In NanoImaging Services' 15+ years of helping pharmaceutical and biotech companies harness the power of cryo-EM, we’ve answered all types of questions. To help get you started in cryo-EM with NIS, we're sharing answers to our most commonly asked questions below. Please reach out if the answer you need isn’t addressed below!
How much sample should I submit?
It is best to discuss this directly with your project manager, but in general most projects require 50-100μl of sample along with ~1.5mL of buffer in which the sample may be diluted in case it is too concentrated for imaging. Although as little as 3uL of sample is necessary to prepare a single grid, multiple grids are typically prepared. In addition, several conditions may have to be tested to optimize the specimen for imaging.
For structural biology projects, typically 1 mg of your protein or complex at 1 - 5 mg/mL concentration is needed. Depending on your sample, we might ask for a higher concentration, however, this can be discussed with our technical team on a call.
For nanoparticle characterization projects, we typically request duplicates of ~50μl of material for each sample, at approximately ~1mg/mL protein, 10-30 mg/mL lipid, or E12 particles/mL.
What buffers can I use for my sample?
A minimal buffer is preferred (i.e. 20 mM HEPES, pH 7.5) to minimize background. If specific components are known to be necessary for sample stability, these should be present in the minimum amount possible. Greater than 1% of glycerol, sucrose, or DMSO in the diluted sample should be avoided. Detergents can be used but should be kept below their CMC, unless they are used to solubilize the sample. For negative stain specimens, phosphate buffers should be avoided.
For nanoparticle characterization samples, the dilution buffer composition can be your formulation buffer, but preferably without sucrose, glycerol, maltose (or other cryo-protectants) or high salt. These components negatively affect the particle contrast In the images. If you cannot get around adding any of these, NIS would suggest keeping the cryo-protectant below 2% and the salt concentration below 500mM.
For structural biology samples, most reagents and buffer conditions are amenable to cryo-EM however not all are compatible with cryo-EM sample preparation. For example, DMSO may interfere with the vitrification process and cause crystal formation. Similarly, excessive sucrose can increase sample viscosity and interfere with particle alignment during data collection.
What sample concentration should I prepare?
For negative stain of protein samples, 0.01-0.5 mg/mL is acceptable. For cryo-EM single particle analyses workflows, 1-5 mg/mL is preferred. In both cases, a sample at the highest possible concentration is preferred since serial dilutions are performed to optimize the TEM grids. For viral or particle concentration, it is best to provide E12-13 particles/mL.
How should I label my samples?
Properly label sample tube(s) and include relevant information such as concentration, buffer, storage requirements etc. Please indicate whether the samples are hazardous and, if so, provide safety and handling precautions.
How should I ship my samples?
Pack samples at the appropriate temperature (room temperature, wet ice, dry ice) with adequate padding to ensure that they will not be damaged during transit. Use a courier service for shipment and delivery. For most samples, overnight delivery is recommended whenever available. Your project manager will provide you with a sample submission form (SSF) - please fill it out entirely, include a hard copy with your shipment, and email the filled form to your project manager when your package is shipped. The tracking information can be sent separately.
NanoImaging Services accepts sample delivery from 8:30am – 5pm (local time at the facility), Monday – Friday. Samples are not received on Saturdays, Sundays, or holidays.
Please confirm with your primary point of contact at NIS to which location you should forward your samples.
NanoImaging Services West
Attn: Sample Receiving
4940 Carroll Canyon Road, Suite 115
San Diego, CA 92121
NanoImaging Services East
Attn: Sample Receiving
4C Gill St
Woburn, MA 01801
What information is needed to fill out your Sample Submission Form?
Our sample submission form asks you to fill out:
- Company Name
- Your Name & Email
- The date you are sending the package
- Tracking number (this can be sent separately to NIS)
- Project number (if not known, please reference the quote for the samples included)
- Shipping method (from a drop down list)
- Total number of aliquots (including samples, buffers, duplicates, etc.)
- Additional items being sent, if any (filtering devices, etc.)
- BSL and OEB levels (from a drop down list)
- List any safety precautions you take while working with these samples
- List information about the samples (protein, virus, lyophilized sample, etc.)
- Temperatures for storing the sample and working with the sample
- How long the samples are stable after thawing (or arrival at NIS)
- Instructions for what to do with the remaining samples after we have prepared your samples for imaging
- Names, concentration, and buffer present for all samples
A paper copy should be included in the shipment and a digital copy can be emailed back to NIS prior to shipment. The tracking information can be sent separately when it becomes available, if needed.
What about international shipments?
Please contact us if you need assistance with documentation required for shipping samples from overseas. NIS will provide an End-User Letter on company letterhead and a Power of Attorney statement prior to shipping. We currently have Power of Attorney statements on file with both World Courier and Marken. All other information regarding the samples needs to be provided by the shipper to the courier.
Please notify us by email when the samples are shipped and include the shipment tracking numbers.
What kind of report should I expect?
A written final report of the study summarizes the imaging and analysis efforts. Representative images are provided as attachments to support key observations and conclusions. The report and entire set of all image files and analysis data produced in the study are available for viewing online using our secure web portal or can be transferred to the client via the portal or on a USB drive. The report includes documentation of sample receipt and handling and details on any analysis or reconstruction.
Is there support for interpreting my data?
Each study is followed by a teleconference call during which we discuss the study’s major findings and give tips on getting the most out of your data. This call is attended by the scientists responsible for conducting your study to ensure that any questions you might have are properly answered.
Will I be able to see my images before I get the final report?
Yes, we provide a secure, web-based system for clients to both view and evaluate the images collected for each experiment. Depending on the type of experiment, images may be available in real-time, allowing remote collaboration to determine the best conditions for your project.
How do I get the results of a project?
As part of your project set up, NIS provides each client a secure account on our web-based portal, which serves as a dashboard for report and data delivery. Results of your study can be quickly downloaded within minutes of notification of a completed project.
What are the options for getting a copy of my images?
NIS supports uploads to Amazon’s Simple Storage Service (AWS S3), and Google’s Cloud Storage Service (GCS). We also can provide images on client-provided encrypted hard drives. If you are collecting images for high resolution single particle processing, we have high bandwidth fiber optic internet to upload images to AWS S3 or GCS concurrent with the data collection to support near real-time data processing.
How will my data be stored during my project?
Results from image collection and data analysis are stored on secure hardware in two different locations (2N redundant), encrypted at rest and isolated from external networks. Our main data center has 24x7x365 on-site security, 2N redundant power, a dedicated plant for cooling, and redundant network connectivity.