Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)
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Curated by eLife
Evaluation Summary:
This manuscript reports a robust and well-characterized expansion method that achieves 10X expansion with a single expansion step using a simple, easy-to-use protocol. The new protocol leads to an enabling methodology for super-resolution imaging of various sub-cellular structures and organelles and is likely to have a high impact.
(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
Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand 10-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes.
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Author Response
Reviewer #2 (Public Review):
In this study, the authors developed a new expansion microscopy (ExM) method called Ten-fold Robust Expansion Microscopy (TREx). This method emphasizes one-round sample expansion of cells by systematically optimizing the monomer recipe. Compared to existing ExM methods which expand samples to similar scale (~ 10 folds), TREx aims for a robust procedure that can be handled more easily. The reviewer experimentally tested the TREx protocol, and validated the TREx 10x gel can be made robustly by researchers who have experience with standard ExM.
We are very pleased that the reviewer tested out our new recipe!
Specific comments:
- The authors claimed in the abstract that "TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with …
Author Response
Reviewer #2 (Public Review):
In this study, the authors developed a new expansion microscopy (ExM) method called Ten-fold Robust Expansion Microscopy (TREx). This method emphasizes one-round sample expansion of cells by systematically optimizing the monomer recipe. Compared to existing ExM methods which expand samples to similar scale (~ 10 folds), TREx aims for a robust procedure that can be handled more easily. The reviewer experimentally tested the TREx protocol, and validated the TREx 10x gel can be made robustly by researchers who have experience with standard ExM.
We are very pleased that the reviewer tested out our new recipe!
Specific comments:
- The authors claimed in the abstract that "TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes". The authors only demonstrated one NHS ester dye, BODIPY-FL NHS dye (lined 151-159) without justification why this dye was selected. Does BODIPY-FL NHS dye work better than other off-the-shelf NHS dyes? The reviewer recommends the authors to validate a few more widely used dyes with TREx, e.g. Cy3/Cy5, Alexa 488, Alexa 568, to guide the readers to choose the appropriate dyes.
We have added text on this issue, "Sim et al (Sim et al., 2021) have shown that highly hydrophobic NHS ester dyes exhibit strong contrast for cytosolic organelles while highly hydrophilic NHS ester dyes strongly stain the nucleus. The moderate hydrophobicity dyes that we used (BODIPY-FL (Zanetti-Domingues, Tynan, Rolfe, Clarke, & Martin-Fernandez, 2013) and AlexaFluor594 (Hughes, Rawle, & Boxer, 2014)) exhibit both nuclear staining and contrast for cytosolic organelles."
- Page 8: The reviewer is happy to see the discussion on the heterogeneous local expansion factors in cells. It is critical for evaluating the expansion isotropy and avoid pitfalls in the applications of TREx. Based on this work and previous work (e.g. U-ExM), organelles with higher protein density may have smaller local expansion factors than the macroscopic expansion factor. The authors discussed the local expansion factor of organelles with different protein density, including centrioles, NPCs, and microtubules. To evaluate the local expansion factors comprehensively, the reviewer asks the authors to add a figure or plot to compare the local expansion factors of different organelles, ideally including centrioles, NPCs, microtubules, clathrin-coated pits, mitochondria, ER, and centromere. The authors have already measured or imaged many of these organelles. For the other organelles, good antibodies are available. Therefore, the additional experiments should be straightforward for the authors. But the comprehensive comparison will make the work much more impactful.
We address this in our response to essential point 3 and agree that the added comparison over multiple organelles has made the work more impactful.
