1. Reviewer #3:

    Labelling strategies for electron microscopy have so far lacked the ability to clearly visualise genetically expressed probes such as GFP for light microscopy. Building on previous studies by the group of Ellisman, the Parton group have made significant adaptations and improvements to the system. Especially addressing the issue of diffusion of the DAB precipitate and the low visibility of it by silver enhancing the particles is a key step forward. The authors have tested their system in a wide variety of EM workflows and show that it works.

    The quantification part of the manuscript is to me potentially the most interesting part. Quantitation of proteins at physiological levels at the ultrastructural level would be a significant achievement. This part is a bit under represented although there are some issues with that. The silver enhanced particles on the added external standard appear to be larger than the ones inside. Does that result in lower detection?

    Overall, this is a manuscript that is very clearly written and very easy to follow for non-experts.

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  2. Reviewer #2:

    In this study, entitled “APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy” Rae et al. describe a method to improve the visualization of the staining for genetically encoded probes that they described in previous studies (namely APEX2 constructs).

    These techniques are very powerful as they increase the sensitivity for detecting low level of expression of the tagged proteins (e.g. compared to GFP tagged proteins). The novelty in this study is that the reaction product (DAB precipitates) is revealed by the nucleation of silver/gold precipitates. Such enhancement has been used extensively in the past for pre-embedding immuno-peroxidase techniques, but has never been combined with the use of APEX2. This has one major advantage: that the contrast of the positive staining can stand out from the contrast of the surrounding ultrastructure, making the sample preparation more adapted to 3D EM techniques, especially volume SEM where contrast is a bottleneck. Moreover, the sensitivity of the technique is shown to be compatible with the detection of endogenous levels of expression.

    The technique is very well detailed and elegantly illustrated by convincing applications on cultured cell systems. The apparent simplicity of use, together with a growing interest in the community for the APEX2 based techniques (also in correlative imaging), significantly raises the potential for it to become a standard in the field, and should thus be shared with the community.

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  3. Reviewer #1:

    The manuscript by Rae et al. reports the development of a new protocol for labeling genetically-tagged proteins of interest with heavy atom particles for visualization by electron microscopy. The optimized protocol builds on the established use of the enzyme APEX fused to the target of interest. APEX oxidizes diaminobenzidine, DAB, which in turn converts silver and gold metal salts to particulates in close proximity to the APEX-fused protein of interest. The optimized protocol is related to the contrast-enhancement method reported by Sedmak et al., 2009 and Mavlyutov et al., 2017. The changes to the method may improve the proportionality of the signal such that the number of APEX tags present in a sample is better correlated with the number of heavy atom particles. While the study appears to be sound, it is an extension of an established labeling method.

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  4. Summary: The manuscript by Rae et al. reports a new protocol for labeling genetically-tagged proteins of interest with heavy atom particles for visualization by electron microscopy. The optimized protocol builds on the use of the enzyme APEX2, fused to the target protein of interest. The contrast enhancement may be useful in diverse 3D EM techniques. Also, reviewers were enthusiastic about the prospects for quantitative studies, even for low-levels of endogenous expression. Semi-quantitative studies may be enabled because the new method appears to improve the proportionality of the signal such that the number of APEX2 tags in a sample correlates with the number of heavy atom particles. The apparent simplicity of the protocol raises the potential for it to become a standard in the field of EM labeling.

    Reviewer #1, Reviewer #2 and Reviewer #3 opted to reveal their name to the authors in the decision letter after review.

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