Hi, I was told that defocus has negative values, but in cryoSPARC, I see all positive values for defocus. Could you please let me know what is the unit of these defocus values and why is defocus so big and positive while listing in CryoSPARC?
positive defocus = negative focus. the terminology is jumbled in the field in part because automated collection programs ask for negative defocus values which is an incorrect double neg. Implicitly, defocus means underfocus, and if you see weird white halo particles it means you are over focus. Cryosparc numbers are in angstroms (+10,000 defocus is 1µm under focus). Yours are -2.8µm, which is on the correct side of focus but significantly far from focus. I would shoot for 5,000-15,000, which is -.5 through -1.5µm defocus.
Everyone: Take a large dataset you collected and plot defocus observed on Y-axis and CTF estimated on X-axis. Close to focus correlates with high resolution, at the expense of contrast / being able to pick every particle.
Defocus just means away from focus. Underfocus is one way (the way we use on all modern TEMs) and overfocus is the other way. On the microscope, and thus in the collection programs, underfocus is negative (you always turn the focus knob to the left).
In processing programs underfocus is often positive to simplify things. In cryoSPARC underfocus is positive and the units are indeed Ångstrom. The “df avg” is the average of the two astigmatic defocus values (often called U and V) that are the major and minor axes of the elliptical CTF.