- Line 388: The authors stated "The strong overlap between NHS ester and mCLING stains was not unexpected, given the reactivity of NHS esters towards both unreacted lysines in the mCLING molecule and antibodies." Since AcX (6-((acryloyl)amino)hexanoic Acid, Succinimidyl Ester) at high concentration was added after the mCLING staining, most of the lysines in the mCLING should be reacted by the AcX. Therefore, NHS ester dye staining should not strongly overlap with the mCLING. The authors should re-evaluate and interpret the overlap. The authors can do simple experiments like increasing the concentration of AcX, or use pH 8 for AcX treatment. If the overlap is reduced, it means the overlap was caused by the unreacted lysines in mCLING, and can be reduced. If the overlap is not reduced, there are other mechanisms which need further examination or interpretation.
The AcX concentration was selected to maximize retention of proteins without hindering gel expansion by cross-linking through multiple AcX modifications on each individual protein. Therefore, it is likely that AcX is not close to saturating the available primary amines. We have explored this further with an AcX competition assay.
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Evaluation Summary:
This manuscript reports a robust and well-characterized expansion method that achieves 10X expansion with a single expansion step using a simple, easy-to-use protocol. The new protocol leads to an enabling methodology for super-resolution imaging of various sub-cellular structures and organelles and is likely to have a high impact.
(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 major strength of the work is that the procedures are enabling, robust, well-characterized, and described in detail. The expansion and staining procedures are therefore likely to be readily adopted by the community. Unlike advances in microscopy hardware that require purchasing new equipment or having specialized expertise for building instrumentation, improvements in sample preparation can be performed for low cost with relatively easily learned skills. So, although the fine tuning of a hydrogel recipe may appear at first as a simple optimization, the end result is a substantial breakthrough that will be useful to the community.
The simple but practical gel deformation methodology for assessing hydrogel sturdiness was executed well. I agree with the authors that although more advanced methodology could …
Reviewer #1 (Public Review):
The major strength of the work is that the procedures are enabling, robust, well-characterized, and described in detail. The expansion and staining procedures are therefore likely to be readily adopted by the community. Unlike advances in microscopy hardware that require purchasing new equipment or having specialized expertise for building instrumentation, improvements in sample preparation can be performed for low cost with relatively easily learned skills. So, although the fine tuning of a hydrogel recipe may appear at first as a simple optimization, the end result is a substantial breakthrough that will be useful to the community.
The simple but practical gel deformation methodology for assessing hydrogel sturdiness was executed well. I agree with the authors that although more advanced methodology could be appropriate in a materials science or polymer physics context, the tilted gel methodology was sufficient to achieve the authors' aims and is simple enough that other researchers can easily adopt it when troubleshooting TREx or possibly when adapting the TREx for new applications.
It is much appreciated that the authors included validation of their new gel recipes using both pre-expansion / post-expansion distortion analysis as well as distance measurements of nuclear pore complexes(NPC) that have a well known size (Fig. 3). The distortion analysis in Figure 3 shows very strong performance with ~3% distortion over a range of length scales. The comparison of NPC diameter to prior published results is good but leaves some room for future improvement and suggests that the local / nanoscale expansion may be smaller for tight complexes than for the overall gel.
The imaging demonstrations in cell and tissue labeling were convincing. The experiments with amine-reactive dyes and membrane-labeling stains were good to see in combination with TREx and help illustrate the potential of the method.
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Reviewer #2 (Public Review):
In this study, the authors developed a new expansion microscopy (ExM) method called Ten-fold Robust Expansion Microscopy (TREx). This method emphasizes one-round sample expansion of cells by systematically optimizing the monomer recipe. Compared to existing ExM methods which expand samples to similar scale (~ 10 folds), TREx aims for a robust procedure that can be handled more easily. The reviewer experimentally tested the TREx protocol, and validated the TREx 10x gel can be made robustly by researchers who have experience with standard ExM.
Specific comments:
The authors claimed in the abstract that "TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes". The authors …
Reviewer #2 (Public Review):
In this study, the authors developed a new expansion microscopy (ExM) method called Ten-fold Robust Expansion Microscopy (TREx). This method emphasizes one-round sample expansion of cells by systematically optimizing the monomer recipe. Compared to existing ExM methods which expand samples to similar scale (~ 10 folds), TREx aims for a robust procedure that can be handled more easily. The reviewer experimentally tested the TREx protocol, and validated the TREx 10x gel can be made robustly by researchers who have experience with standard ExM.
