Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes
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Curated by eLife
Evaluation Summary:
Sample preparation for single-particle electron cryo-microscopy (cryo-EM) remains a bottleneck of this technique. The sample ice thickness cannot be accurately controlled, molecules may display strongly preferred orientations that make more elaborate data collection schemes necessary, and the sample may degrade at the air-water interface before it is finally frozen. In their pioneering work, the authors describe a prototype of a new microfluidic device that addresses some of these problems, including a refreshingly objective and critical discussion about the pros and cons of this novel approach. While some development will be required for this method to become mainstream, it has the potential to become a powerful alternative to the conventional workflow of single-particle cryo-EM, enabling full automation and making sample preparation highly reproducible.
(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 #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.
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Evaluation Summary:
Sample preparation for single-particle electron cryo-microscopy (cryo-EM) remains a bottleneck of this technique. The sample ice thickness cannot be accurately controlled, molecules may display strongly preferred orientations that make more elaborate data collection schemes necessary, and the sample may degrade at the air-water interface before it is finally frozen. In their pioneering work, the authors describe a prototype of a new microfluidic device that addresses some of these problems, including a refreshingly objective and critical discussion about the pros and cons of this novel approach. While some development will be required for this method to become mainstream, it has the potential to become a powerful alternative to the conventional workflow of single-particle cryo-EM, enabling full automation and making …
Evaluation Summary:
Sample preparation for single-particle electron cryo-microscopy (cryo-EM) remains a bottleneck of this technique. The sample ice thickness cannot be accurately controlled, molecules may display strongly preferred orientations that make more elaborate data collection schemes necessary, and the sample may degrade at the air-water interface before it is finally frozen. In their pioneering work, the authors describe a prototype of a new microfluidic device that addresses some of these problems, including a refreshingly objective and critical discussion about the pros and cons of this novel approach. While some development will be required for this method to become mainstream, it has the potential to become a powerful alternative to the conventional workflow of single-particle cryo-EM, enabling full automation and making sample preparation highly reproducible.
(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 #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors present a prototype of a nanofabricated microfluidic device that allows plunge-freezing of sample solutions for high-resolution cryo-EM analysis, specifically single-particle cryo-EM. The design of the device includes cavities (nanochannels) of defined thickness, made of ~10nm thick silicon nitride membranes (SiN). The authors demonstrate the loading of a device consisting of 22 connected cavities with three different samples - apoferritin, 20S proteasome and tobacco mosaic virus - requiring only a few picoliters of sample. Subsequent data collection from the frozen samples produced reconstructions with resolutions between 3 and 5.4 Å.
The new device solves one of the most difficult problems in cryo-EM sample preparation: controlling the sample thickness. This is a major advance as it is a …
Reviewer #1 (Public Review):
The authors present a prototype of a nanofabricated microfluidic device that allows plunge-freezing of sample solutions for high-resolution cryo-EM analysis, specifically single-particle cryo-EM. The design of the device includes cavities (nanochannels) of defined thickness, made of ~10nm thick silicon nitride membranes (SiN). The authors demonstrate the loading of a device consisting of 22 connected cavities with three different samples - apoferritin, 20S proteasome and tobacco mosaic virus - requiring only a few picoliters of sample. Subsequent data collection from the frozen samples produced reconstructions with resolutions between 3 and 5.4 Å.
The new device solves one of the most difficult problems in cryo-EM sample preparation: controlling the sample thickness. This is a major advance as it is a prerequisite for full reproducibility and automation of single-particle cryo-EM, which is an important goal of the field. The device also addresses sample denaturation at the air-water interface, another problem currently limiting single-particle cryo-EM. However, how replacement of the air-water interface with a SiN-water interface affects denaturation and potential preferred orientation of particles remains to be shown. The SiN membranes also add background to the cryo-EM images that may interfere with the high-resolution signal of the particles. Indeed, the additional background may have limited the resolution obtained from the three datasets. The reconstructions also displayed substantially higher B-factors than previously obtained from similar samples. It is possible that this is related to the enclosure of the sample in a cavity: The cavity prevents the escape of gases from the sample that evolve as a result of radiolysis under the electron beam, potentially leading to increased beam-induced motion of the sample. Nevertheless, this work serves as a convincing proof of principle for a promising new sample preparation technique.
