Advancing the application of cryoelectron tomography of intact cells and tissues.

We perform standard cryoelectron tomography (cryoET) sample preparation, provide access to advanced cryoET specimen preparation capabilities, train users in the development of the skills needed for independent research in cryoET, and continually update methods and technologies to stay at the forefront of this field.

WORKFLOW
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APPLICATION PROCESS

Light Microscopy

Light microscopy is done using Zeiss LSM 900 equipped with Airyscan2 detector and Linkam cryostage

Focused Ion Beam Scanning Electron Microscopy

Lamella thinning, block face milling and other FIB needs are accomodated by our Helios and Aquilos FIB-SEMs

Transmission Electron Microscopy

Data from thin specimens will be collected on a energy-filtered Krios TEM

WORKFLOW

Every in-situ workflow is as unique as the biology in question.  It usually starts at sample preparation and terminates with a high-resolution cryoET data collection

Specimen preparation

The ideal specimen for focused ion beam (FIB) thinning is a cell or a group of cells positioned on an EM-grid. However, different questions require different approaches. Bulk tissue, cell suspension and bacterial colonies are all candidates for in-situ analysis. While most of the specimen preparation is expected to done by the user, we will aid and provide expertise when necessary.

Vitrification

Vitrification of biological specimen is achieved by either plunge-freezing into liquid ethane or by high-pressure freezing (HPF). HPF is recommended for tissue samples, large cell aggregates and other bulk specimen, while conventional plunge-freezing is used for smaller cells, like bacteria and yeast, and individual eukaryotic cells.

Light microscopy

Frozen, fluorescently labeled cells can be first analyzed in a dedicated confocal microscope equipped with a cryo stage. Specimens can be analyzed to ensure that the expected phenotype is observed, ice is sufficiently thin and the grids themselves have enough targets for downstream analysis.

FIB-SEM

Vitrified specimen can be FIB-milled to a required thickness to allow data collection in a Transmission Electron Microscope (TEM). Alternatively, volume data can be acquired during the milling process yielding 3D reconstructions on a whole-cell scale.

TEM

Once your samples are done here, they will be shipped to the central hub or back to you, where final imaging experiments, usually tomographic data collection, are conducted.

APPLICATION PROCESS

Application to the NCITU is administered through the joint tomography center portal.

Project meeting

Prior to submitting an application, the in-situ team would meet with you to discuss your project and assess its feasibility. We would be able to provide suggestions and expertise in order for you to get the most out of our center.

Application

A centralized application process will make submitting a project simple: a one-page project description will be distributed somehow someway to the spokes and hub and they will decide if they like you.

Review

When you submit your application, it will be reviewed by a committee which will decide if it is approved based on given scores within the review categories. If your application is approved, it will be added to a queue and time will be allocated accordingly.

Project execution

Once your project is allocated a timeslot, you will be able to send us your samples (i.e. list some types here). The easiest way for us to know when your sample would be arriving is to provide us with a tracking number. We will store your samples as is until your time starts, where we will begin vitrification or milling based on your sample.