The National Cryo-EM Facility
Figure 3: Power spectra provide approximate guidelines for estimating ice thickness. Examples are shown for power spectra of different images collected from a single NCEF imaging session. The average ice thickness of the corresponding images is given in the top right of each power spectrum. The vitreous ice ring at about 0.39 Å (marked with a white arrow in the bottom right image) becomes progressively more visible as the ice thickness in the sample increases. Only in vitreous ice thicker than ∼50 nm is the ring clearly visible.
enough to prevent them from contributing to the final image. An objective aperture can be used to block out electrons with the largest scattering angles, thus increasing image con- trast. An energy filter can block almost all inelastically scat- tered electrons (which only contribute noise to an image) and consequently increase the signal-to-noise ratio in the image. Te amount of scattered electrons is also proportional to the ice thickness and can be described as follows:
t = Λ *ln o Izlp
I
the unfiltered intensity of the beam, and Izlp (1)
with t being the ice thickness, Λ the inelastic mean free path, I0
the fil-
tered intensity. In principle, Λ is dependent only on voltage and the composition of the sample, but in more practical set- tings some elastically scattered electrons are also filtered in the optical path through the electron microscope, specifically when an objective aperture is used. In such cases Λ cannot be described purely as inelastic mean free path and is also depen- dent on the optical path and particularly on the size of the objective aperture.
14 With Itot Determining both the unfiltered and filtered intensity
on each data position is a generally accepted way of measur- ing the ice thickness [4], but it increases the time per image during data collection. However, it is possible to simplify the process on the Krios because the optics on the Krios are such that very few scattered electrons are eliminated without an objective aperture and the energy slit on the energy filter. Tis means that the unfiltered intensity is very close to the intensity of the electron beam without a sample, and in this case we can neglect the difference and change (1) to:
t =ΛΛ *ln*ln Dimage
I I
tot zlp
=
D tot
the total electron dose on the sample and Dimage (2)
the intensity of a beam without sample, Dtot the dose
measured on the image. Tis assumption is only correct for weakly scattering samples like thin ice and carbon films. It does not hold for very thick films (>>200 nm) or other strongly scattering material films (for example, gold foil). To determine average ice thickness for each image from the
www.microscopy-today.com • 2020 May
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