TEM 3D reconstruction and CryEM tomography Flashcards
Cross-correlation coefficient CCC
The Cross Correlation coefficient CCC is the value of the cross correlation function CCF at
a displacement vector of 0,0 for images with normalized grey values
The CCC can have values of: -1 < CCC < 1
formula: sum of (positions_x pixels intensity - average gray value)the same but for another image /(stdev1stdev2)
class averages
Class averages represent different particle views with improved SNR
central section theorem
The central section theorem states that each 2D projection of a 3D object is equivalent to a central section (or central slice) through the 3D Fourier transform of that object. Specifically:
Let π(π₯,π¦,π§)
f(x,y,z) be a 3D object with its Fourier transform πΉ(π’,π£,π€)
F(u,v,w).
A 2D projection of this 3D object along a particular direction (e.g., the
π§
z-axis) corresponds to a 2D slice of the 3D Fourier transform πΉ(π’,π£,0)
F(u,v,0), which lies in a plane through the origin (the central plane) perpendicular to the projection direction.
then taking iFT of the 3D projection should give you a 3D reconstructioon
CEMOVIS
Cryo Electron Microscopy Of Vitreous Sections
very complicated vitrification at high pressure to freeze a whole tissue sample. Then cryomicrotome cuts the tissue to form section with a very sharp diamond knife.
FIB milling
Focused ion beam milling
main priniple of tomography
tilt the sample stage containing 3D object to get multiple anlged projections so you can reconstruct the object. only one copy needed
how to avoid the mechanical drift during tomography
you cant. The beam tracks the position on the sampel to acount for it. You take additional images on the tilt axis close to the ROI and track any changes there (so you dont have to check the ROI too much and damage it)
how can you generate phase contrast
Electron waves passing though a sample will have a phase delay introduced due to the switch of βrefractive indexβ / the charge of the sample. However, if you converge all of the waves in one point akak be in focus you wont be able to distingush this. However, if you are aslightly out of focus now the waves that have passed through the material and have phase contrast will be focused at a different point. THey will constructively or destructively interfere resulting in visible amplitude contrast, but also more modulation by the CTF of course.
How can you account for lost information by the convolution with the CTF?
Use different defocus points to obtian differently oscillating CTF modulated images that contain the missing info.
How can you reduce the amount of inelastic scattering?
Higher Energy, the higher the enrgy the longer the inelastic mean free path will be ( the average distance an electron travel betweeen two scatering events)
How to get rid of inelastically scattered electrons while imagein in cryEM tomography.
Pass electrons through a prims that changes trajectories based on KE. Then you can apply an aperture and seperate the elastic from inelastic ones and improve contrast. q
In what order should you acquire different tilt angle images?
Dose symmetrically. Go through low angles first bothe in + and minus. Not sequentially. THis way your lower tilt angles have better quality sample and sample damage is symmetrically distributrd. This is also better because higher tilts are also generally worse images because the sample will be thicker.
How do you account for higher noise in higher tilt images?
Post processing. YOu weigh down those tilts so they acount less for the final reconstruction.
What will happen if you overlay all tomography projection in one image?
You will obtain very blurry reconstruction. THis is because low frequencies are being overrepresented resulting in blur. Thus weightin is required when getting your final reconstruction.
