We have had lots of questions around how to get the most from HexAuFoil® ultrasmall hole gold sample supports. Check out our FAQs and recent webinars to get all the information you need around HexAuFoil.
We have run a number of webinars discussing some of the first results achieved using these exciting new supports, as well as discovering how to ensure you get the best from your data collection and image processing with HexAuFoil grids.
Hear about some of the first results achieved using these exciting new supports, as well as discovering how to ensure you get the best from your data collection and image processing with HexAuFoil grids.
During the webinar you will learn about:
Discover the optimum sample preparation protocols, microscope settings, and data collection parameters to get the best results from these unique grids.
During the webinar you will learn about:
We've extended our 20% discount to the end of 2024, so you can get your own pack to try as well! Just use the code INTRO20 on your first pack.
Félix, IBS: By visual inspection mainly; but looking at the FFT also helps.
Peter, eBIC: You can use the plasmon imaging feature in EPU V3.8. Check you grids before collecting by taking some images, but we also use on the fly processing to check the data as we are collecting.
Félix, IBS: Most of the pickers work well even with very small proteins (<100 kDa), given the images are good and the ice thin. They definitely worked well for me.
Peter, eBIC: This shouldn't be a problem. I have not had any issue picking particles from HexAuFoil grids. You may need to play with blob pickers or low pass filtering the micrographs. I sometimes revert to using a blob picker in cryosparc and then classifying everything out (not great practice I know). Otherwise I find Cryolo works great.
Peter, eBIC: You only need to do the image shift calibrations for the first grid you are working on. However, if they are far out I would recommend re-doing the atlases.
Julio, TFS: You can use multigrid so long as you are not changing the magnification to collect on other grid-types in the same session. If different magnifications are used for other grids, image shift calibration will need to be repeated for that particular grid session.
Peter, eBIC: This is not an issue that I have had went working with samples with DDM. It may be worth playing with glow-discharge and blotting conditions, as this will probably help with these issues.
Peter, eBIC: I have not experienced any major issues with bending of the grids and have found them to be quite robust (very few blown out of damaged areas). I have not used nickel grids before so cannot speak to them.
Quantifoil: Gold has several properties that make it ideal as a sample support in electron microscopy, including being conductive, nonoxidizing, radiation-hard. In addition, it is does not interfere with the specimens you are studying as it is chemically inert and biocompatible. During the development of UltrAuFoil grids, other metals were tested, but it was found that Gold provided the characteristics that delivered the required performance.
Quantifoil: These grids are available by requesting a quote, and you can obtain a 20% introductory discount with the code INTRO20, when ordering before 31st August 2024. They are in stock and ship within 1-2 weeks of an order being received.
Julio, TFS: The selection of a type of grid and geometry has consequences in the algorithms that we use for foil hole selection and automatic placement of autofocus area. They are not related to optical settings.
Julio, TFS: Yes. Focusing using Z height is supported in EPU 3.8
Quantifoil: The manufacturing details are proprietary, but the method is closely based on that described by the original developers of the grids, Katerina Naydenova and Chris Russo or the MRC Laboratory of Molecular Biology, Cambridge, UK, in their 2022 Ultramicroscopy publication.
Félix, IBS: Nope - we don't have one available to try. The 50 C2 is the smallest available on our system.
Peter, eBIC: We have a 20 C2, but have not tried it with HexAuFoil grids, but it is something that cold be tried. There was no particular reason for using the 50 over the 20.
Julio, TFX: In ThermoFisher we use a 50 μm C2 aperture in Glacios
Félix, IBS: The smallest parrallel illumination on Krios is around 300 nm beam diameter; I actually used 340 nm for this dataset. So, no it was not below the parallel range.
Julio, TFS: The parallel range for Krios G4, is 308nm - 11.5μm, so Félix was within range.
Julio, TFS: We offer EPU 3 for Talos Arctica in conjunction with Gatan Bioquantum/BioContinuum K2/K3 or Glacios 1/2 with Falcon 4/4i cameras. We do not support non-embedded Gatan K3 cameras.
Peter, eBIC: Titan Krios G3i with a Gatan K3 BioQuantumn
Quantifoil: The novel production process developed to manufacture these grids is very flexible, and we are currently considering the next steps on the development pathway - so sign up for our email list and watch this space!
Peter, eBIC: I used 3.7, do not know which version Felix used.
Julio, TFS: We offer support to HexAuFoil grids (gold/hexagonal geometry) since EPU 3.1.
Julio, TFS: Full support of tilted acquisition is granted in EPU 3.8. Félix, which one did you use?
Félix, IBS: EPU 3.6.0; TEM 3.18.1
Peter, eBIC: I have not experienced any issues with alignments when using the latest versions of EPU. I also believe the hexagonal arrangement maximises the number of holes available.
Quantifoil: Peter is right, the hexagonal packing of the holes maximises hole density, to allow more imaging between beam shifts. The hexagonal grid mesh offers the widest open viewing area for any given mesh size, allowing us to take advantage of the support offered by the equivalent of a 400 mesh grid for the very thin gold foil, while delivering plenty of open viewing area.
Quantifoil: The tilted data collection reveals particle movement perpendicular to the surface of the ice, which can't be observed when there is no tilt. It is due to relaxation of strain in the ice during bending: to avoid this phenomenon, the ice thickness must be at least ~1/11 of the hole diameter (Naydenova et al, 2020). The fact that Félix observed this on HexAuFoil grids where the holes are only ~290 nm in diameter suggests that his ice was very thin indeed!
Quantifoil: There is no reason you can't use HexAuFoil grids for cryoET. However, the holes in the gold foil are small (~290 nm), and the mesh size is also small. This will limit the tilt angle you can use quite significantly if you are not FIBmilling. If you are FIB-milling, then the advantages of the grids deliver for SPA, with very thin ice, may not be as helpful to you.
However, as there is very little Carbon in the manufacturing process, so that the grids have low autofluorescence, optimizing the grid format for tomography is one of the options in our development pathway.