Specific comments:
The authors claimed in the abstract that "TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes". The authors only demonstrated one NHS ester dye, BODIPY-FL NHS dye (lined 151-159) without justification why this dye was selected. Does BODIPY-FL NHS dye work better than other off-the-shelf NHS dyes? The reviewer recommends the authors to validate a few more widely used dyes with TREx, e.g. Cy3/Cy5, Alexa 488, Alexa 568, to guide the readers to choose the appropriate dyes.
Page 8: The reviewer is happy to see the discussion on the heterogeneous local expansion factors in cells. It is critical for evaluating the expansion isotropy and avoid pitfalls in the applications of TREx. Based on this work and previous work (e.g. U-ExM), organelles with higher protein density may have smaller local expansion factors than the macroscopic expansion factor. The authors discussed the local expansion factor of organelles with different protein density, including centrioles, NPCs, and microtubules. To evaluate the local expansion factors comprehensively, the reviewer asks the authors to add a figure or plot to compare the local expansion factors of different organelles, ideally including centrioles, NPCs, microtubules, clathrin-coated pits, mitochondria, ER, and centromere. The authors have already measured or imaged many of these organelles. For the other organelles, good antibodies are available. Therefore, the additional experiments should be straightforward for the authors. But the comprehensive comparison will make the work much more impactful.
Line 388: The authors stated "The strong overlap between NHS ester and mCLING stains was not unexpected, given the reactivity of NHS esters towards both unreacted lysines in the mCLING molecule and antibodies." Since AcX (6-((acryloyl)amino)hexanoic Acid, Succinimidyl Ester) at high concentration was added after the mCLING staining, most of the lysines in the mCLING should be reacted by the AcX. Therefore, NHS ester dye staining should not strongly overlap with the mCLING. The authors should re-evaluate and interpret the overlap. The authors can do simple experiments like increasing the concentration of AcX, or use pH 8 for AcX treatment. If the overlap is reduced, it means the overlap was caused by the unreacted lysines in mCLING, and can be reduced. If the overlap is not reduced, there are other mechanisms which need further examination or interpretation.
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Reviewer #3 (Public Review):
Fluorescence imaging has been limited for decades by the diffraction limit. This was first eluded by the invention of different optical methods (e.g. STED, STORM, PAINT), which, however, typically require expensive instrumentation and excellent knowledge of optics. Expansion microscopy, introduced in 2015 by the Boyden laboratory (including the last author of the current manuscript) offers an alternative to this approach, by expanding the sample and then imaging it with conventional optics.
The main strength of the manuscript is that it presents an easily-applied technology for 10-fold expansion, which seems to work both in tissues and in cell cultures. At the same time, a number of problems are apparent with the technique presented. One of the main points of the presented approach (TREx) is to reduce the …
Reviewer #3 (Public Review):
Fluorescence imaging has been limited for decades by the diffraction limit. This was first eluded by the invention of different optical methods (e.g. STED, STORM, PAINT), which, however, typically require expensive instrumentation and excellent knowledge of optics. Expansion microscopy, introduced in 2015 by the Boyden laboratory (including the last author of the current manuscript) offers an alternative to this approach, by expanding the sample and then imaging it with conventional optics.
The main strength of the manuscript is that it presents an easily-applied technology for 10-fold expansion, which seems to work both in tissues and in cell cultures. At the same time, a number of problems are apparent with the technique presented. One of the main points of the presented approach (TREx) is to reduce the gel crosslinker concentration by more than 10-fold, compared to the original expansion microscopy approach of the Boyden laboratory. This may result in expansion errors that still need to be fully explored.
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Excerpt
TREx - a roaring success for visualising cells and tissues
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