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National CryoEM Facility Frequently Asked Questions

 

If our lab has access to our university’s high-end imaging facility, can we still use NCEF?

Yes.

Do I need to have an NCI or other NIH grant to use the NCEF? 

No, all cancer-related projects regardless of grant status are welcome. 

Can I send multiple Sample Information Forms (SIF) for approval? 

Yes.  However, we only allow one PI’s run session in our active run queue at a time.  Once multiple SIFs from the same PI are approved and the corresponding samples arrive at our facility, we will only allow one 48-hour run session in the active run queue and the other approved sessions will be added to our holding queue.

What requirements does NCEF have for the pre-screening process?

Pre-screening is very important as it ensures that we can set up a useful data collection run for each user. Keep in mind that we will need a consistently high-quality grid for imaging, not just a few good areas on a grid. To ensure this high quality, we require different level magnification prescreening images in the SIF.  Please make sure to show representative areas of a typical grid.

Can I use old images as screening images for my SIF? 

No, please submit only images from a grid made in the same freezing session as the grids you plan to send to NCEF.  However, if we are collecting on grids that we have already imaged from a previous session, then yes you may use the old images.

How can I optimize my grids for faster imaging rates at the NCEF?

Imaging speed is dependent on several parameters including number of frames, exposure time, focus frequency, and others. Many of these parameters can be changed and discussed during the run setup (usually video conference). The most significant difference is the choice of collecting data by moving to data locations using image shift versus using stage shift.

As image shift will induce a certain amount of image aberration like astigmatism and coma, we recommend using image shifts only for relatively small distances within one hole on a holey carbon film. Using lacey carbon film or holes with 2-micron diameter (e.g. R 2/2 grids) will allow NCEF to collect data using image shift at much higher speeds.

Can we get help from NCEF with optimizing freezing conditions and protein purification?

We are happy to consult with users and provide advice through email and/or phone conversations.

What shipping service should be used to ship our dry shipper? 

We recommend FedEx or UPS, OVERNIGHT and to please ship out on a Monday, Tuesday, or Wednesday to avoid your dewar arriving on the weekend.

What dry shipper does NCEF recommend?

NCEF does not recommend a specific dry shipper. However, having a hard box for shipping is recommended. We have successfully imaged samples shipped in a variety of shippers and shipping containers.

Should we send more than 8 grids?

NCEF will load up to 8 grids for each imaging session. Usually grid making is reproducibly enough to make one of these grids viable for an imaging session. In the rare event that none of these grids work out, it is unlikely that additional grids made in the same freezing session will work better. So please restrict your shipment to the 8 grids.

Should we send our grids in storage pucks?

Yes, NCEF can handle storage pucks. We will transfer the grid boxes to NCEF storage and return the pucks with your dry shipper.

Can I ship C-flat grids in a dry shipper?

At NCEF we have noticed that shipping C-flat grids in dry shippers according to the standard methods leads to significant damage of grids and makes imaging almost impossible. C-flat grids can either be delivered in person or shipped with special adjustments in the dry shipper. Please contact us before you ship C-flat grids in a dry shipper.

Can we image gold foil grids?

Yes, NCEF can handle imaging on gold foil grids without problems. However, gold foil can present a problem under certain circumstances, e.g. for phase plate imaging. Please discuss with us if you plan on phase plate imaging with gold foil.

Can we ship pre-clipped grids?

Yes, you can send pre-clipped grids to NCEF. We will examine carefully and reject grids that appear badly clipped or bent.

Does our lab have to pay for materials for grid clipping or are there any other fees when we use NCEF?

No, but you are responsible for shipping costs. Please include a pre-paid return shipping label for the return of your dewar from our facility address. 

How soon can our imaging session happen after we send grids?  

We have a dynamic queue with lead times typically under 2 months.

Can we visit for our imaging session?

You are welcome to visit NCEF for your imaging session. However, due to our dynamic queue we cannot absolutely guarantee the exact time of the imaging session. You will also need to make your own travel arrangements.

How soon can I get my data?

We provide a download link for imaging sessions on Tuesdays and Thursdays each week. Based on when your data collection ends, you can typically expect the download link on the next Tuesday or Thursday.

What are the best imaging settings for my data collection?

Settings for the imaging session depend a lot on sample and resolution target. If you have specific ideas, you can let us know already in the SIF. Otherwise we would discuss specifics in the video meeting just before setting up the run.

Can we use the phase plate for our imaging session?

Yes, the NCEF Krios microscopes have phase plates and you can request phase plate imaging for your imaging session. Phase plate imaging with gold foil is problematic – please discuss with us if you are planning on that.

Can I set up my own data collection by visiting NCEF or through remote connection? 

No, NCEF policy is to not let users run the microscope even for setting up targets. We do welcome your input on how to find the best imaging areas for your specific sample.

How long will our grids be stored after the imaging session and can we use them for another session?

We will store grids after imaging sessions for at least 3 months. If you are planning to use grids from the same set for another imaging session, please let us know before the 3 months are over. If your imaging session had several viable grids, we can use another grid for another imaging session. Some small amount of ice contamination is expected, but we have re-imaged several grids without observing problematic contamination. We usually discard unused grids after 3 months. 

Are we required to give feedback to the NCEF about the data processing?

You are not required to give any feedback to NCEF about your data. However, it is extremely useful for us to learn what the outcome of each imaging session, and it helps us to improve our future performance.

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