Cryo-Electron Microscopy of Viruses

Transcription

1 Blockkurs Biophysic and Structural Biology 2013 Praktikumsversuch at C-CINA Cryo-Electron Microscopy of Viruses In this practical we will compare electron microscopy of negatively stained and frozen-hydrated samples. The goal of the exercise is to; - Learn how to prepare and observe frozen-hydrated samples. - Understand the benefits and challenges of observing biological samples under cryo-conditions in vitreous ice with the transmission electron microscope (TEM).

2 A) Sample preparation 1- Negative staining using uranyl-acetate A relatively quick and simple method for the preparation of samples for room temperature observation in the TEM. Protocol; Selection of a suitable sample and sample handling Preparing specimen grids as the sample support Applying the sample to the grid Washing, staining and drying the sample 2- Plunge-freezing to prepare a frozen hydrated sample for observation in the TEM How to vitrify the sample? Liquid ethane is used to vitrify water-rich samples for electron microscopy. The specimen, dispersed in buffer is adsorbed to the sample support carrier (fenestrated grid). After a short incubation time, the sample is then blotted to form a thin film and subsequently plunge-frozen by rapid immersion in liquid ethane at around -180 C.

3 Protocol; We will use a semi-automated plunging device (Vitrobot) Liquefaction method for ethane Glow-discharge of the TEM grids Apply 2-4 µl of concentrated sample Blotting, quick-freezing, transfer to the specimen storage box B) Electron microscopy 1- Imaging negatively stained samples at room temperature We will use a Philips CM10 electron microscope Check the microscope vacuum is sufficient to load the sample Load the sample into the room temperature sample holder Switch on the electron beam to produce an image Screen the grid at low magnification to select suitable grid areas for imaging at higher magnification Record images after correcting the imaging parameters on the TEM (e.g. correct focus and astigmatism, centre the beam)

4 2- Imaging frozen-hydrated samples at low (approx C) temperature We will use a Philips CM200 FEG electron microscope Switch on the High 200kV, increase the extraction voltage in the Field Emission Gun (FEG) Set-up of the cryo-electron microscope for sample observation (check beam settings) Cool the microscope anti-contamination device, cool the GATAN cryo-holder Transfer the frozen grid into the GATAN cryo-holder Insert the cryo-holder with sample into the CM-200 FEG Quickly screen the grid for presence of suitable ice areas for subsequent imaging Collect images on the CMOS CCD camera How to observe the grid without damage to the sample? Imaging the sample on 4K x 4K CMOS camera using lowdose conditions Align the microscope,(eucentric hight, astigmatism, etc), set up the low dose parameters; Search at low magnification (approx x) Focus at high magnification (approx x) away from the region of interest (position S1 or S2) (using the fast Fourier transform (FFT) of the image and observing the effect of defocus on the Contrast Transfer Function (CTF) signal) Exposure, taking a micrograph with a short exposure time on the CMOS camera

5 Questions: How do you measure the electron dose in order to prevent sample damage and what is the maximum tolerable dose? How is the resolution of the image determined?

6 Conclusion: Plunge-freezing versus negative-staining Preparation (time) Grids Sample damage Artifacts Contrast Resolution Negative staining Cryo-EM Instructors: Mohamed Chami, Ken Goldie, Philippe Ringler C-CINA - Center for Cellular Imaging and NanoAnalytics Biozentrum, University Basel at the Department for Biosystems Science and Engineering (D-BSSE) WRO-1058 Mattenstrasse 26 CH-4058 Basel