EZ Clear for simple, rapid, and robust mouse whole organ clearing
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Curated by eLife
Evaluation Summary:
The manuscript reports a new tissue clearing procedure that is faster (clearing within 48 hours), uses less hazardous chemicals, and importantly appears to result in less tissue volume change compared to other methods. The simple protocol adds further to the toolbox of tissue clearing methods and is one that is likely to be even more popular than many current methods, although the scope of tissue on which it can be used and rigorous comparisons to existing protocols have not been fully investigated.
(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. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevent the widespread adoption of these methods. Here, we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hr in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, samples processed with EZ Clear can be subjected to downstream applications, such as tissue embedding and cryosectioning followed by standard histology or immunofluorescent staining without loss of fluorescence signal from endogenous or synthetic reporters. Furthermore, we demonstrate that wholemount adult mouse brains processed with EZ Clear can be successfully immunolabeled for fluorescent imaging while still retaining signal from endogenous fluorescent reporters. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adapt and implement in diverse imaging modalities in biomedical research.
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Evaluation Summary:
The manuscript reports a new tissue clearing procedure that is faster (clearing within 48 hours), uses less hazardous chemicals, and importantly appears to result in less tissue volume change compared to other methods. The simple protocol adds further to the toolbox of tissue clearing methods and is one that is likely to be even more popular than many current methods, although the scope of tissue on which it can be used and rigorous comparisons to existing protocols have not been fully investigated.
(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. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The submitted manuscript describes an optimized tissue clearing protocol with some modest advantages including better preservation of tissue volume, compatibility with traditional histology methods, and simple processing steps. By combining known advantages of organic solvent-based and aqueous-based procedures the authors were able to generate a very simple, efficient, and fast tissue clearing protocol that can preserve endogenous and synthetic fluorescent signals. The manuscript is mostly written well and the fluorescent images are very striking. However, the lack of quantification throughout the manuscript makes it is difficult to assess how robust the results are across many samples and key experimental applications are missing.
1. Immunofluorescent labeling/staining is a very common procedure in whole, …
Reviewer #1 (Public Review):
The submitted manuscript describes an optimized tissue clearing protocol with some modest advantages including better preservation of tissue volume, compatibility with traditional histology methods, and simple processing steps. By combining known advantages of organic solvent-based and aqueous-based procedures the authors were able to generate a very simple, efficient, and fast tissue clearing protocol that can preserve endogenous and synthetic fluorescent signals. The manuscript is mostly written well and the fluorescent images are very striking. However, the lack of quantification throughout the manuscript makes it is difficult to assess how robust the results are across many samples and key experimental applications are missing.
1. Immunofluorescent labeling/staining is a very common procedure in whole, cleared tissues. Given that immunofluorescent labeling works well in tissue sections from EZ Cleared brains, it appears that it should work in the whole tissues after clearing. An extended version of the EZ Clear protocol with immunofluorescent labeling procedures in whole mouse brain tissue should be included along with quantification of fluorescent intensity as a function of depth. If EZ Clear provides more uniform immunofluorescent labeling relative to other protocols, this is a significant advantage.
2. The differences in tissue volume and sample processing steps between EZ Clear and Fast 3D are important, but relatively modest. Additional quantitative comparisons between EZ Clear and Fast 3D/3Disco would considerably strengthen the manuscript. The qualitative differences shown in Figure 1G-J are striking, but it is difficult to determine how robust this effect is across multiple samples without a quantitative comparison. Similar quantitative comparisons should be made for endogenous fluorescent intensity and immunofluorescent labeling as a function of tissue depth between the various protocols.
3. It would be helpful to see how the intensity and contrast of the fluorescent labeling changes as a function of depth (e.g. Lectin-649 labeling in Figures 2E and H). There is a clear improvement with EZ View relative to RIMS, but there are still noticeable changes in the signal as a function of depth. Quantification would help determine the extent of these changes, as well as reproducibility across multiple samples.
4. LSFM imaging should be performed in some of the other mouse tissues to demonstrate sufficient clearing for quantification purposes.
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Reviewer #2 (Public Review):
The manuscript by Hsu and colleagues describes the development of a new clearing reagent/procedure, which they call EZ Clear, that is faster (clearing within 48 hours), uses fewer hazardous chemicals than many others (eg. BABB, 3DISCO and X-CLARITY), and importantly appears to result in less tissue volume change compared to most other methods. The final immersion solution used for imaging is also solvent free and so can be used without specialized objectives. As part of the assessment of the effectiveness and utility of the clearing procedure, the authors present a comparison of imaging quality (including depth of imaging) relative to a few other widely used clearing methods. Further, they assess the suitability of using the tissue cleared using this procedure in other downstream applications such as routine …
Reviewer #2 (Public Review):
The manuscript by Hsu and colleagues describes the development of a new clearing reagent/procedure, which they call EZ Clear, that is faster (clearing within 48 hours), uses fewer hazardous chemicals than many others (eg. BABB, 3DISCO and X-CLARITY), and importantly appears to result in less tissue volume change compared to most other methods. The final immersion solution used for imaging is also solvent free and so can be used without specialized objectives. As part of the assessment of the effectiveness and utility of the clearing procedure, the authors present a comparison of imaging quality (including depth of imaging) relative to a few other widely used clearing methods. Further, they assess the suitability of using the tissue cleared using this procedure in other downstream applications such as routine histology and immunohistochemistry. The simple protocol adds further to the toolbox of tissue clearing methods and is one that is likely to be even more popular than many current methods because of its simplicity (cost and procedural) and speed.
The major strength of the work is that the solutions used are relatively few, does not require specialized equipment, and the procedure is simple with minimal hands-on activity compared to other protocols. Furthermore, the tissue does not appear to significantly change volume which has been a major concern of some of the other methodologies.
Despite the interest that this simplified procedure will no doubt garner in the imaging community, there are numerous weaknesses (a few major and many minor) in the manuscript in its current form. While many of these do not detract from conclusions of the manuscript, there is a concern that the similarity with another procedure (Fast3D) has not clearly been portrayed. There is really only one section describing a comparison between EZ Clear and Fast 3D where the two methods were used on separate hemispheres of the brain from one sample (n=1?). A more rigorous assessment of the differences would seem appropriate given the significant similarity between the composition of the solutions and actual processing steps of both methods. On the surface, it seems that the current method is derived from the Fast3D clearing system and thus the authors should give more credit to this prior work.
Clearing methods also vary somewhat by how well they maintain fluorescence over time. While the authors comment on "preservation of fluorescence", they do so only in reference to it not suppressing fluorescence immediately but not whether fluorescence intensity is maintained, fades or increases following periods of storage in EZ View. This is a key feature noted for other methods and so is a limitation for this paper. Further, the description that there is 'robust retention' of signal is strictly not what is being reported. Rather than retention, what the authors appear to show relates to the efficacy of clearing across the whole tissue.
Compared to papers describing other clearing methods, this manuscript describes less exploration of the breadth of applicability of this method. The authors provide a few example images of cleared mouse organs, but do not present more diversity in tissue types and size, which is where the greatest value of this methodology may lie.
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