2 Motion Correction, Alignment & CTF Theory

Primary goal of motion correction in cryo-EM and cryo-ET

Converting a rapid movie sequence of frames with low signal to noise ratio into a single steady image with corrected sample trajectories

Two main physical causes of sample motion during cryo-EM imaging

Beam-induced motion from beam charging and microlensing, and mechanical drift of the microscope stage arm

Unique advantage of the EER file format

Records exact XY coordinates and timing of individual electron hits on direct electron detectors for enhanced spatial and temporal resolution

Primary standard file formats for storing cryo-EM movie frames and images

MRC, TIF, and EER files

Purpose of tilt series alignment in cryo-electron tomography

Correcting X and Y spatial shifts and angular errors introduced by stage arm movement during sample tilting

Mathematical technique used to compare and align adjacent tilt frames

Cross-correlation, sliding frame sub-regions across each other to find peak overlap scores

Patch tracking method in tilt series alignment

Dividing a 2D frame into a grid of sub-patches, tracking patch movement across the tilt series, and fitting a trajectory model

Role of IMOD software in tilt series alignment

A classical processing software suite offering GUI and manual parameter tuning for tomogram alignment and reconstruction

Primary advantage of AreTomo over traditional IMOD alignment

Automated tilt series alignment without requiring manual parameter adjustments or fiducial tracking

Defocus definition in cryo-transmission electron microscopy

Intentionally positioning the specimen slightly below the focal point of the lens to generate phase contrast

Why imaging perfectly in-focus is problematic in cryo-EM

In-focus imaging produces almost zero contrast, making biological structures nearly invisible against the background

Typical defocus range used in cryo-ET tilt series acquisition

Approximately minus 2 to minus 5 micrometers underfocus

Contrast Transfer Function (CTF) definition

A mathematical function describing how the microscope optics and lensing distort spatial frequencies in the image

Point Spread Function (PSF) vs Contrast Transfer Function (CTF) relationship

PSF describes spatial distortion in real space, whereas CTF describes the same optical distortion in reciprocal Fourier space

Meaning of distance from center in a 2D Fourier transform

Distances close to the center represent low spatial frequencies, while distances far from the center represent high spatial frequencies

Cause of Thon rings in a 2D power spectrum

Oscillations of the Contrast Transfer Function between positive and negative contrast amplitudes in Fourier space

Consequence of CTF zero-crossings

Specific spatial frequencies where information transmission drops to zero and signal cannot be recovered from that single image

Phase flipping procedure in CTF correction

Inverting negative CTF amplitude regions by multiplying by minus one to restore correct phase signs

CTF fitting goal in cryo-ET software

Modeling theoretical CTF curves against experimental Thon ring patterns to accurately estimate specimen defocus

Effect of stage tilting on defocus across the sample

Creates a defocus gradient where different parts of the tilted specimen lie at different heights relative to the focal plan