At Structural Biology Core Facility we can help you with preparing your samples for cyro-EM measurments.
Sample preparation and other services:
SOLARIS will offer application based access to a 300kV Titan Krios G3i cryo electron microscope equipped with two direct electron detectors in the near future. The microscope has been delivered last December and the construction of the facility in Krakow has already started. If you are interested, please follow our websites and/or contact us. After a successful installation and testing period, the Cryo-EM will most likely be added to our regular offer starting from the next call.
One of the most crucial steps in obtaining excellent cryo-EM data is proper sample preparation. In the case of transmission cryo-electron microscopy, preparation process consists of two steps:
1) Depositing sample on the grid;
2) Rapid freezing of the hydrated sample.
First, a sample is applied to microscope grids covered with carbon or polymer film. To reduce the hydrophobic properties of the films, the glow discharge system can be used. In our facility, this step can be done with the use of JEOL HDT-400 unit. Secondly, the sample is plunged into liquid ethane with a cooling rate of about million degrees per second. Fast freezing prevents water from forming crystals and allows to obtain sample locked in a “vitreous” amorphous ice. Because of that, the described process is often called vitrification. At Structural Biology Core Facility a FEI Vitrobot mark IV is available, a device for semi-automated plunge freezing. With the use of Vitrobot, parameters like temperature, humidity, time of application, as well as the number and time of blots can be adjusted for achieving suitable particle distribution and thickness of ice. Moreover, parameters like material of TEM grid, holes in support film, their shape and size, their placement and distance between them have also a great influence on the final quality of the sample. Our mission is to help with optimizing the preparation process to obtain the best results possible.
Negative stain is used to check the quality of already prepared samples. As a relatively fast and easy-to-conduct procedure, it is often the first step to evaluate the quality, homogeneity, and dispersion of the objects of interest on the TEM grid. Determining those parameters is important for optimizing preparation process and for obtaining best results in high-resolution imaging and/or tomography. Negative stain is a preparation method where highly electron-transparent macromolecules, cells or tissues are coated with an amorphous layer of heavy compounds which are dispersing electrons. The above mentioned procedure allows high-contrast imaging of objects consisting mainly of the light elements. Structural Biology Core Facility can help with conducting staining process and of the assessment of its outcome.
Our goal is to make your project go as smoothly and as efficiently as possible. Our staff is ready to help with experiment design, sample preparation and trials recommended for high-resolution data collection. We can guide you to choose appropriate grids and sample preparation conditions, we help with negative staining and we organize samples screening and initial data collection.
If you are interested in vitrification know-how, we can organize a training session with practical use of FEI Vitrobot mark IV located in our Facility.