April 7 Flashcards
What is a tomogram?
It is a movie of images
How does computed tomography (CT) scan work?
It has X ray source, see what shadow it casts. Do this at many angles, gett images
For XRC vs CT scanner, what do they have similarities? Diffs?
Both use X rays, collect data from different angles, add images to get final result.
XRC uses diffraction by crystal, Ct uses transmission through sample. Also XRC gives 3D map, CT scanner gies 2D cross sections, can be joined to give 3D image
What is the general workflow for cryo-tomography?
1.Plunge freezing in liquid ethane
2. Use focused ion beam to get sample thin enough so electrons can be transmitted through (milling)
3. Tilt-series collection
4. Reconstruction
Do proteins, viruses, bacteria, cells, and tissue samples need thinning, what type?
Viruses adn rpoteins do not need thinning. Bacteria may, cells do use focused ion beam. Tissue samples are thick, so cut out and lift out a sample.
Why do we go from -60 to +60 degrees on Cryo-ET, why not more angles?
It is because beam is going through too much sample, too thick for transmission. Also, more electrons interacting with sample gives more damage.
What are data collection limits from cryo-ET?
We don’t know if top or bottom of sample, it is arbirary. We also have higher radiation damage at low angles, and less electron transmission near 60 degrees. So missing info
How do we correct for missing wedges of data at low and high angles (near 0 and 60) for Cryo-ET?
We do a dual tilt. So rotate sample 90 degrees on other axis, then get another tilt series. Solves missing wedge
What is ideal electron dose for Cryo-ET? Why do we have limited resolution in Cryo-ET?
1-10 electrons per angstrom squared. The damage destros high resolution features, so get lower signal;noise.
What does dual-axis tomography do (3)?
We do 2 tilt series at a t 90 degree in-plane rotation
Reduces the missing wedge of info, and mitigates anisotropy (deals with uneveness in signal:noise ratio)
What is dose fractionation (2)?
It is when total electron dose is split over 2 tilt series. Each series gets half the dose. Kind of get missing pyramid shape, better than traditional tilt series with 1 axis
What is sub-tomogram averaging?
It is when put sample in rod holder, so it can be rotated 180 degrees.
If we have many identical copies of protein/particle, then hopefully have them in random orientations.
What is segmentation in Cryo-ET?
It is when you determine what is the mitochondria etc from the picture
