> See also: > - [[Microscopy]] # CryoEM (Cryo-Electron Microscopy) - A molecule is frozen and then placed under an electron microscope - Relies on electron beams rather than light to magnify molecules to an atomic resolution - Basic electron microscopy (without the cryo part) can often ==damage biomolecules== during the analysis. This is especially true being EM can only be performed in a vacuum (to prevent electrical discharge) which are not suitable conditions for biological molecules - - Molecules can be ==frozen via liquid ethane== (nitrogen?) to protect them from the electron beam - Crystals from frozen water often interfered with sensors. However, vitrified water is "glass-like" and randomly ordered as opposed to a crystallized form which is in an ordered, structural pattern (frozen in liquid ethane) - The idea for "protecting" the molecules likely came from a membrane-bound protein that was shielded from the harmful effects of the electron beam due to being embedded in the membrane - Molecules were also coated with a glucose solution to protect them from the vacuum While on its own, this technique often results in blurry/fuzzy/low res images of the molecule, the recent advances of machine learning have made this data useful as an averaged structure can be formed by piecing together the many different images taken of the molecule (from a variety of angles) ![[electron microscopy image anaylsis.mp4|100]] --- **Patch Motion Correction** - The sample is separated into a 10x10 grid to simplify the process of determing the change in motion. - When the ice is hit by the electrons, the corners are essentially "folded in" (doming) on itself due to the impact of the beam. This is observed by the higher degree of movement away from the center of the sensor. When motion is occurring there will be a specific direction of frequency within the micrograph A single beam of electron ![[Pasted image 20230105145412.png|200]] --- **CTF Estimation** - Cross corelation - lowest freq in center (noise) - highest frequency in outer regions (amino acids/protein segments) The end result of CryoEM analysis