures into the main body of the paper since they finally would end up with a total of eight figures.

As part of the supplemental figures describe essential experimental details, we will move them into the main part of the manuscript.

Reviewer #1 In general, the methods and techniques used here are beside some required but important additions described in sufficient detail.

Reviewer #2 Given the identified importance of glow-discharge treatment of precoated tape to the flat deposition of sections during ATUM, a corresponding schematic or appropriate reference(s) providing more information about the custom-built tape plasma device would likely be a prerequisite for effective reproduction of this technique in other laboratories.

Thank you for the valuable comments on the missing experimental details, which could affect the ease of establisihing ATUM-Tomo in other labs. We will clearly highlight the ATUM-Tomo-specific vs. some general EM processing steps of the workflow in the proposed way. A detailed description of the custom-built tape plasma device will be added to the methods section. In addition, we will reference more explicitly our published protocols, which describe the standard electron microscopy embedding steps in great detail (Kislinger et al., STAR protocols, 2020; Kislinger et al., Meth Cell Biol, 2023).

Reviewer #1 Concerning the results section: In my opinion, the results section is a bit unbalanced. There is a mismatch between the detailed description of the methodology (experimental approach) and the scientific findings of the paper. The reviewer can see the enormous methodological impact of the paper, which on the other hand is the major drawback of the paper. To my opinion, the authors should also give a more detailed description of their scientific results.

Concerning the discussion: It would have been nice to give a perspective to which the described methodology can be used not only to describe diverse biological aspects that can be addressed and answered by this experimental approach. For example, how could this method be used to address various questions about the normal and pathologically altered brain?

In my opinion, the paper has one major drawback which is that it is more methodologically based although the authors included a scientific application of the method. The question here is to balance the methodology vs. the scientific achievement of this paper, a decision hard to take. In other words, one could recommend this paper to more methodologically based journals, for example, Nature Methods.

Balancing the technological and biological parts is indeed a difficult issue. We agree that this manuscript mainly describes a technical advancement and demonstrates its power to answer previously unsolved scientific questions. We exemplify this in our model system, neuropathology of the blood-brain barrier. The biological impact of ATUM-SEM has been described in detail in Khalin et al., Small, 2022, and is referenced accordingly. Here we describe how ATUM-Tomo can be applied to reveal biological insights exceeding the capabilities of ATUM-SEM and other volume electron microscopy techniques. However, the description of the methodological development outweighs by far the one of the biological details. We consider eLife‘s Tools and Resources (which, in our view, is in scope similar to Nat Methods) an ideal format for this technically focused manuscript while targeting eLife’s readership with diverse biological fields of interest for potential applications of the method. We will add more suggestions for possible applications to the discussion to accommodate the Reviewer’s concern that having only a single application might seem arbitrary or even suggest a very narrow utility of the technique.

Reviewer #2 Is the separation of sections from permanent marker-treated tape sensitive to the time interval between deposition/SEM imaging and acetone treatment?

Thank you for pointing out this important methodological aspect. We have not systematically investigated whether there is a critical time window between microtomy, SEM, and detachment. From the samples generated for this study, we will try to assess the importance of timing in retrospect.

Reviewer #2 To what extent is slice detachment from permanent marker-treated tape resin-dependent [i.e. has ATUM-Tomo been tested on resin compositions beyond LX112 (LADD)]?

We appreciate this comment addressing the broader technical applicability of ATUM-Tomo. We aim to test the general workflow with tissue embedded in other commonly used resin types.

Reviewer #2 Minor corrections to the text and figures.

Thank you for the detailed corrections. We will apply them accordingly.