Journal club: Adineh et al., 2016
C9940 3-Dimensional Transmission Electron Microscopy
S1007 Doing structural biology with the electron microscope
May 29, 2017
FIRST:
Fourier Shell Correlation, revisited
Fourier Shell Correlation (FSC)
Properties:
- Fourier terms have amplitude + phase.
- Correlation values range from -1 to +1.
- Noise should give an average of 0.
- The comparison is done as a function of spatial frequency (or “resolution”)
Reconstruction2
term 2
Reconstruction1
term A1
Fourier Shell Correlation (FSC)
Reconstruction1
Reconstruction2
term A1 term A2term B1 term B2
Fourier Shell Correlation (FSC)
term A1 term A2term B1 term B2
Fourier Shell Correlation (FSC)
Adineh et al., 2016
Schematic for the cellular sample preparation and APT data acquisition run (a) HeLa cells grown on gold-coated silicon
substrates were coated with a thick layer of gold followed by a strip of platinum for further protection. Using FIB based
protocols, a wedge containing a “sandwiched” cell volume was extracted and a sub-volume was attached to a microtip post in
an orthogonal orientation. Following another protective nickel coat, the sub-volume was shaped into an APT-amenable tip by
further FIB milling. (b) During an APT acquisition run, ions from the tip are extracted and are detected by a position-sensitive
detector; this part of the tip is consumed as a result. Time-of-flight measurements of these detected ions allows their chemical
identification in a mass spectrum, and a reverse-point algorithm can then be used to spatially place the ions in a high resolution
reconstruction of the original tip. Narayan... Subramaniam, 2012, J. Struct. Biol
Figure 1. Schematic of the proposed
approach for site-specific 3D atomic-scale
analysis of bacterial cells. (a) A specimen
from a single bacterial cells was retrieved
using FIB-lift-out technique, and (b) a sitespecific
final needle-shaped specimen tip
was achieved by precise annular FIB milling
and contained a specific region of the
original cell (either cell envelope or
intracellular domain). (c−d) Selected site
specific needle-shaped specimens
from the cell envelope and intracellular
domains were observed with conventional
TEM. (e) Prior to APT, sufficient electrical
conductivity is achieved with a nanoscale
layer of metallic coating, which allows field
evaporation of the specimen tip via pulsedvoltage
APT and leads to (f) a mass-tocharge-state
ratio spectrum of the ionic
species followed by (g) an actual 3D
reconstruction of the tomographic map at
near-atomic resolution.
Supporting Figure 1:
SEM images taken
during the FIB lift-out
process to create APT
specimen from
bacterial cells.
Figure 2. Mass-tocharge-state
ratio
spectra acquired from
the cell envelope of A.
baumannii, revealing
distinct information
from both a susceptible
strain (ATCC 19606 S)
highlighted in blue and
a resistant strain
(ATCC 19606 R)
highlighted in red.
Figure 3. 3D ion species
maps from the APT
reconstructions of (a)
intracellular domain of a
susceptible strain and (b)
intracellular domain of a
resistant strain.
Figure 4. Cryo-TEM images of
the cell envelope from (a) control
and (b) polymyxin B treated A.
baumannii susceptible strain
(ATCC19606 S) and from the
control and polymyxin B treated
resistant strain (ATCC19606 R)
in (c) and (d), respectively.
Figure 5. 3D ion species maps
from the APT reconstructions of
(a) cell envelope of susceptible
strain, and (b) cell envelope of
resistant strain.
DISCUSSION
Cellular tips can be accurately
reconstructed
(a) Mass spectrum of first 1.5 ×
10 6 ions field evaporated from
the tip, plotted in 0.01 Da
bins. The broad peak
corresponding to nickel is boxed,
and relevant ions plotted in the
reconstruction are marked. (b)
Corresponding 3D reconstruction
of this part of the tip. (c) 10 nm
slice from the central plane of the
reconstruction, showing
distribution of selected ions
nickel (red), gallium (green) and
carbon (brown). (d)
Corresponding plot of %
abundance of these ions versus
depth along the z axis. (e) Plot of
% abundance versus depth of
less abundant ions, nitrogen
(blue), potassium (green),
oxygen (orange) and sodium
(pink) of the
same slice. Carbon has been
plotted as a brown dotted line at
1/10 th of its original abundance.
Scale bars: b, 10 nm; c, 5 nm.Narayan... Subramaniam, 2012, J. Struct. Biol