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Reviewer #2 (Public Review):
This is an excellent paper that describes a conceptual advance in specimen preparation methods for cryo-EM. While technically demanding wrt production, these nanofluidic devices may simplify reproducible sample prepapartion from a user perspective. The authors' cryoChip design represents an alternative to conventional well-established methods and adds to a growing number of novel specimen preparation devices. The paper is well written, concise, and comprehensively covers all relevant validations and control experiments. I enjoyed reading it. The discussion was refreshingly objective and critical about the pros and cons of this novel approach. I agree with the authors that the present MEMS devices and their future derivatives are only the beginning of exciting novel applications such as on-chip mixing, high …
Reviewer #2 (Public Review):
This is an excellent paper that describes a conceptual advance in specimen preparation methods for cryo-EM. While technically demanding wrt production, these nanofluidic devices may simplify reproducible sample prepapartion from a user perspective. The authors' cryoChip design represents an alternative to conventional well-established methods and adds to a growing number of novel specimen preparation devices. The paper is well written, concise, and comprehensively covers all relevant validations and control experiments. I enjoyed reading it. The discussion was refreshingly objective and critical about the pros and cons of this novel approach. I agree with the authors that the present MEMS devices and their future derivatives are only the beginning of exciting novel applications such as on-chip mixing, high throughput screening, time resolution and other lab-on-chip experiments.
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Reviewer #3 (Public Review):
This new nanofluidic sample cell for cryo-EM is a revolutionary idea and implementation. It addresses one of the most important and most painful bottlenecks in the cryo-EM workflow. The unreliable and wasteful conventional grid preparation method is the most impactful remaining bottleneck of today's cryo-EM. A better alternative method is desperately needed.
This manuscript describes such a promising alternative method. However, the described method has its own problems and difficulties. All of these are excellently presented and discussed in this outstandingly well written manuscript. But the manuscript fails to convince this reviewer that the new method is (already) better than the alternative methods.As the authors correctly report, quantify and discuss, the new method faces several hurdles:
• The SiNx …
Reviewer #3 (Public Review):
This new nanofluidic sample cell for cryo-EM is a revolutionary idea and implementation. It addresses one of the most important and most painful bottlenecks in the cryo-EM workflow. The unreliable and wasteful conventional grid preparation method is the most impactful remaining bottleneck of today's cryo-EM. A better alternative method is desperately needed.
This manuscript describes such a promising alternative method. However, the described method has its own problems and difficulties. All of these are excellently presented and discussed in this outstandingly well written manuscript. But the manuscript fails to convince this reviewer that the new method is (already) better than the alternative methods.As the authors correctly report, quantify and discuss, the new method faces several hurdles:
• The SiNx membranes on top and bottom of the frozen sample provide additional noise background to the images. The SiNx surfaces lead to massive adsorption of the samples, leading to overcrowding, but at least the sample doesn't seem to denature as much as when adsorbing to an air-water interface. However, the sample will not be in bulk water and for other samples that are not as strongly symmetric as these, preferred orientation is still very likely. CTF estimation on recorded images is difficult due to the signal from the SiNx layers.
• The vitrification of the SiNx grids is difficult and often leads to crystalline ice in the images. But as the authors state, vitrification methods might require adaptation to this new grid type.
• The signal to noise ratio of the recorded images is very low. The calculated ResLog B-factor of 217 Å2 for apoferritin from a Titan Krios with Gif-K2 is actually *not* in range expected for such structures, but rather terrible. The JEOL data with ResLog B-factors of 215 and 490 Å2 are not better. These should be compared to the ResLog B-factors from the same instruments for the same samples, when conventional grids are loaded. But in any case, the authors correctly discuss this issue and correctly estimate that the lower SNR from the SiNx will restrict the method to particles larger than 200kDa.
• The utilized pixel size for these datasets was surprisingly large. If 2.99Å target resolution is achieved, then I'd have expected smaller pixels in the images, such as 0.6Å/px, not 0.8127Å/px.
The most impressive feature about this new cryoChip for me is the tiny amount of sample required to prepare grids. Blotting requires microliters of sample. CryoWriting (your reference 16) requires nanoliters of sample. And your new cryoChip now seems to work with picoliters, which is another reduction of 1000 fold. That is an extremely impressive feature that may enable unique applications in special settings, as for example when imaging (in the future) the entire non-diluted cytosol of a single cell. Also, the possible extensions to prepare multiple different samples on the same nanofluidic chip are very interesting.
In summary, this is an outstandingly well written manuscript of a revolutionary method that is still in its infancy. At its present state, this technology may be useful only in very few exceptional cases, but fails to convince (yet) as general method. However, further development of this young method holds great promises to still strongly improve this novel technology.
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